Serotonin-Storing Secretory Vesicles" HADASSAH TAMIRbC AND MICHAEL D. GERSHONb bDepartment of Anatomy and Cell Biology Columbia University College of Physicians & Surgeons New York, New York 10032 CDivisionof Neuroscience New York State Psychiatric Institute New York. New York 10032
INTRODUCTION Serotonergic neurons appear to play a critical role in the maintenance of mental health. For example, drugs that potentiate the action of serotonin (5-HT) by blocking its re-uptake alleviate depression' and agonists at 5-HTI, receptors are potent anxiolytics.* Other drugs that act on S H T , receptors are hall~cinogens.'~~ Additional evidence has implicated serotonergic neurons in the control of mood, temperature regulation, pain perception, feeding, and sleeping b e h a ~ i o rNot . ~ only are serotonergic neurons important in brain function, but peripheral serotonergic neurons in the gastrointestinal tract also appear to be vital to the normal function of the enteric nervous system (ENS).6 It follows, therefore, that an understanding of the cellular biology of serotonergic neurons, including knowledge of how the transmitter is stored and re-cycled will be of great importance to understanding the role of this subset of neurons in health and disease. In addition this knowledge can be expected to foster the design of new psychotherapeutic and gastrointestinal drugs. A great deal of useful knowledge has already been learned about serotonergic mechanisms, including the location in the brain and P N S of 5-HT-containing neurons and their projections, the biosynthesis and metabolism of 5-HT, the mechanism of uptake of the amine from an ambient medium, and the identification of many subtypes of pre- and post-junctional 5-HT receptor (for a review see ref. 7). Much less is known about the subcellular storage vesicles in which 5-HT is contained than about other components of serotonergic neurons. It is clear that these vesicles resemble in some respects vesicles that store catecholamines (CA). For example, reserpine and tetrabenazine, which inhibit the action of the transporter molecule in the vesicular membrane, deplete 5-HT as well as C A from the brain.* This indicates that the same or a similar transporter is present in both types of synaptic vesicle and that S H T , like CA, is dependent upon vesicular storage to protect intracellular amine from catabolism by monoamine oxidase (MAO).9 Moreover, isolated amine-containing vesicles from several sources take up 5-HT as well as CA." On the other hand, considerable evidence indicates that the synaptic vesicles of serotonergic neurons are also quite different from those of catecholaminergic neurons in a number of important respects. Some of this evidence has been derived from studies of the CNS; however, because neither serotonergic neurons, nor 5-HT- containing synaptic vesicles can be obtained from the brain uncontaminated by other types of neuron or other types of vesicle, respectively, studies of serotonergic vesicular properties have been supplemented by investigations of peripheral serotonergic neurons and paraneurons (thyroid parafollicular cells.") These peripheral sources of 5-HT storage aThis work was supported in part by grants NIMH 37575, NS 12969,and NS15547. 53
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organelles have been valuable models for the study of serotonergic mechanisms in much the same way that research done on chromaffin granules of the adrenal medulla has been helpful in revealing properties of CA storage organelles.I2 Peripheral serotonergic neurons are found in the gut in all vertebrates.6 and serotonergic paraneurons are found in the thyroid gland of a number of mammalian specie^.'^.'^ Platelets, which like serotonergic neurons, take up and store 5-HT have also been used as surrogates for neurons in the study of serotonergic rnechani~ms‘~,‘~; however, in contrast to central and peripheral serotonergic neurons and parafollicular cells, which are embryologically derived from neurectoderm, platelets are mesodermal derivatives. Moreover, platelets do not synthesize 5-HT as do the 5-HT-storing cells of neurectodermal origin, and platelets do not express the specific high affinity 5-HT binding protein (SBP) that characterizes all of the neurectodermal 5-HT-storing cell^."^'^ Furthermore, platelets are not whole cells, but cell fragments; therefore, the amount of cell biology that can be learned from the study of platelets is necessarily limited. In looking for a model relevant to central serotonergic neurons, therefore, it would appear that a neurectoderma1 serotonergic cell would be preferable to platelets. PARAFOLLICULAR CELLS OF THE THYROID During mammalian and avian development cells migrate from the region of the rhombencephalic (vagal) neural crest to the neck where they contribute to the formation of the ultimobranchial bodies.” In avians the ultimobranchial bodies persist, but in mammals they become incorporated into the developing thyroid gland. In both the avian ultimobranchial body and the mammalian thyroid, the crest-derived cells give rise to parafollicular or “C” cells (FIG. l ) , which store 5-HT in concentrations that vary between specie^.'^ The serotonergic neurons of the gut are derived from the same region of the neural crest as are the parafollicular cells.” Parafollicular cells store calcitonin as well as 5-HT.” Recent EM immunocytochemical studies have revealed that calcitonin (FIG. 2) and 5-HT are co-stored in parafollicular cells (FIG.3) in the same subcellular vesicles.2’The relationship of parafollicular cells to neurons remains evident, not only in the structure and properties of the cells, but in their response to nerve growth factor (NGF) and microenvironmental conditions.22 Sheep parafollicular cells, which are particularly rich in 5-HT23, have been isolated.24 When cells from dissociated thyroid glands are treated with thyrotropin (TSH) follicular cells become phagocytic. TSH-activated cell suspensions can then be passed through columns to which thyroglobulin has been coupled. The follicular cells “attempt” to phagocytize the beads and are retained on the columns, while the parafollicular cells pass through. This procedure of “phagocytic chromatography,” coupled with gradient centrifugation, results in a final preparation 97% of which is composed of parafollicular cells. When purified parafollicular cells are cultured in the presence of N G F they extend neurites.22 More significantly, these neurites express neurofilament proteins and synthesize calcitonin gene-related peptide (CGRP), instead of calcitonin. CGRP is produced by alternative splicing of mRNA coded by the calcitonin gene in neurons, while calcitonin is the product of the same gene expressed in endocrine cells. These observations indicate that parafollicular cells can be induced to change their phenotype from that of an endocrine cell to that of a neuron. Within 24-48 h following exposure of parafollicular cells to NGF, the cells that have extended neurites die. In contrast, if isolated parafollicular cells are co-cultured with segments of aneuronal chick hindgut, those parafollicular cells that reach the region where the myenteric plexus of the bowel would normally form (in the external muscular layer) survive indefinitely and even give rise to a neural-appearing plexus.22These observa-
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FIGURE 1. An electron micrograph showing the morphology of typical mammalian parafollicular cells. The illustration was obtained from a bat. Note the large number of dense cores secretory vesicles. PF = parafollicular cells; FC = follicular cells; e = extracellular space; The marker = 1.O pm.
