Dei'elopmental Brain Research, 68 ( 1992) 144-147 Elsevier Science Publishers B.V.
144
BRESD 60462
The pineal adrenergic --> cyclic GMP response develops two weeks after the adrenergic -> cyclic AMP response J o a n L. Weller and David C. Klein Section on Neuroendocrinology, Laboratory of Derelopmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (USA) (Accepted 14 April 1992)
Key words: Cyclic GMP: Pipeal; Guanylate cyclase; Adrenergic
Pineal metabolism is regulated primarily by noradrenergic innervation. Stimulation of the adult gland with norepinephrine elevates both cyclic AMP and cyclic GMP production, through remarkably similar mechanisms requiring activation of both ,8- and al-adrenergic receptors. As described here, however, the adrenergic stimulation of cyclic GMP is first detectable about 2 weeks after the cyclic AMP response can be detected. This indicates there is a profound difference in when cyclic AMP- and cyclic GMP-regulated processes can be adrenergically regulated.
Norepinephrine controls the accumulation of cyclic AMP and cyclic GMP in the rat pineal gland by activating biochemical "AND" gates operated by am- and /31-adrenergic receptors 2'17'2°''~°''~,/3.Adrenergic activation aione produces a 7- to 10.fold increase in cyclic AMP and a 2- to 4-fold increase in cyclic GMP, through mechanisms which are mediated by the GTP binding regul~t~ry protein which stimulates adenylate cyclase; these /3-adrenergic effects are mimicked by both cholera toxin and forskolin il,23,25'~°. Selective stimulation of the al-adrenergic receptor elevates [Ca 2+]i, but has no effect on the accumulation of either cyclic nucleotide 2z2a. However, concurrent stimulation of/3and acadrenergic receptors by norepinephrine increases cyclic AMP and cyclic GMP more than 100fold 20.3~. The post-receptor mechanisms involved in the a~-adrenergic potentiation of/3-adrenergic stimulation of cyclic AMP and cyclic GMP are dependent upon elevation of intracellular Ca 2+ ([Ca 2+]i) and activation of Ca 2+, phospholipid-dependent protein kinase~a0as,25,2~-2~. Although there are a number of similarities in the mechanisms involved in stimulation of cyclic AMP and cyclic GMP, there are notable differences, i.e., the
adrenergic stimulation of cyclic GMP exhibits a distinctly greater requirement for Ca a+ and phospholipase A a activity, and is more sensitive to changes in intracellular pH '~'12'27''~°.In addition, following stimulus deprivation, the cyclic AMP response to adrenergic stimulation increases and the cyclic GMP response markedly decreases 18'~°. One aspect of the adrenergic stimulation of pineal cyclic GMP which has not been studied is development. The mechanisms required for adrenergic stimulation of cyclic AMP appear to be fully matured shortly after birth, prior to adrenergie innervation 1'3'7'16'!9'22'26'32. In the present study we examined the developmental appearance of the adrenergic stimulation of cyclic GMP to determine whether it displayed a similar developmental pattern. This issue is of special interest because if norepinephrine does not increase pineal cyclic GMP early in development it would clearly indicate that tile adrenergi¢--, cyclic GMP response is not required for adrenergic stimulation of pineal Nacetyltransferase, which is fully developed shortly after birth3, 32.
Developmental appearance of the adrenergic stimulation of cyclic GMP. Pineal glands obtained from animals
Correspondence: D.C. Klein, Building 36, Room 4A07, National Institutes of Health, Bethesda, MD, 20892 USA. Fax: (1) (301) 480-3526.
