T cell-dependent mast cell degranulation and release of serotonin in murine delayed-type hypersensitivity.

We have previously suggested that the release of serotonin (5-hydroxytryptamine) (5-HT) by local tissue mast cells is required for the elicitation of delayed-type hypersensitivity (DTH) in mice. In the current study, light microscopic radioautographs from animals treated with [3H]5-HT indicated that local mast cells released 5-HT between 6 and 18 h during the evolution of DTH. Ultrastructural examination of mast cells revealed surface activation, indicated by extension of surface filopodia, and degranulation by fusion and exocytosis. Light and electron microscopic studies of the endothelium of postcapillary venules at sites of DTH revealed the development of gaps between adjacent cells. The development of gaps permitted extravasation of tracers that was abolished by depletion or antagonism of 5-HT. Thus mast cells degranulated and released 5-HT in DTH, and this 5-HT acted on local vessels. Recipients of nonadherent, non-immunoglobulin-bearing sensitized lymphocytes also demonstrated similar mast cell degranulation and the formation of endothelial gaps. This indicated that mast cell degranulation and 5-HT release in murine DTH were probably T cell dependent.

Requirement for vasoactive amines for production of delayed-type hypersensitvity skin reactions.

The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.

Electron microscope radioautographic identification of serotonin-synthesizing cells in the mouse gastric mucosa.

This study correlates the fine structure of mouse gastric endocrine cells with their ability to synthesize serotonin (5-HT) from 5-hydroxytryptophan (5-HTP). Mice were sacrificed 2 hr after the intravenous injection of 5-HTP-(3)H or 5-HT-(3)H. Their stomachs were processed for light- and electron microscope radioautography in a manner which retained labeled 5-HT while washing out other labeled substances. Stomachs from additional mice were incubated in vitro with 5-HT-(3)H and processed similarly. All morphologic types of mouse gastric endocrine cells exhibited a similar facility to incorporate exogenous 5-HTP and to convert it to 5-HT which was bound intracellularly. Differences in densities of silver grains observed over endocrine cells suggested that individual endocrine cells indeed varied in their ability to synthesize and/or to bind 5-HT; such variations, however, were not reflected by differences in fine structure, with the exception that endocrine cells with few granules always contained little newly synthesized 5-HT. The newly synthesized 5-HT was associated with the intracellular granules. The gastric endocrine cells were not labeled by exogenous 5-HT-(3)H, whereas mast cells were labeled by either 5-HT-(3)H or 5-HTP-(3)H administration. The findings of the present study support the position that the gastric endocrine cells represent a single cell type, at least in respect to serotonin metabolism-that the argyrophil or argentaffin reactivity of these cells merely reflects their amine content at a given time.

Subcellular storage organelles for 5-hydroxytryptamine in parafollicular cells of drugs which deplete the amine.

A study was made of the effect of the administration of reserpine and parachlorophenylalanine, an inhibitor of 5-hydroxytryptamine (serotonin; 5-HT), on the capacity of thyroid parafollicular cells to synthesize and store 5-HT. The two drugs were given to nonhibernating bats in doses which produced an equivalent degree of depletion of 5-HT from the thyroid. Tritiated 5-hydroxytryptophan, the precursor of 5-HT, was then given intravenously to assess the ability of parafollicular cell granules to take up and retain newly synthesized 5-HT. Reserpine, but not parachlorophenylalanine, decreased the amount of labeled 5-HT found in the thyroid and prevented autoradiographic labeling of parafollicular cell granules. Quantitative ultrastructural and stereological analysis demonstrated that the granules in untreated animals appeared to be nearly spherical prolate ellipsoids, with a uniformly electron-opaque inner matrix. In animals given reserpine, the axial ratio of the ellipsoidal granules increased greatly and a faint internal striation parallel to the long axis of the granules became apparent. Similar changes were not induced by parachlorophenylalanine. No other morphological changes in the thyroid epithelium were detected after administration of reserpine. This study confirms the association of 5-HT with the mature small secretory granules of thyroid parafollicular cells.

An electron microscope autoradiographic study of the neuronal and extraneuronal localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine.

The localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine (NE) was studied by means of electron microscope autoradiography. Monoamine oxidase was inhibited so that the distribution of amine in both neuronal (Uptake(1)) and extraneuronal (Uptake(2)) sites could be analyzed. Labeling was nonrandom in both the atrial and ventricular myocardium. The highest relative specific activity was found in neural processes which showed morphological criteria of terminal adrenergic axons. Analysis of the distribution of label around the labeled axonal varicosities indicated that the radioactive amine was more concentrated peripherally than centrally in these structures. Label was also found over cardiocytes in both atrium and ventricle. The pattern of this labeling indicated that the radioactive amine was associated with myofilaments. In the ventricle, I bands were most heavily labeled, indicating a probable association of radioactive amine with thin filaments. Labeling was prevented by administration of phenoxybenzamine and decreased only in cardiocytes by normetanephrine. The nonrandom distribution of labeled amine within cardiocytes supports the view that Uptake(2) represents not only a second mechanism of inactivation of the sympathetic neurotransmitter, but may also be involved in the mediation of some of the action of NE on cardiac muscle.

Separation of dissociated thyroid follicular and parafollicular cells: association of serotonin binding protein with parafollicular cells.

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.

Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin.

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.

A nonpeptide morphine-like compound: immunocytochemical localization in the mouse brain.

A nonpeptide morphine-like compound (MLC) which cross reacts with morphine-specific antibodies has been localized with the use of immunocytochemistry. This morphine-like compound is found in neuronal perikarya or processes (or both) in nuclei related to vestibular, cerebellar, and raphe systems.

Neural crest development. Do developing enteric neurons need endothelins?

Recent experiments have led to the unexpected finding that endothelin-3 and the endothelin B receptor are absolutely necessary for the development of the enteric nervous system in the colon, but it is not yet clear why.

Serotonin and neuroprotection in functional bowel disorders.

The 5-HT(4) partial agonist tegaserod is effective in the treatment of chronic constipation and constipation predominant irritable bowel syndrome. 5-HT(4) receptors are located on presynaptic terminals in the enteric nervous system. Stimulation of 5-HT(4) receptors enhances the release of acetylcholine and calcitonin gene related peptide from stimulated nerve terminals. This action strengthens neurotransmission in prokinetic pathways, enhancing gastrointestinal motility. The knockout of 5-HT(4) receptors in mice not only slows gastrointestinal activity but also, after 1 month of age, increases the age-related loss of enteric neurons and decreases the size of neurons that survive. 5-HT(4) receptor agonists, tegaserod and RS67506, increase numbers of enteric neurons developing from precursor cells and/or surviving in culture; they also increase neurite outgrowth and decrease apoptosis. The 5-HT(4) receptor antagonist, GR113808, blocks all of these effects, which are thus specific and 5-HT(4)-mediated. 5-HT(4) receptor agonists, therefore, are neuroprotective and neurotrophic for enteric neurons. Because the age-related decline in numbers of enteric neurons may contribute to the dysmotilities of the elderly, the possibility that the neuroprotective actions of 5-HT agonists can be utilized to prevent the occurrence or worsening of these conditions should be investigated.

Genes and lineages in the formation of the enteric nervous system.

The enteric nervous system is large, complex, and independent of the CNS. Its neural-crest-derived precursors migrate along defined pathways to colonize the bowel. Recent studies of the sequential actions of essential growth and transcription factors have revealed that enteric neuronal development involves a complex interaction of lineage-determined and microenvironmental elements.

Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine.

The enteric nervous system contains intrinsic primary afferent neurons that allow mucosal stimulation to initiate reflexes without CNS input. We tested the hypothesis that submucosal primary afferent neurons are activated by 5-hydroxytryptamine (5-HT) released from the stimulated mucosa. Fast and/or slow EPSPs were recorded in submucosal neurons after the delivery of exogenous 5-HT, WAY100325 (a 5-HT(1P) agonist), mechanical, or electrical stimuli to the mucosa of myenteric plexus-free preparations (+/- extrinsic denervation). These events were responses of second-order cells to transmitters released by excited primary afferent neurons. After all stimuli, fast and slow EPSPs were abolished by a 5-HT(1P) antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, and by 1.0 microM tropisetron, but not by 5-HT(4)-selective antagonists (SB204070 and GR113808A) or 5-HT(3)-selective antagonists (ondansetron and 0.3 microM tropisetron). Fast EPSPs in second-order neurons were blocked by hexamethonium, and most slow EPSPs were blocked by an antagonist of human calcitonin gene-related peptide (hCGRP(8-37)). hCGRP(8-37) also inhibited the spread of excitation in the submucosal plexus, assessed by measuring the uptake of FM2-10 and induction of c-fos. In summary, data are consistent with the hypothesis that 5-HT from enterochromaffin cells in response to mucosal stimuli initiates reflexes by stimulating 5-HT(1P) receptors on submucosal primary afferent neurons. Second-order neurons respond to these cholinergic/CGRP-containing cells with nicotinic fast EPSPs and/or CGRP-mediated slow EPSPs. Slow EPSPs are necessary for excitation to spread within the submucosal plexus. Because some second-order neurons contain also CGRP, primary afferent neurons may be multifunctional and also serve as interneurons.

