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Vasoactive intestinal peptide and secretin produce long-term increases in tyrosine hydroxylase activity in the rat superior cervical ganglion
Brain Research, 607 (1993) 345-348
345
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25602
Vasoactive intestinal peptide and secretin produce long-term increases in tyrosine hydroxylase activity in the rat superior cervical ganglion T.W. McKeon and R.E. Zigmond Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4975 (USA)
(Accepted 5 January 1993)
Key words: Neuropeptide; Non-cholinergic; Transsynaptic; Tyrosine hydroxylase;Secretin; Superior cervical ganglion;
Vasoactive intestinal peptide
Electrical stimulation of the preganglionic fibers innervating the rat superior cervical ganglion (SCG) produces both short-term and long-term increases in tyrosine hydroxylase(TH) activity that are not completely blocked by nicotinic antagonists. Vasoactive intestinal peptide (VIP) and secretin, two neuropeptides known to produce short-term increases in TH activity,were examined for their ability to produce long-term increases in this enzyme activity. Culturing the SCG in the presence of either peptide produced a 30-50% increase in TH activity measured 2 days later. The results raise the possibility that one of these peptides or a related molecule participates in the transsynaptic induction of ganglionic TH.
While synaptic transmission between preganglionic and postganglionic sympathetic neurons or between preganglionic neurons and adrenal chromaffin cells is largely mediated by acetylcholine 31, there is also a non-cholinergic component to this transmission. The most extensively studied example in this regard is the bullfrog 9th and 10th lumbar sympathetic ganglion. In these ganglia, a non-cholinergically mediated slow excitatory postsynaptic potential is seen following preganglionic nerve stimulation 24. This potential has been shown to be mediated by a luteinizing hormone-releasing hormone-like peptide t6'17. The evidence for this conclusion includes m e a s u r e m e n t of the peptide and its release by radioimmunoassay, visualization of the peptide in preganglionic nerve terminals with immunohistochemistry and blockade of this non-cholinergic effect with selective peptide antagonists. Evidence for non-cholinergic transmission in the rat superior cervical ganglion (SCG) and the rat adrenal gland also exists. In the SCG, a non-cholinergic component of the acute transsynaptic activation of tyrosine hydroxylase (TH) activity was found. Thus, stimulation of the preganglionic cervical sympathetic nerve at 10 Hz for 30 min produced an increase in T H activity that
was blocked only about 50% by high concentrations of nicotinic and muscarinic antagonists ~4. Several members of the secretin-glucagon family of peptides (e.g. vasoactive intestinal peptide (VIP) and secretin, but not glucagon) were found to produce an acute activation of T H in the SCG, even in the presence of high concentrations of cholinergic antagonists 13. This effect of VIP and secretin on T H activity is rapidly reversible (i.e. activity is back to control values 30 min after washout of the peptide) ~3 and is accompanied by an increased phosphorylation of the enzyme 3. Neural processes containing VIP-like immunoreactivity (though not secretin- or glucagon-like immunoreactivity) were found in the SCG 9'29. Finally, cell bodies containing VIP-like immunoreactivity were found in the thoracic spinal cord and were identified by retrograde tracing as preganglionic neurons projecting to the SCG 2. Further elucidation of the role of VIP in the acute regulation of T H activity in vivo awaits the discovery of an antagonist that blocks the effects of VIP in this system 3°. Early evidence for the existence of non-cholinergic transmission in the rat adrenal medulla came from studies on the long-term induction of TH. The induction of T H differs from its acute activation in terms of
Correspondence: R.E. Zigmond, Department of Neurosciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue,
Cleveland, OH 44106-4975, USA. Fax: (1) (216) 368-4650.