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tions have led to the hypothesis that there may be a primitive serotonergic precursor cell that gives rise to serotonergic neurons in the gut and to parafollicular cells in the thyroid or ultimobranchial bodies. In any case, because of its strong resemblance to a neuron, the parafollicular cell has been called a paraneuron." It follows that the properties of the 5-HT storage organelles of parafollicular cells are likely to have a great deal in common with the properties of 5-HT-storing synaptic vesicles.
FIGURE 2. A portion of a resting parafollicular cell. The material has been fixed with aldehydes and embedded without osmication in LR White. The sections were incubated with anti-calcitonin sera and sites of immunoreactivity were visualized with a secondary antibody labeled with 5 nm particles of colloidal gold. Virtually every granule shows calcitonin immunoreactivity. The marker = 1.O urn.
PROPERTIES OF 5-HT- AND CATECHOLAMINE (CA)-STORING VESICLES 5-HT-containing secretory vesicles have been isolated from sheep thyroid glands by a two-step process involving separation of subcellular particles, first by size, and then by density." Storage organelles for 5-HT appear to differ from those which contain CA in ATP content, regulation of intravesicular pH, and in their complement of specific intravesicular For example, ATP cannot be demonstrated histochemically in vesicles in known serotonergic axons, such as supraependymal fibers,26or in the 5-HT-storing parafollicular cells of sheep.*' On the other hand, A T P is readily demonstrated in adrenal chromaffin g r a n ~ l e s . ~Moreover, ~-*~ synaptic vesicles isolated from 5-HT-containing synaptosomes are poor in ATP3' and parafollicular cell granules
FIGURE 3. An electron micrograph of a parafollicular cell. The tissue was fixed with paraformaldehyde (4%) and glutaraldehyde (0.1%) and was lightly osrnicated prior to embedding in Epon 812. The sections were picked up on nickel grids and exposed to anti-5-HT sera. Sites of immunoreactivity were visualized with a secondary antibody labeled with 5 nm particles of colloidal gold. About 50% of granules are labeled (+). The marker = 1.0 pm.
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contain virtually none." Additional properties of 5-HT-storing vesicles (isolated from sheep thyroids) which are shared with chromaffin granules include the presence in the vesicles of the same or a similar vacuolar H'ATPase. Antibodies to each of the 4 subunits of the ATPase3' cross react with proteins in parafollicular vesicles and the 72 kDa subunit can be demonstrated in the parafollicular granules of intact cells by EM immunocytochemistry (FIG. 4). The vesicles also contain synaptophysin and chromogranin-B32. The uptake of C A by adrenal chromaffin granules appears to depend upon the chemiosmotic r n e c h a n i ~ m . ~The " ~ ~granular membrane contains a H+-translocating ATPase, which pumps protons into the granules. The activity of this enzyme causes the formation both of a ApH and a potential difference (A*; inside positive) across the granular membrane. The proton gradient drives the transport of C A through an exchange of H+ for CA. The membranes of isolated chromaffin granules are sufficiently permeable to C1- that ATP-dependent acidification of the interior of granules can occur and not be prevented by the development of a limiting A*. In contrast, ATP-dependent acidification of the interior of isolated serotonergic vesicles from parafollicular cells does not occur unless the vesicles are exposed to an ionophore, such as valinomycin (FIG. 5), which dissipates a A*". The C1- conductance of these vesicles is low. These observations suggest that H' transport by isolated serotonergic vesicles is
FIGURE 4. A portion of a resting parafollicular cell. The material has been fixed with aldehydes and embedded without osrnication in LR White. The sections were incubated with antibodies to the 72 kDa subunit of the vacuolar ATPase and sites of immunoreactivity were visualized with a secondary antibody labeled with 40 nm particles of colloidal gold. Many granules are immunolabeled. The marker = 1.Oum.
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&
KCI + Valinomycin
IFccP
I
5 minutes FIGURE 5. Uptake of acridine orange into purified parafollicular cell granules measured by dual wavelength spectroscopy. A downward deflection reflects trapping of acridine orange inside granules and thus H' transport. An upward deflection indicates movement of the dye out of granules. In the control trace little inward movement of acridine orange is initiated by the addition of ATP (1); however, subsequent addition of the H' ionophore, FCCP (I), drives acridine orange out of the granules. In the superimposed tracing (KCI + valinomycin), the addition of ATP, induces inward movement of acridine orange. The addition of FCCP causes rapid efflux of acridine orange from the granules.
electrogenic. With intact resting parafollicular cells the ApH across the membranes of individual granules is highly variable; however, most of the granules acidify greatly when the cells are stimulated to secrete by the addition of secretogogues, such as TSH or increased (Ca2+),. Acidification of vesicles is associated with a rise in a (Ca2+)i and does not occur in CI--free media.25These observations have led to the hypothesis that the internal pH of serotonergic vesicles may be regulated by a Ca*+-dependent CIchannel in the vesicular membrane, the opening of which dissipates the A\k and permits further inward transport of H + . We have recently observed that isolated parafollicular vesicles, and ghosts derived from them, take up 'H-5-HT. This uptake is linear for about 4 min and saturates within 8 min. The uptake of 'H-5-HT by parafollicular vesicles or their ghosts
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resembles the uptake of 3H-CA by chromaffin granules in that the uptake is inhibited by agents that dissipate the ApH across the membranes of the granules. These observations suggest that the transport of 5-HT into vesicles may be regulated by parafollicular cells as they modify the internal pH of their 5-HT storage organelles. Conceivably 5-HT may be loaded into vesicles in preparation for exocytosis when the cells are stimulated by a natural secretogogue.
