Postsynaptic A-Adrenergic Receptors Potentiate the ,8-Adrenergic Stimulation of Pineal Serotonin N-Acetyltransferase

Proc. NatLAcad. Sci. USA Vol. 80, pp. 599-603, January 1983 Neurobiology

Postsynaptic a-adrenergic receptors potentiate the ,8-adrenergic stimulation of pineal serotonin N-acetyltransferase (melatonin/norepinephrine/cyclic AMP/isoproterenol/phenylephrine) DAVID C. KLEIN, DAVID SUGDEN, AND JOAN L. WELLER Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Berta Scharrer, October 12, 1982

ABSTRACT The role played by postsynaptic et-adrenergic activity and melatonin production are also regulated by both a- receptors in the stimulation of pineal N-acetyltransferase (EC and (3adrenergic receptors located on postsynaptic structures. 2.3.1.5) and [3H]melatonin production was investigated in the rat. We have studied this question and our results, presented here, In vivo studies indicated that phenylephrine, an a-adrenergic ag- call for a reexamination of current concepts of the adrenergic onist, potentiated and prolonged the effects ofisoproterenol, a P control of the pineal gland. adrenergic agonist. Similar observations were made in organ culture with glands devoid of functional nerve endings. In addition, a combination of 1 FAM prazosin, an a1-adrenergic blocking agent, MATERIALS AND METHODS and 1 FM propranolol, a -adrenergic blocking agent, was many We used 150- to 250-g male Sprague-Dawley rats (Zivic- times more potent then either agent alone in blocking the stimu- Miller, Allison Park, PA). Theanimals were keptina 14-hrlight/ latory effects of norepinephrine on N-acetyltransferase activity 10-hr dark cycle (lights on at 0500 hr) in our facilities for 7-14 and [3Hlmelatonin production. These findings establish that nor- days prior to treatment or removal of their pineal glands for epinephrine acting through ar- and (3-adrenergic receptors stim- organ culture studies. All animals were allowed food and water ulates rat pineal N-acetyltransferase activity and, as a result, the ad lib. For in vivo studies, drugs were dissolved in 0.9% saline production of melatonin. Apparently, (-adrenergic activation is immediately before use and injected subcutaneously at 1200 hr an absolute requirement, and an et-adrenergic receptor mecha- in a volume of 1 ml/kg ofbody weight. nism potentiates -adrenergic activation. These findings are sig- The methods used forpineal glandculture (16), measurement nificant because they demonstrate a-adrenergic potentiation of ofthe formation of[3H]melatonin from [3H]tryptophan (18, 19), (-adrenergic effects. In addition, they indicate that the widely and assay of N-acetyltransferase activity (18) have been de- held belief that melatonin production is regulated exclusively by scribed. In all in vitro studies, pineal glands were incubated for a postsynaptic -adrenergic mechanism must be revised. 48 hr prior to treatment to allow the presynaptic elements to degenerate. This yields a relatively pure postsynaptic prepa- A daily rhythm in circulating melatonin occurs in all mammals ration ofpinealocytes. Unless otherwise stated, all drug treat- (1). In the rat this is generated by a large rhythm in the activity ments were for 6 hr. ofpineal arylamine N-acetyltransferase (EC 2.3.1.5) (1-4), the The sources of most of the drugs and other materials used enzyme that converts serotonin to the immediate precursor of have been reported (4, 18, 20). We obtained prazosin hydro- melatonin, N-acetylserotonin. chloride from Pfizer (Groton, CT), clonidine hydrochloride and The activity of N-acetyltransferase is regulated by a neural ST 587 as the nitrate salt from Boehringer Ingelheim (Ridge- circuit that stimulates the nocturnal release of norepinephrine field, CT), and yohimbine hydrochloride from