346 its time-course (i.e. induction has a delayed onset and a prolonged duration, while acute activation occurs rapidly and lasts for a short period of time) and its underlying mechanism (induction involves an increase in TH m R N A and TH protein, while acute activation involves phosphorylation of pre-existing enzyme) 38. Mueller et al. 23 in their early studies on the transsynaptic induction of T H produced by administration of the drug reserpine, observed that two nicotinic antagonists, chlorisondamine and pempidine, could block the increase in TH activity in the SCG but not in the adrenal gland. In fact, both antagonists, in themselves, produced increases in TH activity in the adrenal. Slotkin et al. 32 found that the long-term increase in TH activity produced by chlorisondamine was blocked by decentralization of the adrenal gland, suggesting that it was transsynaptically mediated. While the identity of the transmitter mediating these non-cholinergic effects has not yet been established, recent studies have demonstrated that VIP can produce a long-term increase in TH m R N A in PC12 cells 36 and bovine chromaffin cells 25 in culture. Furthermore, VIP has recently been shown to mediate at least part of the non-cholinergic component of the release of catecholamines from the rat adrenal gland evoked by splanchnic nerve stimulation 34. While the transsynaptic induction of TH in the SCG has been shown to be blocked completely by nicotinic antagonists under certain conditions 6'7'8'23'26, under at least one condition it was not. When the cervical sympathetic trunk was stimulated with 40 Hz trains for 90 min, the subsequent increase in TH activity measured 3 days later was only blocked 58% by the nicotinic antagonist hexamethonium 5. We therefore sought to determine whether VIP and related peptides were capable of producing a long-term increase in TH activity in the SCG. A preliminary report of this work was presented to the Society for Neuroscience 22. SCG were removed from adult male Sprague-Dawley rats (150-200 g) and placed in organ culture as previously described 12 except that in certain groups VIP, secretin or glucagon were added to the medium at a concentration of 10/zM together with the protease inhibitor aprotinin (500 k l U / m l ) . After 12 h, the medium was replaced with fresh medium of the same type and, after another 12 h, with medium containing no peptide. After a total of 48 h in culture, the ganglia were frozen and stored at - 80°C. At the time of assay, individual ganglia were homogenized in 5 mM Tris buffer (pH 6.0) containing 0.1% Triton X-100, and an aliquot of the homogenate was incubated for 6 min in a phosphate buffer (pH 6.0) containing the D O P A decarboxylase inhibitor brocresine (300 tzM), and a satu-
6o
4o
Q.
o ,.~
20
E C
0 control
vIP
glucagon
Fig. 1. Effects of V ] P and glucagon on T H activity in the SCG.
Ganglia were exposed to control medium, medium containing VIP (10 ,aM) or medium containing glucagon (10 ~zM) for 24 h. After a further incubation for 24 h in control medium, TH activity was measured at pH 6.0, with saturating pterin cofactor. The data are given as mean values_+S.E.M. for groups of 16 ganglia. * Significantly different from control (P < 0.01).
rating concentration (3.2 mM) of the synthetic pterin cofactor 6-methyltetrahydropterine ~2. Catechols were adsorbed onto alumina and then eluted with acid. D O P A was quantitated by H P L C using electrochemical detection, as previously described 27. The protein content of the ganglia was measured by the bicinchoninic acid method (Pierce, Rockford, IL). Statistical comparisons between groups were made using Student's t-test, two-tailed. SCG incubated with 10 /~M VIP had 41% higher levels of T H activity than did ganglia incubated with control medium (Fig. 1). Incubation of ganglia with 10 txM glucagon, on the other hand, produced no significant change in TH activity (Fig. 1). In a second experiment, in which ganglia were incubated with 10 t~M VIP or 10/xM secretin, T H activity increased 30% and 50%, respectively, over the levels seen in control ganglia (Table I). No significant differences in total protein content of the ganglia were found between any of the groups in either experiment. The fact that VIP can produce a long-term increase in T H activity in organ culture, together with the studies cited above indicating that VIP may be a pre-
TABLE I Tyrosine hydroxylase activity
SCG were maintained in organ culture for 24 h with medium containing VIP (10 p~M) or secretin (10 IxM) or with medium containing no added peptide (control medium), and then all ganglia were transferred to control medium for an additional 24 h. The data are expressed as pM DOPA accumulated per /xg protein per h+ S.E.M. for groups of 8 ganglia. Peptide
pmol DOPA / tz g / h
% control
None VIP Secretin
66 + 3.6 86_+6.5 * 100_+8.3 * *
100 130_+ 8 150+_11
* Significantly different from control (P<0.02); ** Significantly different from control (P < 0.004).