Serotonin and the 5-HT(2B) receptor in the development of enteric neurons.

We tested the hypothesis that 5-HT promotes the differentiation of enteric neurons by stimulating a developmentally regulated receptor expressed by crest-derived neuronal progenitors. 5-HT and the 5-HT(2) agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine(.)HCl (DOI) enhanced in vitro differentiation of enteric neurons, both in dissociated cultures of mixed cells and in cultures of crest-derived cells isolated from the gut by immunoselection with antibodies to p75(NTR). The promotion of in vitro neuronal differentiation by 5-HT and DOI was blocked by the 5-HT(1/2) antagonist methysergide, the pan-5-HT(2) antagonist ritanserin, and the 5-HT(2B/2C)-selective antagonist SB206553. The 5-HT(2A)-selective antagonist ketanserin did not completely block the developmental effects of 5-HT. 5-HT induced the nuclear translocation of mitogen-activated protein kinase. This effect was blocked by ritanserin. mRNA encoding 5-HT(2A) and 5-HT(2B) receptors was detected in the fetal bowel (stomach and small and large intestine), but that encoding the 5-HT(2C) receptor was not. mRNA encoding the 5-HT(2B) receptor and 5-HT(2B) immunoreactivity were found to be abundant in primordial [embryonic day 15 (E15)-E16] but not in mature myenteric ganglia. 5-HT(2B)-immunoreactive cells were found to be a subset of cells that expressed the neuronal marker PGP9.5. These data demonstrate for the first time that the 5-HT(2B) receptor is expressed in the small intestine as well as the stomach and that it is expressed by enteric neurons as well as by muscle. It is possible that by stimulating 5-HT(2B) receptors, 5-HT affects the fate of the large subset of enteric neurons that arises after the development of endogenous sources of 5-HT.

Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation.

The existence of enteric dopaminergic neurons has been suspected; however, the innervation of the gut by sympathetic nerves, in which dopamine (DA) is the norepinephrine precursor, complicates analyses of enteric DA. We now report that transcripts encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the murine bowel (small intestine > stomach or colon; proximal colon > distal colon). Because sympathetic neurons are extrinsic, transcripts encoding TH and DAT in the bowel are probably derived from intrinsic neurons. TH protein was demonstrated immunocytochemically in neuronal perikarya (submucosal >> myenteric plexus; small intestine > stomach or colon). TH, DA, and DAT immunoreactivities were coincident in subsets of neurons (submucosal > myenteric) in guinea pig and mouse intestines in situ and in cultured guinea pig enteric ganglia. Surgical ablation of sympathetic nerves by extrinsic denervation of loops of the bowel did not affect DAT immunoreactivity but actually increased numbers of TH-immunoreactive neurons, expression of mRNA encoding TH and DAT, and enteric DOPAC (the specific dopamine metabolite). The fetal gut contains transiently catecholaminergic (TC) cells. TC cells are the proliferating crest-derived precursors of mature neurons that are not catecholaminergic and, thus, disappear after embryonic day (E) 14 (mouse) or E15 (rat). TC cells appear early in ontogeny, and their development/survival is dependent on mash-1 gene expression. In contrast, the intrinsic TH-expressing neurons of the murine bowel appear late (perinatally) and are mash-1 independent. We conclude that the enteric nervous system contains intrinsic dopaminergic neurons that arise from a mash-1-independent lineage of noncatecholaminergic precursors.

Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility and the expression of cation transporters.