145 150-
I
I
j
o-'i U~
°" i
NTROL
o
o
lO 2o AGE (days)
3o
Fig. 1. Developmental appearance of the adrenergic stimulation of pineal cyclic GMP. Sprague-Dawley rats of the indicated ages were obtained from Zivic Miller Co. (Allison Park, PA) and housed for at least 1 week ill a 14:10 LD lighting cycle. Animals were decapitated and pineal glands were obtained immediately. Glands were incubated on nylon mesh disks (dia.---4 mm) at the gas/medium interface for 1.5 h (2 glands/200/~! B G J b medium with I% bovine serum albumen; 37°C; 95% 0 2, 5% CO2) • The glands were then transferred to fresh medium with or without norepinephrine (1 /tM, Sigma Chemical Corp., St. Louis, MO). Five rain later glands weye transferred to tubes on dry ice. For analysis of pineal cyclic GMP a frozen gland was homogenized in 5% perchloric acid (100 ml) and neutralized with 2 N KHCO 3. The preparation was then centrifuged (12,000× g, 10 rain); samples of the supernatant were acetylated as required and then used for cyclic GMP radioimmunoassay8. The antiserum used to measure cyclic GMP was a gift of Dr. K. Catt (NICHD, NIH, Bethesda, MD). Protein was estimated using published data7. Data are presented as the mean ± S.E.M. of cyclic GMP in three to five pineal glands,
during the first 4 weeks of life were treated with norepinephrine (Fig. 1). There was little or no detectable cyclic GMP response to norepinephrine treatment in pineal glands removed from 2-, 5- and 10-dayold animals. However, norepinephrine produced a 100-fold increase in the amount of cyclic GMP in glands removed from 15.day-old and older animals. This pattern of development was confirmed in two other experiments.
These findings indicate that a 2-week difference exists between the developmental appearance of the adrenergic stimulation of cyclic AMP and cyclic GMP in the rat pineal gland. The molecular basis of this difference may be associated with the known differences between the cyclic AMP and cyclic GMP responses detailed above. For example, the developmental appearance of a hypothetical Ca2+-dependent kinase involved in the regulation of cyclic GMP but not cyclic AMP may determine the developmental appearance of the cyclic GMP response. The absence of the cyclic GMP response early in development, when norepinephrine can stimulate pineal serotonin N-acetyltransferase activity 3'32, indicates that cyclic GMP is not required for the stimulation of this enzyme or other processes which are under adrenergic control. Effects of neonatal denervation. The pineal gland becomes innervated by sympathetic nerves during the first 2 weeks of life 32, with the first indication of innervation detectable shortly after birth and a near adult appearance observed within 10 days after birth. It i~ possible that the developmental appearance of the cyclic GMP response requires the presence of sympathetic nerves. To examine this we studied the developmental appearance of the cyclic GMP response to norepinephrine in 30-day-old animals which had been denervated by superior cervical ganglionectomy (SCGX) 2 days after birth (Table I). The effects of denervation were monitored by measuring [3H]norepinephrine uptake and hydroxyindole-O-methyitransferase activity. The former was markedly decreased, reflecting the absence of nerve endings; the latter was reduced as previously reported u. A cyclic GMP response was detected in glands from SCGX animals, indicating that nerves are not required for the normal development of the cyclic GMP re-
TABLE !
Lffect of neonatal denervation on development of adrenergic stimulation of cyclic GMP accumulation, [3H]norepinephrine uptake, and hydroxyin. dole.O.methyltransferase (HiOMT) activity Glands were obtained from 30-day-old animals that had been superior cervical ganglionectomized (SCGX) at 2 days of age and from age-paired controls. A set of six glands from each surgical treatment group was analyzed for HIOMT activity without culture 24. For other analyses glands were cultured for 2 h under control conditions. For uptake analysis sets of six glands from each surgical treatment group were incubated for 30 rain with 0.1 ~M [3H]norepinephrine (4.5 ~Ci/ml medium, 44.7 Ci/mmol, New England Nuclear, Boston, MA) Is. For cyclic GMP response analysis sets of 6 glands from each surgical treatment group were transferred to fresh medium with or without norepinephrine (1/zM; 15 rain)s. For further details see the legend to Fig. 1.