SEROTONIN-BINDING PROTEIN Serotonergic synaptic vesicles, but not chromaffin granules, appear to contain a specific type of protein that binds 5-HT with high affinity under conditions that approximate the intracellular milieu.33This protein, called SBP, disappears from the forebrain when lesions are made in the nuclei of the median raphe, where the cell bodies of serotonergic neurons are concentrated, and from the distal spinal cord when the cord is t r a n ~ e c t e d . ~ ~These .” observations indicate that SBP is contained within serotonergic neurons. Confirmation of this localization has recently been obtained. SBP has been purified and two forms of the protein have been found.36 These differ from M,; one form of SBP is 45 kDa and the other is 56 kDa. Antibodies have been raised against each. Many of theseantibodies do not distinguish one form of the protein from the other; however, some antibodies specifically recognize only the 45 kDa or the 56 kDa p r ~ t e i n . ~Immunostaining ~.~’ of the rat brain and spinal cord with 45 kDa or 56 kDa SBP-specific antisera has revealed that the immunoreactivities of each is colocalized in cell bodies and processes with the immunoreactivity of 5-HT.37SBP has also been detected in peripheral cells that store 5-HT; however, the protein is found only in those which are derived from neurectoderm, such as the serotonergic neurons of the gut and parafollicular cells,24.38~39 but not in 5-HT-containing cells derived from other germ layers, such as platelets, mast cells, or enterochromaffin cell^.'^^^^ Moreover, SBP accumulates above a spinal cord transection at a rate consistent with the proximo-distal movement of the protein by fast axonal transport.” Fast transport, in turn, is suggestive of an intracellular location in vesicles destined for the synaptic a p p a r a t ~ s . ~This ’ suggestion has been supported by the observations that SBP is concentrated in synaptosomes isolated from the brain and especially in vesicles liberated from these synaptosomes in K+-containing media.30 SBP has also been shown to be released along with 5-HT from stimulated enteric neurons by a Ca2+-dependent mechanism4’ and 45 kDa SBP has been localized chemically and immunocytochemically in the 5-HT storage vesicles of parafollicular cells.2’There is evidence that the 45 kDa form of SBP may predominate in synaptic vesicles, while the 56 kDa SBP is more concentrated in cell bodies and preterminal ax on^.^^.^* Newly taken up 3H-5-HT forms a complex mainly with 45 kDa SBP and during ontogeny the 45 kDa material appears later than 56 kDa SBP, but coincidentally with the period of synapt~genesis.~’ It has been proposed that the 56 kDa form of SBP is a precursor of the 45 kDa material.36 Each of the 12 monoclonal antibodies that have been raised against SBP recognize both 45 and 56 kDa SBP, as does an anti-idiotypic antibody produced from affinity purified antibodies to 5-HT. Moreover, mild trypsinization of the 56 kDa SBP/S-HT complex yields a complex of 45 kDa SBP/S-HT. Finally, peptide maps derived from 45 kDa or 56 kDa SBP labeled with a photoaffinity probe for the 5-HT binding site are similar.44 Fifty-six kDa SBP can be phosphorylated, which reduces its affinity for 5-HT, while 45 kDa SBP is not subject to pho~phorylation.~’ Conceivably, phosphorylation prevents premature binding of 5-HT during the transport of the protein to terminals. This idea remains to be tested.
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MEDULLARY THYROID CARCINOMA (MTC) CELLS Difficulties arise in characterizing SBP when only the brain is investigated. The complexity of the system and its limited ability to survive in vitro complicate studies of the biosynthesis and intracellular transport and processing of the molecule. On the other hand, the purification of SBP and the development of monospecific anti-SBP sera make it possible to investigate these issues. Recently we have characterized a serotonergic cell line derived from parafollicular cells that can be exploited to gain insight into the questions of where and in what form SBP is synthesized, whether 56 kDa SBP is converted to 45 kDa SBP, and if so, where in cells this conversion occurs. This cell line is a human medullary carcinoma of the thyroid (MTC cell^).'^ MTC cells produce 5-HT, both 56-kDa and 45 kDa, and peptides, including calcitonin, calcitonin gene related peptide (CGRP), s ~ m a t o s t a t i n . ~ The ~ - ' ~cells actively take up 'H-5-HT by a process that can be inhibited by fluoxetine and zimelidine, and their ability to store 5-HT is blocked by reserpine.46 Like parafollicular cells, MTC cells secrete 5-HT in response to the secretogogues, T S H (50 m u ) or elevated (Ca2+),(7.0 mM). The effect of TSH is Ca2+-dependentand is associated with the presence on these cells of T S H receptors, which can be demonstrated immunocytochemically by using a monospecific anti-TSH receptor serum (FIG. 6) The response of MTC cells to secretogogues may be coupled to CAMPas a second messenger. MTC cells can be induced to secrete 5-HT by addition of dibutyryl-CAMP (1.0 mM) or forskolin (1pM). It is also known that dibutyryl-CAMP and forskolin induce the secretion of calcitonin from M T C cells.50 The coincident secretion of 5-HT and calcitonin is to be expected, since E M immunocytochemistry reveals that in MTC cells as well as in parafollicular cells 5-HT and calcitonin and co-stored in the same secretory vesicles (FIG. 7). In many respects, therefore, the MTC cells resemble neurons and in fact they can be induced to extend neuritic processes and express neurofilament proteins by manipulating the growth media.46Unlike parafollicular cells, however, M T C cells do not express receptors for N G F and do not respond to this growth factor. The existence of these cells provides a model that can be used to characterize the synthesis, processing, and putative release and re-cycling of SBP. What is learned from MTC cells can be verified in later studies of serotonergic neurons of the gut and brain.