347 ganglionic neurotransmitter in the SCG, raises the possibility that VIP, or a related peptide, is responsible for a non-cholinergic component in the transsynaptic long-term regulation of T H in vivo. Under many experimental conditions (e.g. reserpine administration or cold stress), the long-term increase in T H activity that results can be totally blocked by nicotinic antagonists; however, when the preganglionic nerve is stimulated electrically at 40 Hz trains for 90 min, only a partial block in the T H response is seen. While the seeming inconsistency between these conditions is at first puzzling, it is interesting to note that the extent of the non-cholinergically mediated c o m p o n e n t in the transsynaptic activation of T H produced by nerve stimulation has been found to vary, depending on the pattern of nerve stimulation H. Also, in the adrenal medulla, the extent of the non-cholinergic component in the evoked release of catecholamines has been found to depend on the frequency of stimulation 21. Thus, the role of non-cholinergic transmitters in these systems in vivo may vary depending on the actual stimulus used to evoked these transsynaptic events. As noted above, VIP-like immunoreactivity is present in nerve processes, perhaps of preganglionic origin, in control SCG but virtually no neuronal cell bodies 9'29. Evidence that these nerve processes are not, in fact, postganglionic in origin comes from our recent finding, using in situ hybridization, that almost no postganglionic neurons had detectable levels of VIP message (R.P. Mohney and R.E. Zigmond, unpublished observations). However, under certain conditions adult sympathetic neurons themselves begin to express VIP. For example, when the SCG is placed in organ culture 39 or when it is axotomized in vivo 1°, many postganglionic neurons express VIP-like immunoreactivity and the levels of VIP m R N A increase dramatically. Depolarization of the SCG in organ culture further increases the level of VIP-like immunoreactivity 33. Whether VIP is ever expressed in postganglionic neurons in vivo in the absence of injury, for example, following a period of increased nerve activity, remains to be determined. However, if this is the case, the findings reported here raise the possibility that VIP plays an autocrine role in the regulation of T H activity in sympathetic neurons. Such a role for VIP in the regulation of T H has been proposed for the adrenal medulla 25'36. Secretin also produced a long-term increase in T H activity in the SCG (Table I) and in PC12 cells 36, but not in bovine chromaffin cells 25. Interestingly, a similar pattern of effectiveness of secretin was found in the ability of the peptide to produce an acute activation of T H in these different cell types. While secretin is about
two orders of magnitude more potent than VIP in activating T H in the S C G 13 and in PC12 cells 24, it produced no effect on T H activation in bovine chromaffin cells 35. One possibility is that secretin receptors do not exist in bovine chromaffin ceils. The molecular mechanism of the long-term increase in ganglionic T H activity produced by VIP and secretin remains to be determined. In bovine chromaffin cells and PC12 cells, the effect of VIP on T H has been shown to be via enzyme induction, since increases in the levels of both T H m R N A and T H protein o c c u r 25,36. However, a type of regulation of TH, distinct from both acute activation and long-term induction and called 'delayed activation', has been described previously in two systems, the locus coeruleus 2° and retina 14. In these cases, a delayed increase in T H activity was reported that was not accompanied by an increase in the levels of T H protein. While such a phenomenon could be involved in the long-term increases in ganglionic T H activity produced by VIP and secretin, this seems unlikely given previous studies on the action of these same peptides on other peripheral adrenergic cells. Peptides other than those of the secretin-glucagon family may also be involved in non-cholinergic transmission in sympathetic ganglia and the adrenal medulla. For example, preganglionic neurons innervating the rat SCG have been found which contain calcitonin generelated peptide-like immunoreactivity 37. In addition, neurons in the intermediate gray matter of the lower thoracic cord of the rat have been found to contain both choline acetyltransferase- and enkephalin-like immunoreactivityi8. Finally, evidence has been presented that a neurotensin-like peptide is involved in noncholinergic transmission in the cat stellate ganglion 1'4'~9. Thus, while the results presented here, together with our earlier immunohistochemical studies 2,29, indicate that VIP is a good candidate for the transmitter mediating the non-cholinergic component of the long-term increase in T H activity following nerve stimulation, other peptides could also be involved. This research was supported by a grant from the US Public Health Service (NS12651) and by a Research Scientist award from the National Institutes of Mental Health (MH00162) to R.E.Z. 1 Bachoo, M. and Polosa, C., Cardioaccelerationproduced by close intra-arterial injection of neurotensin into the stellate ganglion of the cat, Can. J. Pharmacol., 66 (1988) 408-412. 2 Baldwin, C., Sasek, C. and Zigmond R.E., Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities, Neuroscience, 40 (1991) 175-184. 3 Cahill A.L. and Perlman R.L., Phosphorylation of tyrosine hydroxylase in the superior cerrvical ganglion, Biochirn. Biophys. Acta, 805 (1984) 217-226.