Group
Surgery
Treatmentin culture
A
None
None Control Norepinephrine (I p.M) None Control Norepinephrine(! ~tM)
B
SCGX
CyclicGMP (pmol/ mg protein)
[JH]norepinephrine (DPM / gland) -
HIOMT (pmol/ gland/ h.) 181.6 ± 19.6
19.3± 5.0 323 + 41
28 620 ± 2 780
49 ±24.8 158 ±26
1210± 140
99.2 ± 17.9
146 sponse to norepinephrine. This finding is somewhat inconsistent with the observation that denervation leads to a loss of the cyclic GMP response in adults. Based on this, one might not expect to detect a cyclic GMP response following neonatal SCGX. However, it is known that the loss of the cyclic GMP response in the adult is reversed by systemic treatment with norepinephrine ~8, which has direct access to pinealocyte receptors in the absence of nerve endings. Accordingly, it would appear likely that the cyclic GMP response is present following neonatal denervation because circulating catecholamines maintain responsiveness. Circulating catecholamines ma~, be elevated to a greater degree in these animals as compared to older animals in response to greater cold stress or increased locomotor activity. Although it seems likely that circulating catecholamines function to maintain the adrenergic--, cyclic GMP response, it seems highly unlikely that norepinephrine released from pineal nerve terminals determines when ,he cyclic GMP response develops. This is because norepinephrine release occurs during the first week of life, as evidenced by the presence of the day/night rhythm in N-acetyltransferase activity 7''~2.Similarly, it is clear that the physical presence of the sympathetic nerves is not required, because the response develops in their absence. Based on this, it seems possible that the development of the cyclic GMP response is controlled by an intrinsic developmental schedule. Accordingly, the factors regulating the adrenergic stimulation of pineal cyclic GMP can be usefully divided into extrinsic and intrinsic elements. Extrinsic elements would include catecholamines released from sympathetic nerves in the pineal gland or diffusing to pinealocytes from the circulation; based on the findings that adenosine and vasoactive intestinal peptide stimulate pineal cyclic GMP 4-*,21, these agonists are also candidates as extrinsic regulating factors. Intrinsic elements can be described only in general terms as signals which trigger the developmental expression of one or more molecules required for maximal stimulation of cyclic GMP. Such molecules would not be involved in the stimulation of cyclic AMP. The elucidation of such molecules might provide some insight into the factors underlying other differences between the adrenergic regulation of cyclic AMP and cyclic GMP, and the development of other features of pineal biochemistry3.14.16.19. 1 Auerbach, D.A.,/3-Adrenergic receptors during development. In D.C. Klein, (Ed.), Melatonin Rhythm Generating System: Developmental Aspects, Karger, Basel, 1982, pp 97-107.
2 Auerbach, D.A., Klein, D.C., Woodard, B. and Aurbach, G.D., Neonatal rat pinealocytes: Typical and atypical characteristics of 1251-iodohydroxybenzylpindolol binding and adenosine 3',5'monophosphate accumulation, Endocrinology, 108 (1981) 559567. 3 Cefia, V., Gonzalez-Garcia, C., Svoboda, P. and Klein, D.C., Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87), J. Biol. Chem., 262 (1987) 14467-14471. 4 Chik, C., He, A. and Klein, D.C., Transmembrane receptor cross-talk: concurrent VIP and a~-adrenergic activation rapidly elevates pinealocyte CGMP > 100-fold, Biochem. Biophys. Res. Commun., 146 (1987) 1478-1484. 5 Chik, C., He, A. and Klein, D.C., al-Adrenergic potentiation of vasoactive intestinal peptide stimulation of rat pinealocyte adenosine Y,5'-monophosphate and guanosine 3',5'-monophosphate: evidence for a role of calcium and protein kinase-C, Endocrinology, 122 (1988) 702-708. 6 Chik, C., He., A. and Klein, D.C., Dual receptor regulation of cyclic nucleotides: a~-adrenergic potentiation of vasoactive intestinal peptide stimulation of pinealocyte adenosine 3',5'-monophosphate, Endocrinology, 122 (1988) 1646-1651. 7 Ellison, N., Weller, J.L. and Klein, D.C., Development of a circadian rhythm in the activity of pineal serotonin Nacetyltransferase, J. Neurochem., 19 (1972) 1335-1341. 8 Harper, J.F. and Brooker, G., Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2-O-acetylation by acetic anhydride in aqueous solution, J. Cycl. Nucl. Res., 1 (1975) 207-218. 9 He, A.K., Chik, C. and Klein, D.C. Protein kinase C is involved in adrenergic stimulation of pineal CGMP accumulation, J. Biol. Chem., 262 (1987) 10059-10064. 