Sigma. Except from sympathetic nerves in the pineal gland (5-8). Norepi- for dl-octopamine, the i-isomer of asymmetric compounds was nephrine acts through an adrenergic mechanism to increase in- used. tracellular cyclic AMP (9, 10) which induces and activates N- Alldata are presented as the mean ± SEM. The vertical error acetyltransferase (3, 4), leading to an increase in melatonin pro- bars in the figures represent the SEM. Statistical comparisons duction (11, 12). used Student's t test. It is now generally thought, based on a number of studies (11-17), that norepinephrine acts exclusively through postsynaptic /3-adrenergic receptors to increase cyclic AMP, N-acetyl- RESULTS transferase- activity, and melatonin production. We were sur- In Vivo Studies. To examine whether N-acetyltransferase prised, therefore, by recent observations (unpublished data) activity is regulated by both a- and f-adrenergic receptors in which seem to indicate that norepinephrine increases pineal vivo, we treated rats with isoproterenol, phenylephrine, or a cyclic AMP through an action involving both a- and (3-adren- combination ofthese drugs. ergic receptors. We found that a combination of isoproterenol Dose-response studies indicated that isoproterenol alone (10 ,tM) and phenylephrine (10 ,uM) produced a stimulation at stimulated N-acetyltransferase activity (Fig. 1), in agreement 10 min, of cyclic AMP and cyclic GMP equivalent to that pro- withearlier observations (21). In-contrast, phenylephrine (1mg/ duced by norepinephrine (10 ,uM) and that the effect of phen- kg) did not increase enzyme activity. The combination ofphenylephrine (10 ,uM) or isoproterenol (10 ,uM) alone was <20% ylephrine (1 mg/kg) and isoproterenol (0.3mg/kg) had a greater of that produced by the combination of isoproterenol (10 UM) effect than isoproterenol (0.3 mg/kg) alone. and phenylephrine (10 MM). Time-course studies confirmed the effects of a combination This raised the obvious possibility that N-acetyltransferase ofphenylephrine (1 mg/kg) and isoproterenol (0.3 mg/kg) (Fig. 2A) and also revealed that phenylephrine (1 mg/kg) prolonged The publication costs ofthis article were defrayed in part by page charge the effect ofa higher dose ofisoproterenol (1 mg/kg) (Fig. 2B). payment. This article must therefore be hereby marked "advertise- These in vivo results are consistent with the conclusion that ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. N-acetyltransferase activity is regulated by both a- and (3ad-

599 Downloaded by guest on September 23, 2021 600 Neurobiology: Klein et al. Proc. Natl. Acad. Sci. USA 80 (1983)

I ,,/ I1 I i I i , 20 CS- 1 ISO/PE A 10 0 ISO/PE (1 mg/kg) ISO Cd I-- I- 10

5c) 4U O 0-~~~~~1§ -/I l * PEt to I A m I I I 0 10-4 10-3 10-2 lo-1 1 10 O 0.1 0.3 1.0 Agonist, ,uM Agonist, mg/kg FIG. 3.. Dose-responsel~~~~~curves for the in vitro stimulation of pineal FIG. 1. Dose-response curves for the in vivo stimulation of pineal N-acetyltransferase activity by phenylephrine (PE), isoproterenol N-acetyltransferase activity by isoproterenol (ISO) alone or in com- (ISO), or isoproterenol plus 1 uM phenylephrine. Pineal glands were bination with phenylephrine (PE). Rats were injected subcutaneously incubated for 48 hr prior to treatment and then transferred to fresh at 1200 hr with the appropriate agonist(s). Two hours later the animals medium containing the agonist(s) at the indicated concentration. were killed, and their pineal glands were removed and placed on solid Glands were treated for 6 hr and then placed on solid CO2. Each value CO2. Each point represents the mean ± SEM of N-acetyltransferase is the mean ± SEM of four determinations. The absence of an error activity in five or six pineal glands. The effect of isoproterenol (0.3 mg/ bar indicates