348 3 Caverson, M.M., Bachoo, M., Ciriello J. and Polosa, C., Effect of preganglionic stimulation or chronic decentralization on neurotensin-[ike immunoreactivity in sympathetic ganglia of the cat, Brain Research, 482 (1989) 365-370. 4 Chalazonitis, A. and Zigmond, R.E., Effects of synaptic and antidromic stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion, J. Physiol., 300 (1980) 525-538. 5 Chalazonitis, A., Rice, P.J. and Zigmond, R.E., Increased ganglionic tyrosine hydroxylase and dopamine-beta-hydroxylase activities following preganglionic nerve stimulation: role of nicotinic receptors, J. Pharmacol. Exp. Ther., 213 (1980) 139-143. 6 Hanbauer, I., Kopin, I.J. and Costa, E., Mechanisms involved in the trans-synaptic increase of tyrosine hydroxylase and dopamine-beta-hydroxylase activity in sympathetic ganglia, Naunyn-Schmiedeberg's Arch. Pharmacol., 280 (1973) 39-48. 7 Hanbauer, 1., Lovenberg, W. Guidotti, A. and Costa, E., Role of cholinergic and glucocorticosteroid receptors in the tyrosine hydroxylase induction elicited by reserpine in the superior cervical ganglion, Brain Res., 96 (1975) 197-200. 8 Hokfelt, T., Elfvin, L.-G., Schultzberg, M., Fuxe, K., Said, S.I., Mutt, V. and Goldstein, M., Immunohistochemical evidence of vasoactive intestinal polypeptide-containing neurons and nerve fibers in sympathetic ganglia, Neuroscience, 2 (1977) 885-896. 9 ttyatt-Sachs, H., McKeon, T.W., Schreiber, R., Piszczkiewicz, S., Sun, Y., Bachoo, M.R. and Zigmond, R.E., Effects of decentralization and axotomy on vasoactive intestinal peptide (VIP)- and neuropeptide Y (NPY)-like immunoreactivity (IR) in the superior cervical ganglion (SCG), Soc. Neurosci. Abstr., 17 (1991) 400. 10 Ip, N.Y. and Zigmond, R.E., Pattern of presynaptic nerve activity can determine the type of neurotransmitter regulating a postsynaptic event, Nature, 3t 1 (1984) 472-474. 11 lp, N.Y. and Zigmond, R.E., Long-term regulation of tyrosine hydroxylase activity in the superior cervical ganglion in organ culture: effects of nerve stimulation and dexamethasone, Brain Res., 338 11985) 61-70. 12 Ip, N.Y., Ho, C.K. and Zigmond, R.E., Secretin and vasoactive intestinal peptide acutely increase tyrosine 3-monooxygenase in the rat superior cervical ganglion, Proc. Natl. Acad. Sci. USA, 79 (1982) 7566-7569. 13 Ip, N.Y., Perlman, R.L. and Zigmond R.E., Acute transsynaptic regulation of tyrosine 3-monooxygenase activity in the rat superior cervical ganglion: Evidence for both cholinergic and noncholinergic mechanisms, Proc. Natl. Acad. Sci. USA, 80 (1983) 21181-21185. 14 luvone, P.M., Joh, T.H., and Neff, N.H., Regulation of retinal tyrosine hydroxylase: long-term exposure to light increased the apparent Vmax without a concomitant increase of immunotitratable enzyme molecules, Brain Res., 178 (1979) 191-195. 15 Jan, Y.N., Jan, L.Y. and Kuffier, S.W., A peptide as a possible transmitter in sympathetic ganglia of the frog, Proc. Natl. Acad. Sci. USA, 76 11979) 1501-1505. 16 Jan, Y.N., Jan, L.Y. and Kuffier, S.W., Further evidence for peptidergic transmission in sympathetic ganglia, Proc. Natl. Acad. Sci. USA. 77 (1980) 5008-5012. 17 Kondo, 1t., Kuramoto, H., Wainer, B.H. and Yanaihara, N., Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats, Brain Res., 335 (1985) 3()9-314. 18 Krukoff, T.L., Ciriello, J. and Calaresu, F.R., Segmental distribution of peptide-like immunoreactivity in cell bodies of the thoracolumbar sympathetic nuclei of the cat, J. Comp. Neurol., 240 (1985) 90-102. 19 Lewander, T., Joh, T.H., and Reis, D.J., Tyrosine hydroxylase: delayed activation in central noradrenergic neurons and induction in adrenal medulla elicited by stimulation of central cholinergic receptors, J. Pharmacol. Exp. Ther., 200 11977) 523-534. 211 Malhotra, R.K. and Wakade, A.R., Non-cholinergic component of rat splanchnic nerves predominates at low neuronal activity and is eliminated by naloxone, J. Physiol., 383 11987) 639-652. 21 McKeon, T.W. and Zigmond, R.E., Long-term regulation of