10 He., A., Chik, C. and Klein, D.C., Effects of protein kinase inhibitor (l-(5-isoquinolinesulfonyl)-2.methylpiperazine (H7) on protein kinase C activity and adrenergic stimulation of CAMP and CGMP in rat pinealocytes, Biochem. Pharmacol., 37 (1988) 1015-1020. I1 He, A.K., Chik, C.L. and Klein, D.C., Forskolin stimulates pinealocyte CGMP accumulation: dramatic potentiation by an a i-adrenergic ~ [Ca" + ]l "* mechanism involving protein kinase C, FEBS Left., 249 (1989) 207-212. 12 He, A.K., Chik, C,L., Weller, J.L., Cragoe Jr,, E.J, and Klein, D.C., Evidence of al-adrenergic ..-, protein kinase C --, N a + / H + antiporter dependent increase in pinealocyte intracellular Ph: Role in the adrenergic stimulation of CGMP accumulation, J. Biol. Chem., 264 (1989) 12983-12988. 13 He, A.K. and Klein, D.C., Activation of aj-adrenoreceptors, protein kinase C, or treatment with intracellular free Ca 2+ elevating agents increases pineal phospholipase A2 activity, J. Biol. Chem., 262 (1987) 11764-11770. 14 He, T., Seiners, R. and Klein, D,C., Development and regulation of rhodopsin kinase in rat pineal and retina, J. Neurochem., 46 (1986) 1176-1179. 15 He, A.K., Thomas, T.P., Anderson, W., Chik, C.L. and Klein, D.C., Protein kinase C: subcellular redistribution by increased Ca 2+ influx, J. Biol. Chem., 263 (1988) 9292-9297. 16 Klein, D.C. (Ed.), The Melatonin Rhythm Generating System: Developmental Aspects, Karger, Basel, 1982, 249 pp. 17 Klein, D.C., Photoneural regulation of the mammalian pineal gland. In D. Evered and S. Clark (Eds.), Photoperiodism, Melatonin and the Pineal. (Ciba Foundation Symposium 117), Pittman, London, 1986, pp. 38-56. 18 Klein, D.C., Auerbach, D. and Weller, J.L., Seesaw signal processing in pineal cells: homologous sensitization of adrenergic stimulation of cyclic GMP accompanies homologous desensitization of/3-adrenergic stimulation of cyclic AMP, Prec. Natl. Acad. Sci. U.S.A., 78 (1981) 4625-4629. 19 Klein, D.C., Namboodiri, M.A.A. and Auerbach, D.A., The melatonin rhythm generating system: developmental aspects, Life Sci., 28 (1981) 1975-1986. 20 Klein, D.C., Sugden, D. and Weller, J.L., Postsynaptic a-adrenergic receptors potentiate the/3-adrenergic stimulation of pineal
147
21
22 23 24 25 26
27
serotonin N-acetyltransferase, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 599-603. Nikodijevic, O. and Klein, D.C., Adenosine stimulates adenosine Y,5'-monophosphate and guanosine Y,5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission, Endocrinology, 125 (1989) 2150-2157. Sugden, D. and Klein, D.C., Development of the rat pineal at-adrenoceptor, Brain Res., 325 (1985)345-348. Sugden, D. and Klein, D.C., Activators of protein kinase C act at a post-receptor site to amplify CAMP production in rat pinealocytes, ./. Neurochem., 32 (1988) 149-155. Sugden, D. and Klein, D.C., Regulation of rat pineal hydroxyindole-O-methyitransferase in neonatal and adult rats, J. Neurochem., 40 (1983) 1647-1653. Sugden, D. and Klein, D.C., A cholera toxin substrate regulates cyclic GMP content of rat pinealocytes, J. Biol. Chem., 262 (1987) 7447-7450. Sugden, D., Namboodiri, M.A.A., Klein, D.C., Pierce, J.E., Grady Jr., R.K. and Mefford, I., Ovine pineal at-adrenoreceptors: characterization and evidence for a functional role in the regulation of serum melatonin, Endocrinology, 116 (1985)1960-1967. Sugden, L., Sugden, D. and Klein, D.C., Essential role of calcium
28 29
30
31
32
influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes, J. Biol. Chem., 261 (1986) 11608-11612. Sugden, L., Sugden, D. and Klein, D.C., al-Adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx, J. Biol. Chem., 262 (1987) 741-745. Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P. and Anderson, W.B., Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes, Nature, 314 (1985) 359-361. Vanecek, J., Sugden, D. and Klein, D.C., See-saw signal processing in pinealocytes involves reciprocal changes in the a i-adrenergic component of the cyclic GMP response and the fl.adrenergic component of the cyclic AMP responses, J. Neurochem., 47 (1986) 678-686. Vanecek, J., Sugden, D., Weller, J.L. a,.~ Klein, D.C., Atypical synergistic a l- and /3-adrenergic regulatio.1 of adenosine 3',5'monophosphate and guanosine 3',~ .monophosphate in rat pinealocytes, Endocrinology, 116 (1985) 2167-2173. Yuwiler, A., Klein, D.C., Buda, M. and Weller, J.L., Adrenergic control of pineal N-acetyltransferase activity: developmental aspects, Am. J. Physiol., 233 (1977) EI41-EI46.
