A convenient fluorimetry-based degranulation assay using RBL-2H3 cells.

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as ß-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of ß-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation.

Immunohistochemical distribution of proteins involved in glutamate release in subepithelial sensory nerve endings of rat epiglottis.

To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.

Morphology and chemical characteristics of taste buds associated with P2X3-immunoreactive afferent nerve endings in the rat incisive papilla.

The present study investigated the cellular components and afferent innervations of taste buds in the rat incisive papilla by immunohistochemistry using confocal scanning laser microscopy. Taste buds containing guanine nucleotide-binding protein G(t), subunit α3 (GNAT3)-imunoreactive cells were densely distributed in the lateral wall of incisive papilla forming the opening of nasoincisor ducts. GNAT3-immunoreactive cells in the taste buds were slender in shape and the tips of apical processes gathered at one point at the surface of the epithelium. The number of taste buds was 56.8 ± 4.5 in the incisive papilla. The incisive taste buds also contained ectonucleoside triphosphate diphosphohydrolase 2-immunoreactive cells and synaptotagmin-1-immunoreactive cells in addition to GNAT3-immunoreactive cells. Furthermore, GNAT3-immunoreactive cells were immunoreactive to taste transduction molecules such as phospholipase C, ß2-subunit, and inositol 1,4,5-trisphosphate receptor, type 3. P2X3-immunoreactive subepithelial nerve fibers intruded into the taste buds and terminated with hederiform or calix-like nerve endings attached to GNAT3-immunoreactive cells and synaptosomal-associated protein, 25 kDa-immunoreactive cells. Some P2X3-immunoreactive endings were also weakly immunoreactive for P2X2. Furthermore, a retrograde tracing method using fast blue dye indicated that most of the P2X3-immunoreactive nerve endings originated from the geniculate ganglia (GG) of the facial nerve. These results suggest that incisive taste buds are morphologically and cellularly homologous to lingual taste buds and are innervated by P2X3-immunoreactive nerve endings derived from the GG. The incisive papilla may be the palatal taste papilla that transmits chemosensory information in the oral cavity to the GG via P2X3-immunoreactive afferent nerve endings.

GluN2A- and GluN2B-immunoreactive type I cells attached to vesicular glutamate transporter 2-immunoreactive afferent nerve terminals of the rat carotid body.

We previously reported the immunoreactivity for the vesicular glutamate transporter 2 (VGLUT2) in afferent nerve terminals attached to chemoreceptor type I cells of the carotid body (CB), suggesting that glutamate is released from afferent terminals to stimulate these cells. In the present study, we examined the immunoreactivity for the glutamate-binding subunits of N-methyl-D-aspartate (NMDA) receptors, GluN2A and GluN2B in the rat CB, and the immunohistochemical relationships between these subunits and VGLUT2. Immunoreactivities for GluN2A and GluN2B were predominant in a subpopulation of tyrosine hydroxylase-immunoreactive type I cells rather than those of dopamine beta-hydroxylase-immunoreactive cells. Punctate VGLUT2-immunoreactive products were attached to GluN2A- and GluN2B-immunoreactive type I cells. Bassoon-immunoreactive products were localized between VGLUT2-immunoreactive puncta and type I cells immunoreactive for GluN2A and GluN2B. These results suggest that afferent nerve terminals release glutamate by exocytosis to modulate chemosensory activity of a subpopulation of type I cells via GluN2A- and GluN2B subunits-containing NMDA receptors.

Morphology of GNAT3-immunoreactive chemosensory cells in the nasal cavity and pharynx of the rat.

Solitary chemosensory cells and chemosensory cell clusters are distributed in the pharynx and larynx. In the present study, the morphology and reflexogenic function of solitary chemosensory cells and chemosensory cell clusters in the nasal cavity and pharynx were examined using immunofluorescence for GNAT3 and electrophysiology. In the nasal cavity, GNAT3-immunoreactive solitary chemosensory cells were widely distributed in the nasal mucosa, particularly in the cranial region near the nostrils. Solitary chemosensory cells were also observed in the nasopharynx. Solitary chemosensory cells in the nasopharyngeal cavity were barrel like or slender in shape with long lateral processes within the epithelial layer to attach surrounding ciliated epithelial cells. Chemosensory cell clusters containing GNAT3-immunoreactive cells were also detected in the pharynx. GNAT3-immunoreactive cells gathered with SNAP25-immunoreactive cells in chemosensory clusters. GNAT3-immunoreactive chemosensory cells were in close contact with a few SP- or CGRP-immunoreactive nerve endings. In the pharynx, GNAT3-immunoreactive chemosensory cells were also attached to P2X3-immunoreactive nerve endings. Physiologically, the perfusion of 10 mM quinine hydrochloride (QHCl) solution induced ventilatory depression. The QHCl-induced reflex was diminished by bilateral section of the glossopharyngeal nerve, suggesting autonomic reflex were evoked by chemosensory cells in pharynx but not in nasal mucosa. The present results indicate that complex shape of nasopharyngeal solitary chemosensory cells may contribute to intercellular communication, and pharyngeal chemosensory cells may play a role in respiratory depression.