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37
38
tyrosine hydroxylase activity in the superior cervical ganglion by vasoactive intestinal peptide and secretin, Soc. Neurosci. Abstr., 17 (1991) 400. Mueller, R.A., Thoenen, H. and Axelrod, J., Inhibition of neuronally induced tyrosine hydroxylase by nicotinic receptor blockade, Eur. J. Pharmacol., 10 11970) 51-56. Nishi S and Koketsu K., Early and late after discharges of amphibian sympathetic ganglion cells, Z Neurophysiol., 31 (1968) 109-12l. Olasmaa, M., Guidotti, A. and Costa, E., Vasoactive intestinal polypeptide facilitates tyrosine hydroxylase induction by cholinergic agonists in bovine adrenal chromaffin cells, MoL Pharmacol., 41 (1992) 456-464. Otten, U. and Thoenen, H., Mechanisms of tyrosine hydroxylase and dopamine beta-hydroxylase induction in organ cultures of rat sympathetic ganglia by potassium depolarization and cholinomimetics, Naunyn-Schmiedeberg's Arch. Pharmacol., 292 (19761 153-159. Rittenhouse, A.R., Schwarzschild, M.A. and Zigmond, R.E., Both synaptic and antidromic stimulation of neurons in the rat superior cervical ganglion acutely increase tyrosine hydroxylase activity, Neuroscience, 25 11988) 207-215. Roskowski Jr., R., White, L., Knowlton, R. and Roskoski, L.M., Regulation of tyrosine hydroxylase activity in rat PC12 cells by neuropeptides of the secretin family, Mol. Pharmacol., 36 (1991) 925-931. Sasek, C.A. and Zigmond, R.E. Localization of vasoactive intestinal peptide and peptide histidine isoleucine amide-like immunoreactivities in the rat superior cervical ganglion and its nerve trunks, J. Comp. Neurol., 280 (1989)522-532. Schwarzschild, M.A., Vale, W., Corigliano-Murphy, A.C., Pisano, J.J., Ip, N.Y. and Zigmond, R.E., Activation of ganglionic tyrosine hydroxylase by peptides of the secretin-glucagon family: Structure-function studies, Neuroscience, 31 (1989) 159-167. Skok,V.I., Physiology of Autonomic Ganglia, Igaku Shoin Ltd., Tokyo, 1973. Slotkin, T.A., Seidler, F.J., Lau, C., Bartoome. J. and Schanberg, S.M., Effects of chronic chlorisondamine administration on the sympatho-adrenal axis, Biochem. Pharmacol., 25 (1976) 13111315. Sun, Y., Rao, M.S., Landis, S.C. and Zigmond, R.E., Depolarization increases vasoactive intestinal peptide- and substance P-like immunoreactivities in neonatal and adult sympathetic neurons, J. Neurosci., 12 (19921 3717-3728. Wakade, T.D., Blank, M.A., Malhotra, R.K., Poucho, R. and Wakade, A.R., The peptide VIP is a neurotransmitter in rat adrenal medulla: physiological role in controlling catecholamine secretion, J. Physiol., 444 (1991) 349-362. Waymire, J.C., Craviso, G.L., Lichteig, K., Johnston, J.P., Baldwin, C. and Zigmond, R.E., Vasoactive intestinal peptide modulates catecholamine biosynthesis in isolated adrenal chromaffin cells through a cyclic AMP-dependent phosphorylation and activation of tyrosine hydroxylase, J. Neurochem., 57 (1991) 13131324. Wessels-Reiker, M., Haycock, J.W., Howlett, A.C. and Strong, R., Vasoactive intestinal polypeptide induces tyrosine hydroxylase in PC12 cells, J. Biol. Chem., 266 (1991) 9347-9350. Yamamoto, K., Senba, E., Matsunaga, T. and Tohyama, M., Calcitonin gene-related peptide containing sympathetic preganglionic and sensory neurons projecting to the superior cervical ganglion of the rat, Brain Res., 487 (1989) 158-164. Zigmond, R.E., A comparison of the long-term and short-term regulations of tyrosine hydroxylase activity, J. Physiol., Paris, 83 (19891 267-271. Zigmond, R.E., Hyatt-Sachs, H., Baldwin, C., Qu, X.M., Sun, Y., McKeon, T.W., Schreiber, R.C. and Vaidyanathan, U., Phenotypic plasticity in adult sympathetic neurons: Changes in neuropeptide expression in organ culture, Proc. Natl. Acad. ScL USA, 89 (1992) 1507-1511.