144
BRESD 60462
The pineal adrenergic --> cyclic GMP response develops two weeks after the adrenergic -> cyclic AMP response J o a n L. Weller and David C. Klein Section on Neuroendocrinology, Laboratory of Derelopmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (USA) (Accepted 14 April 1992)
Key words: Cyclic GMP: Pipeal; Guanylate cyclase; Adrenergic
Pineal metabolism is regulated primarily by noradrenergic innervation. Stimulation of the adult gland with norepinephrine elevates both cyclic AMP and cyclic GMP production, through remarkably similar mechanisms requiring activation of both ,8- and al-adrenergic receptors. As described here, however, the adrenergic stimulation of cyclic GMP is first detectable about 2 weeks after the cyclic AMP response can be detected. This indicates there is a profound difference in when cyclic AMP- and cyclic GMP-regulated processes can be adrenergically regulated.
Norepinephrine controls the accumulation of cyclic AMP and cyclic GMP in the rat pineal gland by activating biochemical "AND" gates operated by am- and /31-adrenergic receptors 2'17'2°''~°''~,/3.Adrenergic activation aione produces a 7- to 10.fold increase in cyclic AMP and a 2- to 4-fold increase in cyclic GMP, through mechanisms which are mediated by the GTP binding regul~t~ry protein which stimulates adenylate cyclase; these /3-adrenergic effects are mimicked by both cholera toxin and forskolin il,23,25'~°. Selective stimulation of the al-adrenergic receptor elevates [Ca 2+]i, but has no effect on the accumulation of either cyclic nucleotide 2z2a. However, concurrent stimulation of/3and acadrenergic receptors by norepinephrine increases cyclic AMP and cyclic GMP more than 100fold 20.3~. The post-receptor mechanisms involved in the a~-adrenergic potentiation of/3-adrenergic stimulation of cyclic AMP and cyclic GMP are dependent upon elevation of intracellular Ca 2+ ([Ca 2+]i) and activation of Ca 2+, phospholipid-dependent protein kinase~a0as,25,2~-2~. Although there are a number of similarities in the mechanisms involved in stimulation of cyclic AMP and cyclic GMP, there are notable differences, i.e., the
adrenergic stimulation of cyclic GMP exhibits a distinctly greater requirement for Ca a+ and phospholipase A a activity, and is more sensitive to changes in intracellular pH '~'12'27''~°.In addition, following stimulus deprivation, the cyclic AMP response to adrenergic stimulation increases and the cyclic GMP response markedly decreases 18'~°. One aspect of the adrenergic stimulation of pineal cyclic GMP which has not been studied is development. The mechanisms required for adrenergic stimulation of cyclic AMP appear to be fully matured shortly after birth, prior to adrenergie innervation 1'3'7'16'!9'22'26'32. In the present study we examined the developmental appearance of the adrenergic stimulation of cyclic GMP to determine whether it displayed a similar developmental pattern. This issue is of special interest because if norepinephrine does not increase pineal cyclic GMP early in development it would clearly indicate that tile adrenergi¢--, cyclic GMP response is not required for adrenergic stimulation of pineal Nacetyltransferase, which is fully developed shortly after birth3, 32.
Developmental appearance of the adrenergic stimulation of cyclic GMP. Pineal glands obtained from animals
Correspondence: D.C. Klein, Building 36, Room 4A07, National Institutes of Health, Bethesda, MD, 20892 USA. Fax: (1) (301) 480-3526.
145 150-
I
I
j
o-'i U~
°" i
NTROL
o
o
lO 2o AGE (days)
3o
Fig. 1. Developmental appearance of the adrenergic stimulation of pineal cyclic GMP. Sprague-Dawley rats of the indicated ages were obtained from Zivic Miller Co. (Allison Park, PA) and housed for at least 1 week ill a 14:10 LD lighting cycle. Animals were decapitated and pineal glands were obtained immediately. Glands were incubated on nylon mesh disks (dia.---4 mm) at the gas/medium interface for 1.5 h (2 glands/200/~! B G J b medium with I% bovine serum albumen; 37°C; 95% 0 2, 5% CO2) • The glands were then transferred to fresh medium with or without norepinephrine (1 /tM, Sigma Chemical Corp., St. Louis, MO). Five rain later glands weye transferred to tubes on dry ice. For analysis of pineal cyclic GMP a frozen gland was homogenized in 5% perchloric acid (100 ml) and neutralized with 2 N KHCO 3. The preparation was then centrifuged (12,000× g, 10 rain); samples of the supernatant were acetylated as required and then used for cyclic GMP radioimmunoassay8. The antiserum used to measure cyclic GMP was a gift of Dr. K. Catt (NICHD, NIH, Bethesda, MD). Protein was estimated using published data7. Data are presented as the mean ± S.E.M. of cyclic GMP in three to five pineal glands,
during the first 4 weeks of life were treated with norepinephrine (Fig. 1). There was little or no detectable cyclic GMP response to norepinephrine treatment in pineal glands removed from 2-, 5- and 10-dayold animals. However, norepinephrine produced a 100-fold increase in the amount of cyclic GMP in glands removed from 15.day-old and older animals. This pattern of development was confirmed in two other experiments.