SUMMARY Advances have been made in the characterization of 5-HT-storing organelles of neurectodermal cells. The parafollicular cell of the thyroid has been used as a model. This cell stores 5-HT, shares many properties with neurons, and can be induced to change its phenotype from endocrine to neuronal by exposure in vitro to NGF. The membranes of isolated parafollicular 5-HT storage vesicles appear to contain a chloride channel that is gated in response to stimulation of the cells by secretogogues. Opening of this channel permits the interior of the vesicle to acidify in response to the action of a H'ATPase in the vesicular membrane. Development of a AiP appears to limit acidification of the vesicular interior when the chloride conductance is low. Transmembrane transport of 'H-5-HT into parafollicular vesicle is inhibited by dissipating the ApH across the granular membranes. The physiological significance of the ability of parafollicular vesicles to modify the internal pH of their 5-HT-storing organelles remains to be determined. Like the synaptic vesicles of central and peripheral serotonergic neurons parafollicular vesicles contain a specific 5-HT binding protein, SBP. 5-HT storage organelles and SBP have been found in medullary thyroid
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FIGURE 7. An EM showing a portion of an MTC cell. The preparation has been exposed to anti-calcitonin sera and processed as in FIG. 2. Virtually every granule displays calcitonin immunoreactivity. The inset shows the colocalization of 5-HT ( 5 nm gold) and calcitonin (1 5 nM gold) immunoreactivites in the same granule of an MTC cell. The marker = 1.OFm.
carcinoma (MTC) cells, a tumor line derived from parafollicular cells. The cell biology of SBP is now under study utilizing the M T C cells. REFERENCES 1.
2. 3. 4. 5. 6.
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31. CIDON,S. & N. NELSON.1986. Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membrane. J. Biol. Chem. 261: 9222-9227. unpublished observation. 32. WINKLER, M. D. & H. TAMIR.1985. Peripheral sources of serotonin and serotonin binding 33. GERSHON, proteins. In Serotonin and Cardiovascular System. P. M. Vanhoutte, Ed. New York: Raven Press, pp. 15-26. H. & M. J. KUHAR.1975. Association of serotonin binding protein with projections 34. TAMIR, of the midbrain raphe nuclei. Brain Res. 83: 169-172. M. D., K. P. LIU.,S. E. KARPIAK & H. TAMIR.1983. Storage of serotonin in vivo 35. GERSHON, as a complex with serotonin binding protein in central and peripheral serotonergic neurons. J. Neurosci. 3: 1901-191 1. & H. TAMIR. 1985. Identification, purification, and character36. LIU,K. P., M. D. GERSHON ization of two forms of serotonin binding protein from rat brain. J. Neurochem. 44: 1289-1301. 37. KIRCHGESSNER, A. L., M. D. GERSHON,K. P. Liu & H. TAMIR.1988. Co-storage of serotonin binding protein with serotonin in the rat CNS. J . Neurosci. 8: 3879-3890. 38. JONAKAIT, G. M., H. TAMIR.,M. M. RAPPORT& M. D. GERSHON.1977. Detection of a soluble serotonin binding protein in the mammalian myenteric plexus and other peripheral sites of storage. J. Neurochem. 2 8 277-284. 39. BERND,P., E. A. NUNEZ,M. D. GERSHON& H. TAMIR.1979. Serotonin binding protein: Characterization and localization in the parafollicular cells of the sheep thyroid. Anat. Rec. 193: 257-268. 40. GRAFSTEIN, 9. 1977. Axonal transport: The intracellular traffic of the neuron. In Handbook of Physiology: The Nervous System. J. M. Brookhard, V. 9. Mountcastle, E. R. Kandel, and S . R. Geiger, Eds. Bethesda, MD: Am. Physiol. SOC.,pp. 691-717. J. M., H. TAMIR,A. R. GINTZLER& M. D. GERSHON.1979. Release of 41. JONAKAIT, serotonin and its binding protein from enteric neurons. Brain Res 174 55-69. 42. ADLERSBERG, M., H. TAMIR& K. P. LIU. 1984. Photoaffinity labeling of 45 and 56Kd forms of serotonin binding protein(S9P); Evidence that 45 Kd SBP is stored in synaptic vesicles. Abs. 1 0 364. 43. LIU,K. P., H. TAMIR,S. H. HSIUNG,M. ADLERSBERG & M. D. GERSHON. 1987. Prenatal development of serotonin binding protein in relation to other transmitter-related characteristics of central serotonergic neurons. Dev. Brain Res. 32: 31-41. 1990. Photoaffinity labeling of two forms of 44. LIU, K. P., H. TAMIR& M. ADLERSBERG. serotonin binding protein: Peptide mapping of the binding sites. J. Neurochem. 5 4 963970. M., K. P. LIU, Y. EHRLICH& H. TAMIR.1987. A Ca” dependent protein 45. ADLERSBERG, kinase activity associated with serotonin binding protein. J Neurochem. 4 9 1105-1 1 1 5. S . H. HSIUNG,M. ADLERSBERG, E. A. NUNEZ& 46. TAMIR,H., K. P. LIU,R. F. PAYETTE, M. D. GERSHON.1989. Human medullary thyroid carcinoma: Characterization of the serotonergic and neuronal properties of a neuroectodermally derived cell line. J. Neuroscience. 9 1199-1212. ~ . J. S . HOROSZEWICZ, 47. L E O N G ,S., K. SHIMAOKA, M. FRIEDMAN, E. KAWINSKI, M. J. SONG, R. ZIEGEL,T. M. CHU, S. BAYLIN, & E. A. MIRAND. A new cell line for study of human medullary thyroid carcinoma. In Advances in Thyroid Neoplasia. M. Andreoli, F. Monaco, and J. Robbins, Eds., Rome: Field Education, Italia, pp. 95-108. H. T. KEUTMANN, R. S. 48. GKONOS,P. J., W. BORN,9. N . JONES,J. B. PETERMANN, BIRNBAUM, J. A. FISCHER & 9. A. Roos. 1986. Biosynthesis of calcitonin gene-related peptide and cacitonin by a human medullary thyroid carcinoma cell line. J. Biol. Chem. 261: 14386-14391. Y., M. ITO, & K. OGAWA. 1985. Immunoreactive somatostatin in thyroid C cells. 49. KAMEDA, An immunohistochemical study. Virchow’s Arch. (A) 4 1 0 97-101. A,, S. 9. BAYLIN,M. A. LEVINE& 9. D. NELKIN.1986. Cyclic AMP and 50. DEBUSTROS, Phorbol esters separately induced growth inhibition, calcitonin secretion, and the calcitonin gene transcription in cultured human medullary thyroid carcinoma. J. Biol. Chem. 261: 8036-8041.