Distribution and morphology of baroreceptors in the rat carotid sinus as revealed by immunohistochemistry for P2X3 purinoceptors.

The morphological characteristics of baroreceptors in the rat carotid sinus were reevaluated by whole-mount preparations with immunohistochemistry for P2X3 purinoceptors using confocal scanning laser microscopy. Immunoreactive nerve endings for P2X3 were distributed in the internal carotid artery proximal to the carotid bifurcation, particularly in the region opposite the carotid body. Some pre-terminal axons in nerve endings were ensheathed by myelin sheaths immunoreactive for myelin basic protein. Pre-terminal axons ramified into several branches that extended two-dimensionally in every direction. The axon terminals of P2X3-immunoreactive nerve endings were flat and leaf-like in shape, and extended hederiform- or knob-like protrusions in the adventitial layer. Some axons and axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2, and axon terminals were closely surrounded by terminal Schwann cells with S100 or S100B immunoreactivity. These results revealed the detailed morphology of P2X3-immunoreactive nerve endings and suggested that these endings respond to a mechanical deformation of the carotid sinus wall with their flat leaf-like terminals.

Stimulation of dopamine D2-like receptors in the lumbosacral defaecation centre causes propulsive colorectal contractions in rats.

KEY POINTS: The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure. In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum. The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2-like dopamine receptors. Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons. ABSTRACT: We have recently demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum. We hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. Because dopamine is one of the major neurotransmitters of descending pain inhibitory pathways in the spinal cord, we examined the effects of the intrathecal application of dopamine to the spinal defaecation centre on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anaesthetized rats. Slice patch clamp and immunohistochemistry were used to confirm the existence of dopamine-sensitive neurons in the sacral parasympathetic nuclei. Intrathecal application of dopamine into the L6-S1 spinal cord, where the lumbosacral defaecation centre is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons using TTX blocked the effect of dopamine. Although thoracic spinal transection had no effect on the enhancement of colorectal motility by intrathecal dopamine, the severing of the pelvic nerves abolished the enhanced motility. Pharmacological experiments revealed that the effect of dopamine is mediated primarily by D2-like dopamine receptors. Neurons labelled with retrograde dye injected at the colorectum showed an inward current in response to dopamine in slice patch clamp recordings. Furthermore, immunohistochemical analysis revealed that neurons immunoreactive to choline acetyltransferase express D2-like dopamine receptors. Taken together, our findings demonstrate that dopamine activates sacral parasympathetic preganglionic neurons via D2-like dopamine receptors and causes propulsive motility of the colorectum in rats. The present study supports the hypothesis that descending pain inhibitory pathways regulate defaecation reflexes.

Three-dimensional architectures of P2X2-/P2X3-immunoreactive afferent nerve terminals in the rat carotid body as revealed by confocal laser scanning microscopy.

We investigated the three-dimensional architectures of P2X2-/P2X3-immunoreactive nerve terminals in the rat carotid body using immunohistochemistry with confocal laser microscopy. Nerve endings immunoreactive for P2X2 and P2X3 were associated with clusters of type I cells, whereas some nerve endings were sparsely distributed in a few clusters. Most nerve endings surrounding type I cells were hederiform in shape and extended several flattened axon terminals, which were polygonal or pleomorphic in shape and contained P2X2-/P2X3-immunoreactive products. Three-dimensional reconstruction views revealed that some flattened nerve endings with P2X3 immunoreactivity formed arborized, sac- or goblet-like terminal structures and were attached to type I cells immunoreactive for tyrosine hydroxylase (TH). However, P2X3-immunoreactive axon terminals were sparsely distributed in type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for P2X2, S100, and TH revealed that P2X2-immunoreactive axon terminals were attached to TH-immunoreactive type I cells on the inside of type II cells with S100 immunoreactivity. These results revealed the detailed morphology of P2X2-/P2X3-immunoreactive nerve terminals and suggest that sensory nerve endings may integrate chemosensory signals from clustered type I cells with their variform nerve terminals.