345
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25602
Vasoactive intestinal peptide and secretin produce long-term increases in tyrosine hydroxylase activity in the rat superior cervical ganglion T.W. McKeon and R.E. Zigmond Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4975 (USA)
(Accepted 5 January 1993)
Key words: Neuropeptide; Non-cholinergic; Transsynaptic; Tyrosine hydroxylase;Secretin; Superior cervical ganglion;
Vasoactive intestinal peptide
Electrical stimulation of the preganglionic fibers innervating the rat superior cervical ganglion (SCG) produces both short-term and long-term increases in tyrosine hydroxylase(TH) activity that are not completely blocked by nicotinic antagonists. Vasoactive intestinal peptide (VIP) and secretin, two neuropeptides known to produce short-term increases in TH activity,were examined for their ability to produce long-term increases in this enzyme activity. Culturing the SCG in the presence of either peptide produced a 30-50% increase in TH activity measured 2 days later. The results raise the possibility that one of these peptides or a related molecule participates in the transsynaptic induction of ganglionic TH.
While synaptic transmission between preganglionic and postganglionic sympathetic neurons or between preganglionic neurons and adrenal chromaffin cells is largely mediated by acetylcholine 31, there is also a non-cholinergic component to this transmission. The most extensively studied example in this regard is the bullfrog 9th and 10th lumbar sympathetic ganglion. In these ganglia, a non-cholinergically mediated slow excitatory postsynaptic potential is seen following preganglionic nerve stimulation 24. This potential has been shown to be mediated by a luteinizing hormone-releasing hormone-like peptide t6'17. The evidence for this conclusion includes m e a s u r e m e n t of the peptide and its release by radioimmunoassay, visualization of the peptide in preganglionic nerve terminals with immunohistochemistry and blockade of this non-cholinergic effect with selective peptide antagonists. Evidence for non-cholinergic transmission in the rat superior cervical ganglion (SCG) and the rat adrenal gland also exists. In the SCG, a non-cholinergic component of the acute transsynaptic activation of tyrosine hydroxylase (TH) activity was found. Thus, stimulation of the preganglionic cervical sympathetic nerve at 10 Hz for 30 min produced an increase in T H activity that
was blocked only about 50% by high concentrations of nicotinic and muscarinic antagonists ~4. Several members of the secretin-glucagon family of peptides (e.g. vasoactive intestinal peptide (VIP) and secretin, but not glucagon) were found to produce an acute activation of T H in the SCG, even in the presence of high concentrations of cholinergic antagonists 13. This effect of VIP and secretin on T H activity is rapidly reversible (i.e. activity is back to control values 30 min after washout of the peptide) ~3 and is accompanied by an increased phosphorylation of the enzyme 3. Neural processes containing VIP-like immunoreactivity (though not secretin- or glucagon-like immunoreactivity) were found in the SCG 9'29. Finally, cell bodies containing VIP-like immunoreactivity were found in the thoracic spinal cord and were identified by retrograde tracing as preganglionic neurons projecting to the SCG 2. Further elucidation of the role of VIP in the acute regulation of T H activity in vivo awaits the discovery of an antagonist that blocks the effects of VIP in this system 3°. Early evidence for the existence of non-cholinergic transmission in the rat adrenal medulla came from studies on the long-term induction of TH. The induction of T H differs from its acute activation in terms of
Correspondence: R.E. Zigmond, Department of Neurosciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue,
Cleveland, OH 44106-4975, USA. Fax: (1) (216) 368-4650.