These findings indicate that a 2-week difference exists between the developmental appearance of the adrenergic stimulation of cyclic AMP and cyclic GMP in the rat pineal gland. The molecular basis of this difference may be associated with the known differences between the cyclic AMP and cyclic GMP responses detailed above. For example, the developmental appearance of a hypothetical Ca2+-dependent kinase involved in the regulation of cyclic GMP but not cyclic AMP may determine the developmental appearance of the cyclic GMP response. The absence of the cyclic GMP response early in development, when norepinephrine can stimulate pineal serotonin N-acetyltransferase activity 3'32, indicates that cyclic GMP is not required for the stimulation of this enzyme or other processes which are under adrenergic control. Effects of neonatal denervation. The pineal gland becomes innervated by sympathetic nerves during the first 2 weeks of life 32, with the first indication of innervation detectable shortly after birth and a near adult appearance observed within 10 days after birth. It i~ possible that the developmental appearance of the cyclic GMP response requires the presence of sympathetic nerves. To examine this we studied the developmental appearance of the cyclic GMP response to norepinephrine in 30-day-old animals which had been denervated by superior cervical ganglionectomy (SCGX) 2 days after birth (Table I). The effects of denervation were monitored by measuring [3H]norepinephrine uptake and hydroxyindole-O-methyitransferase activity. The former was markedly decreased, reflecting the absence of nerve endings; the latter was reduced as previously reported u. A cyclic GMP response was detected in glands from SCGX animals, indicating that nerves are not required for the normal development of the cyclic GMP re-
TABLE !
Lffect of neonatal denervation on development of adrenergic stimulation of cyclic GMP accumulation, [3H]norepinephrine uptake, and hydroxyin. dole.O.methyltransferase (HiOMT) activity Glands were obtained from 30-day-old animals that had been superior cervical ganglionectomized (SCGX) at 2 days of age and from age-paired controls. A set of six glands from each surgical treatment group was analyzed for HIOMT activity without culture 24. For other analyses glands were cultured for 2 h under control conditions. For uptake analysis sets of six glands from each surgical treatment group were incubated for 30 rain with 0.1 ~M [3H]norepinephrine (4.5 ~Ci/ml medium, 44.7 Ci/mmol, New England Nuclear, Boston, MA) Is. For cyclic GMP response analysis sets of 6 glands from each surgical treatment group were transferred to fresh medium with or without norepinephrine (1/zM; 15 rain)s. For further details see the legend to Fig. 1.