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DISCUSSION OF T H E PAPER QUESTION:Does the serotonin vesicle differ in different locales or species? TAMIR:It varies with the species. Some species contain serotonin, but if they do have serotonin they will have it all along. In humans the embryo has serotonin and then later on in life the amount diminishes. But we haven’t seen changes from serotonin to yet another phenotypic expression. In terms of locale, for example from gut to brain, we are just beginning to look at that. QUESTION: What is the size of the serotonin vesicles? Is there only one kind or are there two kinds? TAMIR:You see only one kind. They look like the dense core vesicles, about a 0.1 km, that is quite large, and they all contain calcitonin. Almost all of them contain serotonin, whereas serotonin binding protein is seen in about half of them.
INTRODUCTION Serotonergic neurons appear to play a critical role in the maintenance of mental health. For example, drugs that potentiate the action of serotonin (5-HT) by blocking its re-uptake alleviate depression' and agonists at 5-HTI, receptors are potent anxiolytics.* Other drugs that act on S H T , receptors are hall~cinogens.'~~ Additional evidence has implicated serotonergic neurons in the control of mood, temperature regulation, pain perception, feeding, and sleeping b e h a ~ i o rNot . ~ only are serotonergic neurons important in brain function, but peripheral serotonergic neurons in the gastrointestinal tract also appear to be vital to the normal function of the enteric nervous system (ENS).6 It follows, therefore, that an understanding of the cellular biology of serotonergic neurons, including knowledge of how the transmitter is stored and re-cycled will be of great importance to understanding the role of this subset of neurons in health and disease. In addition this knowledge can be expected to foster the design of new psychotherapeutic and gastrointestinal drugs. A great deal of useful knowledge has already been learned about serotonergic mechanisms, including the location in the brain and P N S of 5-HT-containing neurons and their projections, the biosynthesis and metabolism of 5-HT, the mechanism of uptake of the amine from an ambient medium, and the identification of many subtypes of pre- and post-junctional 5-HT receptor (for a review see ref. 7). Much less is known about the subcellular storage vesicles in which 5-HT is contained than about other components of serotonergic neurons. It is clear that these vesicles resemble in some respects vesicles that store catecholamines (CA). For example, reserpine and tetrabenazine, which inhibit the action of the transporter molecule in the vesicular membrane, deplete 5-HT as well as C A from the brain.* This indicates that the same or a similar transporter is present in both types of synaptic vesicle and that S H T , like CA, is dependent upon vesicular storage to protect intracellular amine from catabolism by monoamine oxidase (MAO).9 Moreover, isolated amine-containing vesicles from several sources take up 5-HT as well as CA." On the other hand, considerable evidence indicates that the synaptic vesicles of serotonergic neurons are also quite different from those of catecholaminergic neurons in a number of important respects. Some of this evidence has been derived from studies of the CNS; however, because neither serotonergic neurons, nor 5-HT- containing synaptic vesicles can be obtained from the brain uncontaminated by other types of neuron or other types of vesicle, respectively, studies of serotonergic vesicular properties have been supplemented by investigations of peripheral serotonergic neurons and paraneurons (thyroid parafollicular cells.") These peripheral sources of 5-HT storage aThis work was supported in part by grants NIMH 37575, NS 12969,and NS15547. 53
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organelles have been valuable models for the study of serotonergic mechanisms in much the same way that research done on chromaffin granules of the adrenal medulla has been helpful in revealing properties of CA storage organelles.I2 Peripheral serotonergic neurons are found in the gut in all vertebrates.6 and serotonergic paraneurons are found in the thyroid gland of a number of mammalian specie^.'^.'^ Platelets, which like serotonergic neurons, take up and store 5-HT have also been used as surrogates for neurons in the study of serotonergic rnechani~ms‘~,‘~; however, in contrast to central and peripheral serotonergic neurons and parafollicular cells, which are embryologically derived from neurectoderm, platelets are mesodermal derivatives. Moreover, platelets do not synthesize 5-HT as do the 5-HT-storing cells of neurectodermal origin, and platelets do not express the specific high affinity 5-HT binding protein (SBP) that characterizes all of the neurectodermal 5-HT-storing cell^."^'^ Furthermore, platelets are not whole cells, but cell fragments; therefore, the amount of cell biology that can be learned from the study of platelets is necessarily limited. In looking for a model relevant to central serotonergic neurons, therefore, it would appear that a neurectoderma1 serotonergic cell would be preferable to platelets. PARAFOLLICULAR CELLS OF THE THYROID During mammalian and avian development cells migrate from the region of the rhombencephalic (vagal) neural crest to the neck where they contribute to the formation of the ultimobranchial bodies.” In avians the ultimobranchial bodies persist, but in mammals they become incorporated into the developing thyroid gland. In both the avian ultimobranchial body and the mammalian thyroid, the crest-derived cells give rise to parafollicular or “C” cells (FIG. l ) , which store 5-HT in concentrations that vary between specie^.'