346 its time-course (i.e. induction has a delayed onset and a prolonged duration, while acute activation occurs rapidly and lasts for a short period of time) and its underlying mechanism (induction involves an increase in TH m R N A and TH protein, while acute activation involves phosphorylation of pre-existing enzyme) 38. Mueller et al. 23 in their early studies on the transsynaptic induction of T H produced by administration of the drug reserpine, observed that two nicotinic antagonists, chlorisondamine and pempidine, could block the increase in TH activity in the SCG but not in the adrenal gland. In fact, both antagonists, in themselves, produced increases in TH activity in the adrenal. Slotkin et al. 32 found that the long-term increase in TH activity produced by chlorisondamine was blocked by decentralization of the adrenal gland, suggesting that it was transsynaptically mediated. While the identity of the transmitter mediating these non-cholinergic effects has not yet been established, recent studies have demonstrated that VIP can produce a long-term increase in TH m R N A in PC12 cells 36 and bovine chromaffin cells 25 in culture. Furthermore, VIP has recently been shown to mediate at least part of the non-cholinergic component of the release of catecholamines from the rat adrenal gland evoked by splanchnic nerve stimulation 34. While the transsynaptic induction of TH in the SCG has been shown to be blocked completely by nicotinic antagonists under certain conditions 6'7'8'23'26, under at least one condition it was not. When the cervical sympathetic trunk was stimulated with 40 Hz trains for 90 min, the subsequent increase in TH activity measured 3 days later was only blocked 58% by the nicotinic antagonist hexamethonium 5. We therefore sought to determine whether VIP and related peptides were capable of producing a long-term increase in TH activity in the SCG. A preliminary report of this work was presented to the Society for Neuroscience 22. SCG were removed from adult male Sprague-Dawley rats (150-200 g) and placed in organ culture as previously described 12 except that in certain groups VIP, secretin or glucagon were added to the medium at a concentration of 10/zM together with the protease inhibitor aprotinin (500 k l U / m l ) . After 12 h, the medium was replaced with fresh medium of the same type and, after another 12 h, with medium containing no peptide. After a total of 48 h in culture, the ganglia were frozen and stored at - 80°C. At the time of assay, individual ganglia were homogenized in 5 mM Tris buffer (pH 6.0) containing 0.1% Triton X-100, and an aliquot of the homogenate was incubated for 6 min in a phosphate buffer (pH 6.0) containing the D O P A decarboxylase inhibitor brocresine (300 tzM), and a satu-
6o
4o
Q.
o ,.~
20
E C
0 control
vIP
glucagon
Fig. 1. Effects of V ] P and glucagon on T H activity in the SCG.
Ganglia were exposed to control medium, medium containing VIP (10 ,aM) or medium containing glucagon (10 ~zM) for 24 h. After a further incubation for 24 h in control medium, TH activity was measured at pH 6.0, with saturating pterin cofactor. The data are given as mean values_+S.E.M. for groups of 16 ganglia. * Significantly different from control (P < 0.01).
rating concentration (3.2 mM) of the synthetic pterin cofactor 6-methyltetrahydropterine ~2. Catechols were adsorbed onto alumina and then eluted with acid. D O P A was quantitated by H P L C using electrochemical detection, as previously described 27. The protein content of the ganglia was measured by the bicinchoninic acid method (Pierce, Rockford, IL). Statistical comparisons between groups were made using Student's t-test, two-tailed. SCG incubated with 10 /~M VIP had 41% higher levels of T H activity than did ganglia incubated with control medium (Fig. 1). Incubation of ganglia with 10 txM glucagon, on the other hand, produced no significant change in TH activity (Fig. 1). In a second experiment, in which ganglia were incubated with 10 t~M VIP or 10/xM secretin, T H activity increased 30% and 50%, respectively, over the levels seen in control ganglia (Table I). No significant differences in total protein content of the ganglia were found between any of the groups in either experiment. The fact that VIP can produce a long-term increase in T H activity in organ culture, together with the studies cited above indicating that VIP may be a pre-
TABLE I Tyrosine hydroxylase activity
SCG were maintained in organ culture for 24 h with medium containing VIP (10 p~M) or secretin (10 IxM) or with medium containing no added peptide (control medium), and then all ganglia were transferred to control medium for an additional 24 h. The data are expressed as pM DOPA accumulated per /xg protein per h+ S.E.M. for groups of 8 ganglia. Peptide
pmol DOPA / tz g / h
% control
None VIP Secretin
66 + 3.6 86_+6.5 * 100_+8.3 * *
100 130_+ 8 150+_11
* Significantly different from control (P<0.02); ** Significantly different from control (P < 0.004).