that the error fell within the area covered by the symbol. kg) plus phenylephrine (1 mg/kg) was significantly greater (P < 0.05) than that of isoproterenol (0.3 mg/kg) alone. Time-course studies indicated that phenylephrine at 0.1 /uM, a concentration that had no effect on N-acetyltransferase activ- renergic receptors in the pineal gland but do not provide de- ity, significantly enhanced the effect of0.01 ,uM isoproterenol finitive proof because the drugs could be acting at any site in (Fig. 4). the neural circuit regulating the gland. To examine this question To determine whether norepinephrine stimulated N-acetyl- directly organ culture studies were done. transferase activity through a dual a- plus P3-adrenergic mech- In Vitro Studies. Dose-response studies indicated that iso- anism, we used a-adrenergic blockers in combination with the proterenol was about 1,000-fold more effective than phenyleph- 0-adrenergic blocking agent propranolol (Table 2; Fig. 5). The rine in stimulating N-acetyltransferase activity (Fig. 3), in con- a-adrenergic antagonists used were prazosin and WB 4101 (two firmation of previous observations (4, 22). However, a1-adrenergic antagonists), yohimbine (an a2-adrenergic antag- phenylephrine at 1 /.tM clearly potentiated the effect of isopro- onist); and phentolamine and phenoxybenzamine (two less-spe- terenol over a broad range ofconcentrations; this effect was also cific a-adrenergic antagonists). Propranolol consistently pro- seen at a phenylephrine concentration of 0.1 ,tM (Table 1). In duced a partial inhibition of the norepinephrine-stimulation of regard to the specificity of the effect on the isoproterenol- N-acetyltransferase activity, as previously observed (13, 16). induced stimulation of N-acetyltransferase activity, phenyl- Prazosin had either a partial inhibitory effect or no effect. Other ephrine at 1 liM was effective but 5-hydroxytryptamine, dopa- a-adrenergic blocking agents were without effect at the con- mine, and octopamine (1 uM) were not (Table 1). The a- centrations tested. However, the combination of propranolol agonists methoxamine and ST 587 (23) were less effective than (10 tLM) and prazosin (1 piM) or WB 4101 (1 tkM) consistently phenylephrine; clonidine, an a2-agonist, was ineffective. produced a nearly complete inhibition of the norepinephrine- stimulation of N-acetyltransferase activity. Phentolamine (1 ,uM) in combination with propranolol (10 ,M) produced a sig- $-I < AfII II nificant (P 0.05) reduction. Neither yohimbine nor phenoxy- A B 0C 15 benzamine (1 juM) markedly decreased N-acetyltransferase ac- I-$10 ISO + PE I-- tivity ofglands treated with norepinephrine and propranolol in ISO + PE experiments in which the same concentration of prazosin was 10 effective. A combination of propranolol (1 /.LM) and prazosin F5. (1 4M) also was more effective than either drug alone in block- a 4 ing the stimulation of N-acetyltransferase activity by combi- cd H lIOll nation ofisoproterenol (0.01 ,uM) and phenylephrine (1 MM). ¢ I.O llISO The effect ofadrenergic blocking agents on the norepineph- 80 PE P rine stimulation ofmelatonin production was examined by mea- suring [3H]melatonin production by glands that had been in- .I cubated with [3H]tryptophan for 24 hr prior to and then during 0 2 4 0 2 4 the 6-hr treatment period. A combination of propranolol (10 Time, hr ,uM) and prazosin (1 pLM) completely inhibited the norepinephrine-stimulation of [3H]melatonin production. Either of FIG. 2. Time-course of the in vivo stimulation of pineal N-acetyl- the drugs alone was clearly less effective (Table 3). Similar re- transferase activityby isoproterenol (ISO) or phenylephrine (PE) alone sults were obtained with WB 4101 and phentolamine. or in combination. For details see Fig. 1. (A) ISO at 0.3 mg/kg; PE at 1 mg/kg. The 2-hr value for the combination is significantly greater DISCUSSION than all other points (P < 0.05). (B) ISO at 1 mg/kg; PE at 1 mg/kg. The 3-hr value for the combination is significantly greater than the These findings provide evidence that pineal N-acetyltransferase 3-hr value for ISO alone (P < 0.05). is regulated by norepinephrine through postsynaptic a- and Downloaded by guest on September 23, 2021 Neurobiology: Klein et aL Proc. NatL Acad. Sci. USA 80 (1983) 601