^ The serotonergic neurons of the gut are derived from the same region of the neural crest as are the parafollicular cells.” Parafollicular cells store calcitonin as well as 5-HT.” Recent EM immunocytochemical studies have revealed that calcitonin (FIG. 2) and 5-HT are co-stored in parafollicular cells (FIG.3) in the same subcellular vesicles.2’The relationship of parafollicular cells to neurons remains evident, not only in the structure and properties of the cells, but in their response to nerve growth factor (NGF) and microenvironmental conditions.22 Sheep parafollicular cells, which are particularly rich in 5-HT23, have been isolated.24 When cells from dissociated thyroid glands are treated with thyrotropin (TSH) follicular cells become phagocytic. TSH-activated cell suspensions can then be passed through columns to which thyroglobulin has been coupled. The follicular cells “attempt” to phagocytize the beads and are retained on the columns, while the parafollicular cells pass through. This procedure of “phagocytic chromatography,” coupled with gradient centrifugation, results in a final preparation 97% of which is composed of parafollicular cells. When purified parafollicular cells are cultured in the presence of N G F they extend neurites.22 More significantly, these neurites express neurofilament proteins and synthesize calcitonin gene-related peptide (CGRP), instead of calcitonin. CGRP is produced by alternative splicing of mRNA coded by the calcitonin gene in neurons, while calcitonin is the product of the same gene expressed in endocrine cells. These observations indicate that parafollicular cells can be induced to change their phenotype from that of an endocrine cell to that of a neuron. Within 24-48 h following exposure of parafollicular cells to NGF, the cells that have extended neurites die. In contrast, if isolated parafollicular cells are co-cultured with segments of aneuronal chick hindgut, those parafollicular cells that reach the region where the myenteric plexus of the bowel would normally form (in the external muscular layer) survive indefinitely and even give rise to a neural-appearing plexus.22These observa-
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FIGURE 1. An electron micrograph showing the morphology of typical mammalian parafollicular cells. The illustration was obtained from a bat. Note the large number of dense cores secretory vesicles. PF = parafollicular cells; FC = follicular cells; e = extracellular space; The marker = 1.O pm.
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tions have led to the hypothesis that there may be a primitive serotonergic precursor cell that gives rise to serotonergic neurons in the gut and to parafollicular cells in the thyroid or ultimobranchial bodies. In any case, because of its strong resemblance to a neuron, the parafollicular cell has been called a paraneuron." It follows that the properties of the 5-HT storage organelles of parafollicular cells are likely to have a great deal in common with the properties of 5-HT-storing synaptic vesicles.
FIGURE 2. A portion of a resting parafollicular cell. The material has been fixed with aldehydes and embedded without osmication in LR White. The sections were incubated with anti-calcitonin sera and sites of immunoreactivity were visualized with a secondary antibody labeled with 5 nm particles of colloidal gold. Virtually every granule shows calcitonin immunoreactivity. The marker = 1.O urn.
PROPERTIES OF 5-HT- AND CATECHOLAMINE (CA)-STORING VESICLES 5-HT-containing secretory vesicles have been isolated from sheep thyroid glands by a two-step process involving separation of subcellular particles, first by size, and then by density." Storage organelles for 5-HT appear to differ from those which contain CA in ATP content, regulation of intravesicular pH, and in their complement of specific intravesicular For example, ATP cannot be demonstrated histochemically in vesicles in known serotonergic axons, such as supraependymal fibers,26or in the 5-HT-storing parafollicular cells of sheep.*' On the other hand, A T P is readily demonstrated in adrenal chromaffin g r a n ~ l e s . ~Moreover, ~-*~ synaptic vesicles isolated from 5-HT-containing synaptosomes are poor in ATP3' and parafollicular cell granules
FIGURE 3. An electron micrograph of a parafollicular cell. The tissue was fixed with paraformaldehyde (4%) and glutaraldehyde (0.1%) and was lightly osrnicated prior to embedding in Epon 812. The sections were picked up on nickel grids and exposed to anti-5-HT sera. Sites of immunoreactivity were visualized with a secondary antibody labeled with 5 nm particles of colloidal gold. About 50% of granules are labeled (+). The marker = 1.0 pm.
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contain virtually none." Additional properties of 5-HT-storing vesicles (isolated from sheep thyroids) which are shared with chromaffin granules include the presence in the vesicles of the same or a similar vacuolar H'ATPase. Antibodies to each of the 4 subunits of the ATPase3' cross react with proteins in parafollicular vesicles and the 72 kDa subunit can be demonstrated in the parafollicular granules of intact cells by EM immunocytochemistry (FIG. 4). The vesicles also contain synaptophysin and chromogranin-B32. The uptake of C A by adrenal chromaffin granules appears to depend upon the chemiosmotic r n e c h a n i ~ m . ~The " ~ ~granular membrane contains a H+-translocating ATPase, which pumps protons into the granules. The activity of this enzyme causes the formation both of a ApH and a potential difference (A*; inside positive) across the granular membrane. The proton gradient drives the transport of C A through an exchange of H+ for CA. The membranes of isolated chromaffin granules are sufficiently permeable to C1- that ATP-dependent acidification of the interior of granules can occur and not be prevented by the development of a limiting A*. In contrast, ATP-dependent acidification of the interior of isolated serotonergic vesicles from parafollicular cells does not occur unless the vesicles are exposed to an ionophore, such as valinomycin (FIG. 5), which dissipates a A*". The C1- conductance of these vesicles is low. These observations suggest that H' transport by isolated serotonergic vesicles is
FIGURE 4. A portion of a resting parafollicular cell. The material has been fixed with aldehydes and embedded without osrnication in LR White. The sections were incubated with antibodies to the 72 kDa subunit of the vacuolar ATPase and sites of immunoreactivity were visualized with a secondary antibody labeled with 40 nm particles of colloidal gold. Many granules are immunolabeled. The marker = 1.Oum.