Induction of Sd(a)-sialomucin and sulfated H-sulfomucin in mouse small intestinal mucosa by infection with parasitic helminth.

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, and sulphated H type 2 antigen, Fucα1-2Galß1-4GlcNAc(6SO3H)ß-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N. brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H. polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V. nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat.

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.

Immunohistochemical localization of P2Y12 purinoceptors in the rat carotid body.

The present study investigated the localization of the adenosine 5'-diphosphate (ADP)-selective P2Y12 purinoceptors in the rat carotid body using multilabeling immunofluorescence. Punctate immunoreactive products for P2Y12 were distributed in chemoreceptive type I cells immunoreactive to vesicular nucleotide transporter (VNUT) or dopamine beta-hydroxylase, but not in S100B-immunoreactive glial-like type II cells. P2Y12 immunoreactivity was localized in cell clusters containing VNUT-immunoreactive type I cells surrounded by the perinuclear cytoplasm and cytoplasmic processes of type II cells immunoreactive for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, which hydrolyze extracellular nucleotide tri- and/or di-phosphates. In ATP bioluminescence assays using carotid bodies, the degradation of extracellular ATP was attenuated in the presence of the selective NTPDases inhibitor ARL67156, suggesting ATP-degrading activity by NTPDases in the tissue. These results suggest that ATP released from type I cells is degraded into ADP and adenosine 5'-monophosphate by NTPDases expressed in type II cells, and that ADP modulates type I cells via P2Y12 purinoceptors.

Distribution of P2X3 purinoceptor-immunoreactive sensory nerve endings in the carotid body of Japanese macaque (Macaca fuscata).

In the carotid body of laboratory rodents, adenosine 5'-triphosphate (ATP)-mediated transmission is regarded as critical for transmission from chemoreceptor type I cells to P2X3 purinoceptor-expressing sensory nerve endings. The present study investigated the distribution of P2X3-immunoreactive sensory nerve endings in the carotid body of the adult male Japanese monkey (Macaca fuscata) using multilabeling immunofluorescence. Immunoreactivity for P2X3 was detected in nerve endings associated with chemoreceptor type I cells immunoreactive for synaptophysin. Spherical or flattened terminal parts of P2X3-immunoreactive nerve endings were in close apposition to the perinuclear cytoplasm of synaptophysin-immunoreactive type I cells. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), which hydrolyzes extracellular ATP, was localized in the cell body and cytoplasmic processes of S100B-immunoreactive cells. NTPDase2-immunoreactive cells surrounded P2X3-immunoreactive terminal parts and synaptophysin-immunoreactive type I cells, but did not intrude into attachment surfaces between terminal parts and type I cells. These results suggest ATP-mediated transmission between type I cells and sensory nerve endings in the carotid body of the Japanese monkey, as well as those of rodents.

Immunohistochemical localization of proteins involved in exocytosis of glutamate from P2X3 purinoceptor-expressing subserosal afferent nerve endings in the rat gastric antrum.

We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-ß,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat.

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X3 purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.

Immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II subunits in the rat carotid body.

Carotid body (CB) activity stimulated by a lower partial oxygen pressure in rats is enhanced by exposure to chronic intermittent hypoxia. However, the mechanisms that modulate CB activity remain unclear. In the present study, the expression and distribution of one of the candidate molecules to modulate reactivity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) were examined in the rat CB using reverse transcriptional polymerase chain reaction and immunofluorescence with isoform-specific antibodies. CaMKIIγ and CaMKIIδ were distributed in CB chemoreceptor cells, and exhibited intense immunoreactivity in dopamine ß-hydroxylase-positive chemoreceptor cells. CaMKIIß and CaMKIIγ were distributed in sensory nerve endings attached to chemoreceptor cells of the CB. In the petrosal ganglion, immunoreactivities for CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ were detected in the perinuclear region of ganglion cells. The present results indicate that CaMKIIγ and CaMKIIδ in chemoreceptor cells and CaMKIIß and CaMKIIγ in sensory nerve endings enhanced reciprocal synaptic transmission, i.e., noradrenaline and ATP for cells to neurons and glutamate for neurons to cells.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.

Immunohistochemical analysis of glutamatergic and serotonergic signaling pathways in chemosensory cell clusters in the pharynx and larynx of rats.