Table 1. Effects of a-adrenergic agonists and other Table 2. Effect of adrenergic antagonists on the in vitro amines on the P-adrenergic stimulation of pineal stimulation of pineal N-acetyltransferase activity by N-acetyltransferase activity adrenergic agonists N-Acetyltransferase, N-Acetyltransferase, Treatment nmol/gland/hr Treatment nmol/gland/hr Exp. 1: Exp. 1: Control 0.10 + 0.05 Control 0.1 ± 0.02 Phenylephrine (0.01 ,uM) 0.10 ± 0.04 Norepinephrine (1 MM) 9.6 ± 1.9 Phenylephrine (0.1 XM) 0.18 ± 0.18 + prazosin (1pM) 11.3 ± 0.8 Phenylephrine (1.0 pM) 1.9 ± 0.5 + phentolamine (1 pM) 9.5 ± 1.9 Isoproterenol (0.01 pM) 6.9 ± 1.5 + WB 4101 (1 uM) 9.3 ± 2.3 + phenylephrine (0.01 pM) 5.6 ± 1.33 + propranolol (10 pM) 5.7 ± 0.4* + phenylephrine (0.1 pM) 12.8 ± 1.07* + propranolol (10 pM) + phenylephrine (1.0 pM) 10.5 ± 1.4* + prazosin (1 pM) 0.3 + 0.03t + propranolol (10 ,M) Exp. 2: + phentolamine (1 pM) 1.5 ± 0.3t Control 0.2 ± 0.06 + propranolol (10 MM) Phenylephrine (1 pM) 2.4 ± 1.0 + WB 4101 (1 uM) 0.4 ± O.lt 5-Hydroxytryptamine (1 pM) 1.1 ± 0.9 Dopamine (1 pAM) 0.2 ± 0.1 Exp. 2: Octopamine (1 ,uM) 0.6 ± 0.2 Control 0.1 ± 0.01 Isoproterenol (0.01 ,M) 4.9 ± 1.1 Norepinephrine (1 pM) 9.4 ± 1.4 + phenylephrine (1 uM) 13.4 ± 1.2t + yohimbine (1 pM) 5.7 ± 1.5 + 5-hydroxytryptamine (1 AM) 6.6 ± 1.1 + phenoxybenzamine (1 pM) 8.0 ± 1.9 + dopamine (1 ,M) 4.8 ± 1.1 + propranolol (10 pM) 2.5 ± 0.4* + octopamine (1 pM) 5.1 ± 0.8 + propranolol (10 pM) + yohimbine (1 pM) 1.9 ± 0.5 Exp. 3: + propranolol (10 ,uM) Control 0.4 ± 0.1 + phenoxybenzamine (1 ,M) 2.5 ± 0.5 Phenylephrine (1 uM) 2.0 ± 0.2 Methoxamine (1 ,uM) 0.9 ± 0.3 Exp. 3: Clonidine (1 pM) 2.7 ± 0.4 Control 0.1 + 0.01 ST 587 (1 AM) 2.7 ± 0.8 Norepinephrine (1 pM) 9.2 ± 2.0 Isoproterenol (0.01 pM) 3.6 ± 0.7 + propranolol (0.1 uM) 10.7 ± 1.1 + phenylephrine (1 ELM) 11.0 ± 0.4t + prazosin (1 pM) 8.7 ± 0.9 + methoxamine (1 PM) 6.3 ± 1.6 + yohimbine (1 PM) 9.4 ± 2.6 + clonidine (1 PM) 5.6 ± 1.7 + propranolol (0.1 pM) + ST 587 (1 ,M) 7.4 ± 1.2 + prazosin (1 lM) 3.9 ± 0.7t + propranolol (0.1 pM) Each value is based on the activity of N-acetyltransferase in four to + yohimbine (1 pM) 13.3 ± 1.3 six rat pineal glands that had been incubated for 48 hr prior to a 6-hr treatment with the indicated compounds (14). Exp. 4: * Significantly greater than isoproterenol value, P < 0.05. Control t Significantly greater than all other values in the experiment, P < Not detectable 0.05. Isoproterenol (0.01 pM) + phenylephrine (1 pM) 10.5 ± 1.4 + propranolol (1 WM) 1.3 ± 1.8* + prazosin (1 pM) 8.1 ± 1.8 + propranolol (1 ,uM) 10 ISO/PE + prazosin (1 cc"Sc pM) 0.04 ± 0.OOlt 1- For details, see the legends to Figs. 3 and 5. 10 * Significantly (P < 0.05) lowerthan the norepinephrine value in Exps. 1 and 2 and the isoproterenol/phenylephrine value in Exp. 4. ccs t Significantly (P < 0.05) lower than the norepinephrine/propranolol a. group in Exps. 1 and 3 and the isoproterenol/phenylephrine/pro- 5 _ pranolol group in Exp. 4. a)E ¢,cc adrenergic receptors. Previous investigations have established that the ,B-adrenergic receptor involved is a ,f1 subtype (16, 20). The observations that yohimbine does not enhance the effects 0 ofpropranolol and that prazosin and WB 4101 are potent in this I regard