Group
Surgery
Treatmentin culture
A
None
None Control Norepinephrine (I p.M) None Control Norepinephrine(! ~tM)
B
SCGX
CyclicGMP (pmol/ mg protein)
[JH]norepinephrine (DPM / gland) -
HIOMT (pmol/ gland/ h.) 181.6 ± 19.6
19.3± 5.0 323 + 41
28 620 ± 2 780
49 ±24.8 158 ±26
1210± 140
99.2 ± 17.9
146 sponse to norepinephrine. This finding is somewhat inconsistent with the observation that denervation leads to a loss of the cyclic GMP response in adults. Based on this, one might not expect to detect a cyclic GMP response following neonatal SCGX. However, it is known that the loss of the cyclic GMP response in the adult is reversed by systemic treatment with norepinephrine ~8, which has direct access to pinealocyte receptors in the absence of nerve endings. Accordingly, it would appear likely that the cyclic GMP response is present following neonatal denervation because circulating catecholamines maintain responsiveness. Circulating catecholamines ma~, be elevated to a greater degree in these animals as compared to older animals in response to greater cold stress or increased locomotor activity. Although it seems likely that circulating catecholamines function to maintain the adrenergic--, cyclic GMP response, it seems highly unlikely that norepinephrine released from pineal nerve terminals determines when ,he cyclic GMP response develops. This is because norepinephrine release occurs during the first week of life, as evidenced by the presence of the day/night rhythm in N-acetyltransferase activity 7''~2.Similarly, it is clear that the physical presence of the sympathetic nerves is not required, because the response develops in their absence. Based on this, it seems possible that the development of the cyclic GMP response is controlled by an intrinsic developmental schedule. Accordingly, the factors regulating the adrenergic stimulation of pineal cyclic GMP can be usefully divided into extrinsic and intrinsic elements. Extrinsic elements would include catecholamines released from sympathetic nerves in the pineal gland or diffusing to pinealocytes from the circulation; based on the findings that adenosine and vasoactive intestinal peptide stimulate pineal cyclic GMP 4-*,21, these agonists are also candidates as extrinsic regulating factors. Intrinsic elements can be described only in general terms as signals which trigger the developmental expression of one or more molecules required for maximal stimulation of cyclic GMP. Such molecules would not be involved in the stimulation of cyclic AMP. The elucidation of such molecules might provide some insight into the factors underlying other differences between the adrenergic regulation of cyclic AMP and cyclic GMP, and the development of other features of pineal biochemistry3.14.16.19. 1 Auerbach, D.A.,/3-Adrenergic receptors during development. In D.C. Klein, (Ed.), Melatonin Rhythm Generating System: Developmental Aspects, Karger, Basel, 1982, pp 97-107.
2 Auerbach, D.A., Klein, D.C., Woodard, B. and Aurbach, G.D., Neonatal rat pinealocytes: Typical and atypical characteristics of 1251-iodohydroxybenzylpindolol binding and adenosine 3',5'monophosphate accumulation, Endocrinology, 108 (1981) 559567. 3 Cefia, V., Gonzalez-Garcia, C., Svoboda, P. and Klein, D.C., Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87), J. Biol. Chem., 262 (1987) 14467-14471. 4 Chik, C., He, A. and Klein, D.C., Transmembrane receptor cross-talk: concurrent VIP and a~-adrenergic activation rapidly elevates pinealocyte CGMP > 100-fold, Biochem. Biophys. Res. Commun., 146 (1987) 1478-1484. 5 Chik, C., He, A. and Klein, D.C., al-Adrenergic potentiation of vasoactive intestinal peptide stimulation of rat pinealocyte adenosine Y,5'-monophosphate and guanosine 3',5'-monophosphate: evidence for a role of calcium and protein kinase-C, Endocrinology, 122 (1988) 702-708. 6 Chik, C., He., A. and Klein, D.C., Dual receptor regulation of cyclic nucleotides: a~-adrenergic potentiation of vasoactive intestinal peptide stimulation of pinealocyte adenosine 3',5'-monophosphate, Endocrinology, 122 (1988) 1646-1651. 7 Ellison, N., Weller, J.L. and Klein, D.C., Development of a circadian rhythm in the activity of pineal serotonin Nacetyltransferase, J. Neurochem., 19 (1972) 1335-1341. 8 Harper, J.F. and Brooker, G., Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2-O-acetylation by acetic anhydride in aqueous solution, J. Cycl. Nucl. Res., 1 (1975) 207-218. 9 He, A.K., Chik, C. and Klein, D.C. Protein kinase C is involved in adrenergic stimulation of pineal CGMP accumulation, J. Biol. Chem., 262 (1987) 10059-10064. 