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&
KCI + Valinomycin
IFccP
I
5 minutes FIGURE 5. Uptake of acridine orange into purified parafollicular cell granules measured by dual wavelength spectroscopy. A downward deflection reflects trapping of acridine orange inside granules and thus H' transport. An upward deflection indicates movement of the dye out of granules. In the control trace little inward movement of acridine orange is initiated by the addition of ATP (1); however, subsequent addition of the H' ionophore, FCCP (I), drives acridine orange out of the granules. In the superimposed tracing (KCI + valinomycin), the addition of ATP, induces inward movement of acridine orange. The addition of FCCP causes rapid efflux of acridine orange from the granules.
electrogenic. With intact resting parafollicular cells the ApH across the membranes of individual granules is highly variable; however, most of the granules acidify greatly when the cells are stimulated to secrete by the addition of secretogogues, such as TSH or increased (Ca2+),. Acidification of vesicles is associated with a rise in a (Ca2+)i and does not occur in CI--free media.25These observations have led to the hypothesis that the internal pH of serotonergic vesicles may be regulated by a Ca*+-dependent CIchannel in the vesicular membrane, the opening of which dissipates the A\k and permits further inward transport of H + . We have recently observed that isolated parafollicular vesicles, and ghosts derived from them, take up 'H-5-HT. This uptake is linear for about 4 min and saturates within 8 min. The uptake of 'H-5-HT by parafollicular vesicles or their ghosts
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resembles the uptake of 3H-CA by chromaffin granules in that the uptake is inhibited by agents that dissipate the ApH across the membranes of the granules. These observations suggest that the transport of 5-HT into vesicles may be regulated by parafollicular cells as they modify the internal pH of their 5-HT storage organelles. Conceivably 5-HT may be loaded into vesicles in preparation for exocytosis when the cells are stimulated by a natural secretogogue.
SEROTONIN-BINDING PROTEIN Serotonergic synaptic vesicles, but not chromaffin granules, appear to contain a specific type of protein that binds 5-HT with high affinity under conditions that approximate the intracellular milieu.33This protein, called SBP, disappears from the forebrain when lesions are made in the nuclei of the median raphe, where the cell bodies of serotonergic neurons are concentrated, and from the distal spinal cord when the cord is t r a n ~ e c t e d . ~ ~These .” observations indicate that SBP is contained within serotonergic neurons. Confirmation of this localization has recently been obtained. SBP has been purified and two forms of the protein have been found.36 These differ from M,; one form of SBP is 45 kDa and the other is 56 kDa. Antibodies have been raised against each. Many of theseantibodies do not distinguish one form of the protein from the other; however, some antibodies specifically recognize only the 45 kDa or the 56 kDa p r ~ t e i n . ~Immunostaining ~.~’ of the rat brain and spinal cord with 45 kDa or 56 kDa SBP-specific antisera has revealed that the immunoreactivities of each is colocalized in cell bodies and processes with the immunoreactivity of 5-HT.37SBP has also been detected in peripheral cells that store 5-HT; however, the protein is found only in those which are derived from neurectoderm, such as the serotonergic neurons of the gut and parafollicular cells,24.38~39 but not in 5-HT-containing cells derived from other germ layers, such as platelets, mast cells, or enterochromaffin cell^.'^^^^ Moreover, SBP accumulates above a spinal cord transection at a rate consistent with the proximo-distal movement of the protein by fast axonal transport.” Fast transport, in turn, is suggestive of an intracellular location in vesicles destined for the synaptic a p p a r a t ~ s . ~This ’ suggestion has been supported by the observations that SBP is concentrated in synaptosomes isolated from the brain and especially in vesicles liberated from these synaptosomes in K+-containing media.30 SBP has also been shown to be released along with 5-HT from stimulated enteric neurons by a Ca2+-dependent mechanism4’ and 45 kDa SBP has been localized chemically and immunocytochemically in the 5-HT storage vesicles of parafollicular cells.2’There is evidence that the 45 kDa form of SBP may predominate in synaptic vesicles, while the 56 kDa SBP is more concentrated in cell bodies and preterminal ax on^.^^.^* Newly taken up 3H-5-HT forms a complex mainly with 45 kDa SBP and during ontogeny the 45 kDa material appears later than 56 kDa SBP, but coincidentally with the period of synapt~genesis.~’ It has been proposed that the 56 kDa form of SBP is a precursor of the 45 kDa material.36 Each of the 12 monoclonal antibodies that have been raised against SBP recognize both 45 and 56 kDa SBP, as does an anti-idiotypic antibody produced from affinity purified antibodies to 5-HT. Moreover, mild trypsinization of the 56 kDa SBP/S-HT complex yields a complex of 45 kDa SBP/S-HT. Finally, peptide maps derived from 45 kDa or 56 kDa SBP labeled with a photoaffinity probe for the 5-HT binding site are similar.44 Fifty-six kDa SBP can be phosphorylated, which reduces its affinity for 5-HT, while 45 kDa SBP is not subject to pho~phorylation.~’ Conceivably, phosphorylation prevents premature binding of 5-HT during the transport of the protein to terminals. This idea remains to be tested.