indicate that the a-adrenergic receptor involved in the 0 3 6 9 12 regulation of pineal N-acetyltransferase probably is an a, sub- hr type. Time, It is interesting that there has been some indication in the FIG. 4. Time-course of the in vitro stimulation of pineal N-acet- literature that rat pineal N-acetyltransferase and cyclic AMP yltransferase activity by phenylephrine (PE) at 0.1 pM or isoproter- might be regulated by both a- and ,B-adrenergic receptors (4, enol (ISO) at 0.01 pM alone or in combination. Glands were treated 13, 20, 22, 24-26). However, this interpretation has received for the indicated time. For further details see Fig. 3. little interest or experimental support. We believe the reason Downloaded by guest on September 23, 2021 Pu-VA2 Neurobiology: Klein et aL Proc. Natd Acad. Sci. USA 80 (1983) where, it seems likely that this does occur and that it may have 15 been overlooked for the same reasons it has been overlooked in the pineal gland. An issue ofcentral importance is the mechanism of a-adren- 10 o-, ergic potentiation off3-adrenergic effects. We have constructed Cd 10 F a working hypothesis focused on cyclic AMP to explain the a- adrenergic/,B-adrenergic interaction. This hypothesis makes use of the knowledge that (3-adrenergic agonists stimulate rat bof 0) pineal N-acetyltransferase through a cyclic AMP mechanism Uas (3, 4, 11) and that a-adrenergic agonists potentiate the p3-ad- 5k renergic stimulation ofcyclic AMP (unpublished data). Accord- ing to this, 3-adrenergic activation is an absolute requirement Propranolol/ for an increase in cyclic AMP, presumably reflecting the acti- prazosin vation ofadenylate cyclase (27, 28), and a-adrenergic activation 0)I enhances -adrenergic stimulation ofintracellular cyclic AMP. Al Several possible mechanisms of action exist, including an in- 0 0.1 1 10 crease in the affinity of the (-adrenergic receptor for 83-adren- Antagonist, uM ergic agonists, an increase in the efficiency of the receptor- adenylate cyclase coupling, a decrease in the activity of cyclic FIG. 5. Effect of propranolol or prazosin alone or in combination nucleotide phosphodiesterase, or acombination ofthese effects. (1:1) on the in vitro stimulation of pineal N-acetyltransferase activity This hypothesis would explain the dose-response and time- by 1 MM norepinephrine. Glands were treated for 30 min with the an- course results we obtained. Also, taken togetherwith the known tagonists prior to their transfer into medium containing norepineph- weak a-adrenergic agonist effect of isoproterenol and a weak rine and the antagonists for a 6-hr treatment. For further details see the legend to Fig. 3. The 1 MM and 10 MuM propranolol/prazosin values ,(3adrenergic agonist action of phenylephrine, this hypothesis are significantly lower than the 1 uM and 10 MM propranolol or 1 pM would explain a number ofpuzzling observations including the and 10 MM prazosin values (P < 0.05). atypical biphasic dose-response curve describing the isoproterenol stimulation ofcyclic AMP (18) and the potencies offlu- oro derivatives ofnorepinephrine (29) in neonatal pinealocytes for this is that (3-adrenergic agonists alone are effective in the in suspension culture. The blockade of rat and a-adrenergic agonists are effective only at high concen- a-adrenergic phenyl- have ephrine stimulation of neonatal rat pinealocyte N-acetyltrans- trations (22). Also, a-adrenergic antagonists generally ferase by WB 4101 (25), which does not fit well with this hy- been found to be either without effect or to have stimulatory pothesis, may reflect atypical characteristics acquired during effects (4, 13, 24, 26). An exception to this is the finding that long-term culture. WB 4101 can block phenylephrine stimulation ofN-acetyltrans- monolayer