The enteric serotonin reuptake transporter (SERT) has been proposed to play a critical role in serotonergic neurotransmission and in the initiation of peristaltic and secretory reflexes. We analyzed potential compensatory mechanisms and enteric function in the bowels of mice with a targeted deletion of SERT. The guts of these animals were found to lack mRNA encoding SERT; moreover, high-affinity uptake of 5-HT into epithelial cells, mast cells, and enteric neurons was present in the SERT +/+ bowel but absent in the SERT -/- bowel. However, both the SERT +/+ gut and the -/- gut expressed molecules capable of transporting 5-HT, but with affinities and selectivity much lower than those of SERT. These included the dopamine transporter (DAT) and polyspecific organic cation transporters OCT-1 and OCT-3. DAT and OCT immunoreactivities were present in both the submucosal and myenteric plexuses, and the OCTs were also located in the mucosal epithelium. 5-HT was found in all of its normal sites in the SERT -/- bowel, which contained mRNA encoding tryptophan hydroxylase, but no 5-HT was present in the blood of SERT -/- animals. Stool water and colon motility were increased in most SERT -/- animals; however, the increase in motility (diarrhea) occasionally alternated irregularly with decreased motility (constipation). The watery diarrhea is probably attributable to the potentiation of serotonergic signaling in SERT -/- mice, whereas the transient constipation may be caused by episodes of enhanced 5-HT release leading to 5-HT receptor desensitization.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Thyrotropin-induced thyroidal release of 5-hydroxytryptamine and accompanying ultrastructural changes in parafollicular cells.

The action of TSH on parafollicular cells of the bat thyroid was examined. Parafollicular cells were loaded with [3H]5-hydroxytryptamine ([3H]5-HT) by incubation with the precursor [3H]5-hydroxytryptophan in vitro. Subsequent exposure to TSH released [3H]5-HT from the glands, but not [3H]5-hydroxytryptophan. When thyroids were loaded with [3H]norepinephrine, TSH failed to release that amine. The [3H]5-HT-releasing effects of TSH were blocked by dinitrophenol and antimycin A and so were energy dependent. [3H]5-HT was not released by pentagastrin or calcitonin. Electron microscopic examination of thyroid glands exposed to TSH in vitro revealed degranulation of some parafollicular cells as well as the presence of abnormal appearing microgranules and large intracisternal (within rough endoplasmic reticulum) accumulations of secretory material. These results demonstrate for the first time direct effects of TSH on parafollicular cells. The results are consistent with the hypothesis that 5-HT, a normal constituent of bat parafollicular cells and an activator of follicular cells, may act as an intrathyroid local hormone.

Calcium-induced release of 5-hydroxytryptamine from thyroid lobes in vitro and accompanying ultrastructural changes in parafollicular and follicular cells.

Biogenic amines, including serotonin (5-HT), have been shown to activate follicular cells of the thyroid. 5-HT is stored in bat's parafollicular cells. Previous radioautographical evidence indicates that this 5-HT is present in calcitonin granules. The present study was done to determine if Ca++, the natural stimulus to calcitonin release, would also release parafollicular cell 5-HT and, if so, whether this release would be accompanied by activation of follicular cells. Parafollicular cells were filled with labeled 5-HT by incubation of thyroid lobes of bats or mice with 5-[3H]hydroxytryptophan, the precursor of [3H]5-HT. Thyroid lobes were incubated in vitro in Ca++-free medium containing a chelating agent and were then challenged with Ca++ (0-30 mM). Release of [3H]5-HT was defected beginning at 5 mM Ca++. [3H]5-HT release was roughly proportional to the Ca++ concentration. Calcium challenge also affected thyroid ultrastructure in bats. After 10-min exposure to 30 mM Ca++, there were an increased number and centripetal movement of follicular cell lysosomes, development of apical pseudopods, and formation of colloid droplets. Many lysosomes also developed a crystalline-like matrix. An unique membrane-enclosed, rod-shaped organelle appeared in a small number of follicular cells. Parafollicular cells exposed to high concentrations of Ca++ were often degranulated, although many appeared unchanged. These changes confirm that 5-HT is released from parafollicular cells by elevating the external Ca++ concentration, supporting the hypothesis that 5-HT and calcitonin share storage granules. 5-HT release may mediate the activation of follicular cells by Ca++.