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MEDULLARY THYROID CARCINOMA (MTC) CELLS Difficulties arise in characterizing SBP when only the brain is investigated. The complexity of the system and its limited ability to survive in vitro complicate studies of the biosynthesis and intracellular transport and processing of the molecule. On the other hand, the purification of SBP and the development of monospecific anti-SBP sera make it possible to investigate these issues. Recently we have characterized a serotonergic cell line derived from parafollicular cells that can be exploited to gain insight into the questions of where and in what form SBP is synthesized, whether 56 kDa SBP is converted to 45 kDa SBP, and if so, where in cells this conversion occurs. This cell line is a human medullary carcinoma of the thyroid (MTC cell^).'^ MTC cells produce 5-HT, both 56-kDa and 45 kDa, and peptides, including calcitonin, calcitonin gene related peptide (CGRP), s ~ m a t o s t a t i n . ~ The ~ - ' ~cells actively take up 'H-5-HT by a process that can be inhibited by fluoxetine and zimelidine, and their ability to store 5-HT is blocked by reserpine.46 Like parafollicular cells, MTC cells secrete 5-HT in response to the secretogogues, T S H (50 m u ) or elevated (Ca2+),(7.0 mM). The effect of TSH is Ca2+-dependentand is associated with the presence on these cells of T S H receptors, which can be demonstrated immunocytochemically by using a monospecific anti-TSH receptor serum (FIG. 6) The response of MTC cells to secretogogues may be coupled to CAMPas a second messenger. MTC cells can be induced to secrete 5-HT by addition of dibutyryl-CAMP (1.0 mM) or forskolin (1pM). It is also known that dibutyryl-CAMP and forskolin induce the secretion of calcitonin from M T C cells.50 The coincident secretion of 5-HT and calcitonin is to be expected, since E M immunocytochemistry reveals that in MTC cells as well as in parafollicular cells 5-HT and calcitonin and co-stored in the same secretory vesicles (FIG. 7). In many respects, therefore, the MTC cells resemble neurons and in fact they can be induced to extend neuritic processes and express neurofilament proteins by manipulating the growth media.46Unlike parafollicular cells, however, M T C cells do not express receptors for N G F and do not respond to this growth factor. The existence of these cells provides a model that can be used to characterize the synthesis, processing, and putative release and re-cycling of SBP. What is learned from MTC cells can be verified in later studies of serotonergic neurons of the gut and brain.
SUMMARY Advances have been made in the characterization of 5-HT-storing organelles of neurectodermal cells. The parafollicular cell of the thyroid has been used as a model. This cell stores 5-HT, shares many properties with neurons, and can be induced to change its phenotype from endocrine to neuronal by exposure in vitro to NGF. The membranes of isolated parafollicular 5-HT storage vesicles appear to contain a chloride channel that is gated in response to stimulation of the cells by secretogogues. Opening of this channel permits the interior of the vesicle to acidify in response to the action of a H'ATPase in the vesicular membrane. Development of a AiP appears to limit acidification of the vesicular interior when the chloride conductance is low. Transmembrane transport of 'H-5-HT into parafollicular vesicle is inhibited by dissipating the ApH across the granular membranes. The physiological significance of the ability of parafollicular vesicles to modify the internal pH of their 5-HT-storing organelles remains to be determined. Like the synaptic vesicles of central and peripheral serotonergic neurons parafollicular vesicles contain a specific 5-HT binding protein, SBP. 5-HT storage organelles and SBP have been found in medullary thyroid
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FIGURE 7. An EM showing a portion of an MTC cell. The preparation has been exposed to anti-calcitonin sera and processed as in FIG. 2. Virtually every granule displays calcitonin immunoreactivity. The inset shows the colocalization of 5-HT ( 5 nm gold) and calcitonin (1 5 nM gold) immunoreactivites in the same granule of an MTC cell. The marker = 1.OFm.
carcinoma (MTC) cells, a tumor line derived from parafollicular cells. The cell biology of SBP is now under study utilizing the M T C cells. REFERENCES 1.
2. 3. 4. 5. 6.
CURZON, G. 1988. Serotonergicmechanisms ofdepression. Clin. Neuropharmacol. 11: S11s20. PEDIGO,N. J. W., H. I. YAMAMURA & D. L. NELSON.1981. Discrimination of multiple ('H) 5-hydroxytrptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem. 3 6 220-226. PEROUTKA, S. J. & S. H. SNYDER. 1979. Multiple serotonin receptors: differential binding of ('H-)5-hydroxytryptamine, ('H)lysergic acid diethylamide and ('H)spiroperidol, Mol. Pharmacol. 1 6 687-699. JACOBS,B. L. 1983. Mechanism of action of hallucinogenic drugs: focus upon postsynaptic serotonergic receptors. In Psychopharmacology, 1, D. G. Grahame-Smith, Ed. Excerpta Medica, pp. 344-76. VOGT, M. 1982. Some functional aspects of central serotonergic neurons. In Biology of Serotonergic Transmission N. N. Osborne, Ed. New York: John Wiley & Sons, pp. 299-315. GERSHON,M. D., G. M. MAWE& T. M. BRANCHECK. 1989. 5-hydroxytryptamine and enteric neurons. In Peripheral Action of 5-Hydroxytryptamine.J . R. Fozard, Ed. Oxford: Oxford University Press. pp. 247-265.
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DISCUSSION OF T H E PAPER QUESTION:Does the serotonin vesicle differ in different locales or species? TAMIR:It varies with the species. Some species contain serotonin, but if they do have serotonin they will have it all along. In humans the embryo has serotonin and then later on in life the amount diminishes. But we haven’t seen changes from serotonin to yet another phenotypic expression. In terms of locale, for example from gut to brain, we are just beginning to look at that. QUESTION: What is the size of the serotonin vesicles? Is there only one kind or are there two kinds? TAMIR:You see only one kind. They look like the dense core vesicles, about a 0.1 km, that is quite large, and they all contain calcitonin. Almost all of them contain serotonin, whereas serotonin binding protein is seen in about half of them.