ferase in cultures It should be added that norepinephrine is known to stimulate activity monolayer containing pinealocytes phospholipid turnover in the pineal gland through an a1-ad- derived from neonatal rat pineal glands (25). The stimulatory renergic mechanism (30-32). Based on observations, in a num- effects of a-adrenergic antagonists have been attributed by ber of tissues, which indicate that sys- some to or central effects of these a-adrenergic regulatory investigators presynaptic tems may involve phospholipids, calcium, and calmodulin (33, drugs rather than direct effects on pinealocytes (4, 13, 26) and 34), we suspect that a similar mechanism could be involved in also to undifferentiated adrenergic pineal receptors having a- and characteristics the effects described in this report. It also seems possible that (3adrenergic (25). pinealocyte cyclic GMP could be involved in the control of N- We know of no other example of a-adrenergic potentiation because it to be in view of the wide occur- acetyltransferase (35) appears regulated through of -adrenergic effects. However, an a- and similar to that rence in the brain and else- ,3-adrenergic system regulating cyclic of P-adrenergic control systems AMP (unpublished data). that a- are in- Table 3. Effects of adrenergic antagonists on the norepinephrine The evidence and ,B-adrenergic receptors stimulation of [3H]melatonin production by pineal glands in volved in the regulation of pineal N-acetyltransferase activity organ culture now makes it imperative to examine the relative sensitivity and number of each type of receptor, especially as it relates to con- [3H]Melatonin, trol of N-acetyltransferase activity. Treatment pmol/gland As a final comment, we would like to point out that, although Control 29 ± 5 the hypothesis of exclusive ,3-adrenergic regulation of pineal Norepinephrine (10 ,uIM) 360 ± 59 melatonin production has been supported by observations in + prazosin (1 MuM) 415 ± 120 the rat, this hypothesis has not been confirmed in other species. + phentolamine (1 uM) 278; 242 For example, in humans, isoproterenol treatment does not in- + WB 4101 (1 AM) 255; 307 crease serum melatonin (36) and, in hamsters, only a partial in- + propranolol (10 MLM) 385 ± 59 crease in pineal melatonin has been produced with a relatively + propranolol (10 MuM) + prazosin (1 MM) 51 ± 6 high dose (20 mg/kg) of isoproterenol (37). The common ex- + propranolol (10 AM) + phentolamine (1 MiM) 145; 162 planation for these observations may be that, unlike the rat in + propranolol (10 ,M) + WB 4101 (1 AM) 51; 71 which (3-adrenergic activation appears to be sufficient to pro- Pairs of glands were incubated for 48 hr prior to a 6-hr treatment duce an increase in melatonin production, other species require period (14). [3H]Tryptophan (10 MCi/ml; 50 jCi/Amol; 1 Ci = 3.7 x either a combination of a- and f3-adrenergic activation or a-ad- 1010 Bq) was present during the 24- to 48-hr pretreatment and 6-hr renergic activation alone. Ifthis is correct, then treatment with treatment periods. The [3H]melatonin in a 10-,l sample of culture and or with alone medium was isolated by two-dimensional thin-layer chromatography isoproterenol phenylephrine phenylephrine and measured by routine methods (19). Single values represent the might produce an increase in serum melatonin in some species mean of duplicate assays of a single medium. Values ± SEM represent in which isoproterenol alone is not effective. the mean of duplicate assays of four different culture media. A logical extension ofthis would be efforts to develop a pineal Downloaded by guest on September 23, 2021 Neurobiology: Klein et aL Proc. NatL Acad. Sci. USA 80 (1983) 603 function test in humans by using phenylephrine and isoproter- 18. Parfitt, A., Weller, J. L., Sakai, K. K., Marks, B. H. & Klein, D. enol alone and in combination. This would be ofspecial interest C. (1975) MoL PharmacoL 11, 241-255. because clinical investigators now have no method to charac- 19. Klein, D. C. & Notides, A. (1969) Biochemistry 31, 480-483. 