10 He., A., Chik, C. and Klein, D.C., Effects of protein kinase inhibitor (l-(5-isoquinolinesulfonyl)-2.methylpiperazine (H7) on protein kinase C activity and adrenergic stimulation of CAMP and CGMP in rat pinealocytes, Biochem. Pharmacol., 37 (1988) 1015-1020. I1 He, A.K., Chik, C.L. and Klein, D.C., Forskolin stimulates pinealocyte CGMP accumulation: dramatic potentiation by an a i-adrenergic ~ [Ca" + ]l "* mechanism involving protein kinase C, FEBS Left., 249 (1989) 207-212. 12 He, A.K., Chik, C,L., Weller, J.L., Cragoe Jr,, E.J, and Klein, D.C., Evidence of al-adrenergic ..-, protein kinase C --, N a + / H + antiporter dependent increase in pinealocyte intracellular Ph: Role in the adrenergic stimulation of CGMP accumulation, J. Biol. Chem., 264 (1989) 12983-12988. 13 He, A.K. and Klein, D.C., Activation of aj-adrenoreceptors, protein kinase C, or treatment with intracellular free Ca 2+ elevating agents increases pineal phospholipase A2 activity, J. Biol. Chem., 262 (1987) 11764-11770. 14 He, T., Seiners, R. and Klein, D,C., Development and regulation of rhodopsin kinase in rat pineal and retina, J. Neurochem., 46 (1986) 1176-1179. 15 He, A.K., Thomas, T.P., Anderson, W., Chik, C.L. and Klein, D.C., Protein kinase C: subcellular redistribution by increased Ca 2+ influx, J. Biol. Chem., 263 (1988) 9292-9297. 16 Klein, D.C. (Ed.), The Melatonin Rhythm Generating System: Developmental Aspects, Karger, Basel, 1982, 249 pp. 17 Klein, D.C., Photoneural regulation of the mammalian pineal gland. In D. Evered and S. Clark (Eds.), Photoperiodism, Melatonin and the Pineal. (Ciba Foundation Symposium 117), Pittman, London, 1986, pp. 38-56. 18 Klein, D.C., Auerbach, D. and Weller, J.L., Seesaw signal processing in pineal cells: homologous sensitization of adrenergic stimulation of cyclic GMP accompanies homologous desensitization of/3-adrenergic stimulation of cyclic AMP, Prec. Natl. Acad. Sci. U.S.A., 78 (1981) 4625-4629. 19 Klein, D.C., Namboodiri, M.A.A. and Auerbach, D.A., The melatonin rhythm generating system: developmental aspects, Life Sci., 28 (1981) 1975-1986. 20 Klein, D.C., Sugden, D. and Weller, J.L., Postsynaptic a-adrenergic receptors potentiate the/3-adrenergic stimulation of pineal
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serotonin N-acetyltransferase, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 599-603. Nikodijevic, O. and Klein, D.C., Adenosine stimulates adenosine Y,5'-monophosphate and guanosine Y,5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission, Endocrinology, 125 (1989) 2150-2157. Sugden, D. and Klein, D.C., Development of the rat pineal at-adrenoceptor, Brain Res., 325 (1985)345-348. Sugden, D. and Klein, D.C., Activators of protein kinase C act at a post-receptor site to amplify CAMP production in rat pinealocytes, ./. Neurochem., 32 (1988) 149-155. Sugden, D. and Klein, D.C., Regulation of rat pineal hydroxyindole-O-methyitransferase in neonatal and adult rats, J. Neurochem., 40 (1983) 1647-1653. Sugden, D. and Klein, D.C., A cholera toxin substrate regulates cyclic GMP content of rat pinealocytes, J. Biol. Chem., 262 (1987) 7447-7450. Sugden, D., Namboodiri, M.A.A., Klein, D.C., Pierce, J.E., Grady Jr., R.K. and Mefford, I., Ovine pineal at-adrenoreceptors: characterization and evidence for a functional role in the regulation of serum melatonin, Endocrinology, 116 (1985)1960-1967. Sugden, L., Sugden, D. and Klein, D.C., Essential role of calcium
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influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes, J. Biol. Chem., 261 (1986) 11608-11612. Sugden, L., Sugden, D. and Klein, D.C., al-Adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx, J. Biol. Chem., 262 (1987) 741-745. Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P. and Anderson, W.B., Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes, Nature, 314 (1985) 359-361. Vanecek, J., Sugden, D. and Klein, D.C., See-saw signal processing in pinealocytes involves reciprocal changes in the a i-adrenergic component of the cyclic GMP response and the fl.adrenergic component of the cyclic AMP responses, J. Neurochem., 47 (1986) 678-686. Vanecek, J., Sugden, D., Weller, J.L. a,.~ Klein, D.C., Atypical synergistic a l- and /3-adrenergic regulatio.1 of adenosine 3',5'monophosphate and guanosine 3',~ .monophosphate in rat pinealocytes, Endocrinology, 116 (1985) 2167-2173. Yuwiler, A., Klein, D.C., Buda, M. and Weller, J.L., Adrenergic control of pineal N-acetyltransferase activity: developmental aspects, Am. J. Physiol., 233 (1977) EI41-EI46.