347 ganglionic neurotransmitter in the SCG, raises the possibility that VIP, or a related peptide, is responsible for a non-cholinergic component in the transsynaptic long-term regulation of T H in vivo. Under many experimental conditions (e.g. reserpine administration or cold stress), the long-term increase in T H activity that results can be totally blocked by nicotinic antagonists; however, when the preganglionic nerve is stimulated electrically at 40 Hz trains for 90 min, only a partial block in the T H response is seen. While the seeming inconsistency between these conditions is at first puzzling, it is interesting to note that the extent of the non-cholinergically mediated c o m p o n e n t in the transsynaptic activation of T H produced by nerve stimulation has been found to vary, depending on the pattern of nerve stimulation H. Also, in the adrenal medulla, the extent of the non-cholinergic component in the evoked release of catecholamines has been found to depend on the frequency of stimulation 21. Thus, the role of non-cholinergic transmitters in these systems in vivo may vary depending on the actual stimulus used to evoked these transsynaptic events. As noted above, VIP-like immunoreactivity is present in nerve processes, perhaps of preganglionic origin, in control SCG but virtually no neuronal cell bodies 9'29. Evidence that these nerve processes are not, in fact, postganglionic in origin comes from our recent finding, using in situ hybridization, that almost no postganglionic neurons had detectable levels of VIP message (R.P. Mohney and R.E. Zigmond, unpublished observations). However, under certain conditions adult sympathetic neurons themselves begin to express VIP. For example, when the SCG is placed in organ culture 39 or when it is axotomized in vivo 1°, many postganglionic neurons express VIP-like immunoreactivity and the levels of VIP m R N A increase dramatically. Depolarization of the SCG in organ culture further increases the level of VIP-like immunoreactivity 33. Whether VIP is ever expressed in postganglionic neurons in vivo in the absence of injury, for example, following a period of increased nerve activity, remains to be determined. However, if this is the case, the findings reported here raise the possibility that VIP plays an autocrine role in the regulation of T H activity in sympathetic neurons. Such a role for VIP in the regulation of T H has been proposed for the adrenal medulla 25'36. Secretin also produced a long-term increase in T H activity in the SCG (Table I) and in PC12 cells 36, but not in bovine chromaffin cells 25. Interestingly, a similar pattern of effectiveness of secretin was found in the ability of the peptide to produce an acute activation of T H in these different cell types. While secretin is about
two orders of magnitude more potent than VIP in activating T H in the S C G 13 and in PC12 cells 24, it produced no effect on T H activation in bovine chromaffin cells 35. One possibility is that secretin receptors do not exist in bovine chromaffin ceils. The molecular mechanism of the long-term increase in ganglionic T H activity produced by VIP and secretin remains to be determined. In bovine chromaffin cells and PC12 cells, the effect of VIP on T H has been shown to be via enzyme induction, since increases in the levels of both T H m R N A and T H protein o c c u r 25,36. However, a type of regulation of TH, distinct from both acute activation and long-term induction and called 'delayed activation', has been described previously in two systems, the locus coeruleus 2° and retina 14. In these cases, a delayed increase in T H activity was reported that was not accompanied by an increase in the levels of T H protein. While such a phenomenon could be involved in the long-term increases in ganglionic T H activity produced by VIP and secretin, this seems unlikely given previous studies on the action of these same peptides on other peripheral adrenergic cells. Peptides other than those of the secretin-glucagon family may also be involved in non-cholinergic transmission in sympathetic ganglia and the adrenal medulla. For example, preganglionic neurons innervating the rat SCG have been found which contain calcitonin generelated peptide-like immunoreactivity 37. In addition, neurons in the intermediate gray matter of the lower thoracic cord of the rat have been found to contain both choline acetyltransferase- and enkephalin-like immunoreactivityi8. Finally, evidence has been presented that a neurotensin-like peptide is involved in noncholinergic transmission in the cat stellate ganglion 1'4'~9. Thus, while the results presented here, together with our earlier immunohistochemical studies 2,29, indicate that VIP is a good candidate for the transmitter mediating the non-cholinergic component of the long-term increase in T H activity following nerve stimulation, other peptides could also be involved. This research was supported by a grant from the US Public Health Service (NS12651) and by a Research Scientist award from the National Institutes of Mental Health (MH00162) to R.E.Z. 1 Bachoo, M. and Polosa, C., Cardioaccelerationproduced by close intra-arterial injection of neurotensin into the stellate ganglion of the cat, Can. J. Pharmacol., 66 (1988) 408-412. 2 Baldwin, C., Sasek, C. and Zigmond R.E., Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities, Neuroscience, 40 (1991) 175-184. 3 Cahill A.L. and Perlman R.L., Phosphorylation of tyrosine hydroxylase in the superior cerrvical ganglion, Biochirn. Biophys. Acta, 805 (1984) 217-226.
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