20. Auerbach, D., Klein, D. C., Woodard, B. & Aurbach, G. D. terize adrenergic responsiveness ofthe human pineal gland. A (1981) Endocrinology 108, 559-567. pineal function test would help elucidate the role ofthe pineal 21. Deguchi, T. & Axelrod, J. (1972) Proc. Natl Acad. Sci. USA 69, gland in health and disease and could be used as a diagnostic 2208-2212. tool. 22. Buda, M. & Klein, D. C. (1978) Endocrinology 103, 1483-1493. 23. DeJonge, A., Van Meel, C. A., Timmermans, P. B. & Van Zwie- We would like to express our appreciation to Pfizer Inc. and Boeh- ten, P. A. (1981) Life Sci. 28, 2009-2016. ringer Ingelheim for their generous gifts of some of the drugs used in 24. Klein, D. C. & Parfitt, A. (1976) in Catecholamines and Stress, this investigation. eds. Kvetnansky, R. & Usdin, E. (Pergamon, New York), pp. 119-128. 1. Klein, D. C. (1979) in Endocrine Rhythms; Comprehensive En- 25. Rowe, V. & Parr, J. (1981)J. PharmacoL Exp. Ther. 218, 97-102. docrinology I, ed. Kreiger, D. (Raven, New York), pp. 203-224. 26. Alphs, L., Heller, A. & Lovenberg, W. (1980)J. Neurochem. 34, 2. Klein, D. C. & Weller, J. L. (1970) Science 169, 1093-1095. 83-90. 3. Klein, D. C. & Berg, T. R. (1970) Adv. Biochem. Psychophar- 27. Weiss, B. & Costa, E. (1968) J. Pharmacol Exp. Ther. 101, 310- macol 3, 241-263. 316. 4. Klein, D. C. & Weller, J. L. (1973)J. Pharmacol Exp. Ther. 186, 28. Zatz, M., Kebabian, J. W., Romero, J. A., Lefkowitz, R. J. & 516-527. Axelrod, J. (1976) J. Pharmacol Exp. Ther. 196, 714-722. 5. Klein, D. C., Weller, J. L. & Moore, R. Y. (1971) Proc. NatL 29. Auerbach, D., Klein, D. C., Kirk, K. & Creveling, C. R. (1981) Acad. Sci. USA 68, 3107-3110. Biochem. Pharmacol 30, 1085-1089. 6. Moore, R. Y. & Klein, D. C. (1974) Brain Res. 71, 17-33. 30. Berg, G. R. & Klein, D. C. (1972)J. Neurochem. 19, 2519-2532. 7. Klein, D. C. & Moore, R. Y. (1979) Brain Res. 174, 245-262. 31. Smith, T. L., Eichberg, J. & Hauser, G. (1979) Life Sci. 24, 8. Brownstein, M. & Axelrod, J. (1974) Science 184, 173-175. 2179-2184. 9. Strada, S., Klein, D. C., Weller, J. & Weiss, B. (1972) Endocri- 32. Hauser, G., Nijjar, M. S., Smith, T. L. & Eichberg, J. (1978) in nology 90, 1470-1475. Cyclitols and Phosphoinositides, eds. Wells, W. W. & Eisen- 10. Deguchi, T. & Axelrod, J. (1973) Mol Pharmacol 9, 612-619. berg, G., Jr. (Academic, New York), pp. 167-182. 11. Klein, D. C., Berg, G. R. & Weller, J. (1970) Science 168, 979- 33. Fain, J. N. & Garcia-Sainz, J. A. (1980) Life Sci. 26, 1183-1194. 980. 34. Cheung, W. Y. (1982) Sci. Am. 246 (6), 62-70. 12. Shein, H. M. & Wurtman, R. J. (1969) Science 166, 519-520. 35. Klein, D. C., Auerbach, D. & Weller, J. L. (1981) Proc. NatL 13. Deguchi, T. & Axelrod, J. (1972) Proc. NatL Acad. Sci. USA 69, Acad. Sci. USA 78, 4625-4628. 2547-2550. 36. Vaughan, G. M., Pelham, R. W., Pang, S. F., Loughlin, L. L., 14. Deguchi, T. (1973) Mol, Pharmacol 9, 184-189. Wilson, K. M., Sandrock, K. L., Vaughan, M. K., Koslow, S. H. 15. Brownstein, M., Saavedra, J. M. & Axelrod, J. (1973) Mol, Phar- & Heiter, R. J. (1976) J. Clin. Endocrinol Metab. 42, 752-764. macoL 9, 605-607. 37. Tamarkin, L., Reppert, S. M. & Klein, D. C. (1979) Endocri- 16. Parfitt, A., Weller, J. L. & Klein, D. C. (1976) Neuropharma- nology 104, 385-389. cology 15, 353-358. 17. Zatz, M., Kebabian, J. W., O'Dea, R. F. (1978) in Receptors and Hormone Action, eds. O'Malley, B. W. & Birnbauer, L. (Aca- demic, New York), Vol. 3, pp. 169-219. Downloaded by guest on September 23, 2021