The present study examined cellular components and the localization of vesicular glutamate transporter (VGLUT) 1 and 2 and serotonin (5-HT) in chemosensory cell clusters in the rat pharynx and larynx. Triple immunolabeling for guanine nucleotide-binding protein G (t), subunit ⍺3 (GNAT3) and nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) with synaptotagmin-1 (Syt1) revealed NTPDase2-immunoreactive type I-like cells in addition to GNAT3-immunoreactive type II-like and Syt1-immunoreactive type III-like cells in pharyngolaryngeal chemosensory cell clusters. Therefore, these clusters appear to comprise similar cell types to those in the lingual taste buds with slight morphological modifications. An immunofluorescence analysis of VGLUT1 or VGLUT2 and GNAT3 with P2X3 purinoceptors revealed that VGLUTs co-localized to P2X3-immunoreactive spherical nerve terminals closely associated with GNAT3-immunoreactive type II-like cells. Moreover, triple immunolabeling for Syt1/synaptosomal-associated protein, 25 kDa (SNAP25) and P2X3 with VGLUT1 or VGLUT2 revealed punctate immunoreactive products for VGLUT1 and VGLUT2 within P2X3-immunoreactive flat axon terminals wrapped around Syt1/SNAP25-immunoreactive type III-like cells. The afferent nerve fibers innervating cell clusters may contain glutamate and release it by exocytosis. On the other hand, immunoreactive products for 5-HT and dopa decarboxylase were detected in Syt1-immunoreactive cells, indicating the release of 5-HT by these cells. The present results suggest that chemosensory cell clusters in the pharynx and larynx may be modulated by intrinsic glutamate and 5-HT.

Expression patterns of the transcription factors Fezf1, Fezf2, and Bcl11b in the olfactory organs of turtle embryos.

Many tetrapod vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and vomeronasal organ (VNO). In turtles, the olfactory organ consists of two types of sensory epithelia, the upper chamber epithelium (UCE; corresponding to the OE) and the lower chamber epithelium (LCE; corresponding to the VNO). In many turtle species, the UCE contains ciliated olfactory receptor cells (ORCs) and the LCE contains microvillous ORCs. To date, several transcription factors involved in the development of the OE and VNO have been identified in mammals. Fez family zinc-finger protein 1 and 2 (Fezf1 and 2) are expressed in the OE and VNO, respectively, of mouse embryos, and are involved in the development and maintenance of ORCs. B-cell lymphoma/leukemia 11B (Bcl11b) is expressed in the mouse embryo OE except the dorsomedial parts of the nasal cavity, and regulates the expression of odorant receptors in the ORCs. In this study, we examined the expression of Fezf1, Fezf2, and Bcl11b in the olfactory organs of embryos in three turtle species, Pelodiscus sinensis, Trachemys scripta elegans, and Centrochelys sulcata, to evaluate their involvement in the development of reptile olfactory organs. In all three turtle species, Bcl11b was expressed in the UCE, Fezf2 in the LCE, and Fezf1 in both the UCE and LCE. These results imply that the roles of the transcription factors Fezf1, Fezf2, and Bcl11b in olfactory organ development are conserved among mammals and turtles.

3-Iodothyronamine, a trace amine-associated receptor agonist, regulates intracellular Ca2+ increases via CaMK II through Epac2 in rat cerebral arterioles.

Trace amines (TAs) in the nervous system bind to TA-associated receptors (TAARs) and are involved in the regulation of monoaminergic functions. Among TAAR subtypes, TAAR1 has been implicated in the development of neurological disorders, such as schizophrenia. The present study investigated the effects of the TAAR1 agonist, 3-iodothyronamine (T1AM) on cerebral arterioles using fluctuations in the intracellular concentration of Ca2+ ([Ca2+]i) as an index of contractile responses. In cerebral arterioles, most of the TAAR agonists did not increase [Ca2+]i, while only T1AM elevated [Ca2+]i in vascular smooth muscle cells. This increase involved extracellular Ca2+ influx through T-type Ca2+ channels and inositol trisphosphate- and ryanodine-receptor-mediated Ca2+ release from intracellular stores. The inhibition of the cAMP sensor, exchange protein directly activated by cAMP (Epac) 2, and calmodulin kinase (CaMK) II strongly inhibited Ca2+ elevations. The present study revealed that T1AM acted not only on the TAAR1 receptor as previously suggested, but also on other G-protein coupled receptors and/or signal transduction systems to increase intracellular Ca2+ in cerebral arteriole smooth muscle cells. These results suggest that when using T1AM in clinical practice, attention should be paid to the early rise in blood pressure.