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.

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.

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.

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).

Suitability of Polymyxin B as a Mucosal Adjuvant for Intranasal Influenza and COVID-19 Vaccines.

Polymyxin B (PMB) is an antibiotic that exhibits mucosal adjuvanticity for ovalbumin (OVA), which enhances the immune response in the mucosal compartments of mice. Frequent breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants indicate that the IgA antibody levels elicited by the mRNA vaccines in the mucosal tissues were insufficient for the prophylaxis of this infection. It remains unknown whether PMB exhibits mucosal adjuvanticity for antigens other than OVA. This study investigated the adjuvanticity of PMB for the virus proteins, hemagglutinin (HA) of influenza A virus, and the S1 subunit and S protein of SARS-CoV-2. BALB/c mice immunized either intranasally or subcutaneously with these antigens alone or in combination with PMB were examined, and the antigen-specific antibodies were quantified. PMB substantially increased the production of antigen-specific IgA antibodies in mucosal secretions and IgG antibodies in plasma, indicating its adjuvanticity for both HA and S proteins. This study also revealed that the PMB-virus antigen complex diameter is crucial for the induction of mucosal immunity. No detrimental effects were observed on the nasal mucosa or olfactory bulb. These findings highlight the potential of PMB as a safe candidate for intranasal vaccination to induce mucosal IgA antibodies for prophylaxis against mucosally transmitted infections.

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.

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.

Immunolocalization of vesicular glutamate transporter 2 and exocytosis-related proteins in afferent nerve endings innervating taste buds in the rat incisive papilla.

The present study aimed to investigate the immunolocalization of vesicular glutamate transporter (VGLUT) 1 and 2, and proteins associated with exocytosis, i.e., core components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2) and synaptotagmin-1 (Syt1), in incisive papillary taste buds of rats using double-indirect immunofluorescence. No VGLUT1 immunoreactivity was observed, whereas VGLUT2-immunoreactive punctate products were closely associated with guanine nucleotide-binding protein G(t) subunit α3-immmunoreactive cells in taste buds. VGLUT2 was immunolocalized in P2X3 purinoceptor-expressing afferent nerve endings. Synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2 were immunolocalized in nerve endings containing VGLUT2-immunoreactive products as well as a few cells in taste buds. VGLUT2 was co-immunolocalized in some intragemmal nerve endings immunoreactive for Syt1, a calcium sensor implicated in vesicle membrane fusion. The present results suggest that afferent nerve endings innervating incisive taste buds release glutamate by exocytosis to modulate taste cell function.

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.

Morphology of P2X3-immunoreactive basket-like afferent nerve endings surrounding serosal ganglia and close relationship with vesicular nucleotide transporter-immunoreactive nerve fibers in the rat gastric antrum.

We previously reported P2X3 purinoceptor (P2X3)-expressing vagal afferent nerve endings with large web-like structures in the subserosal tissue of the antral lesser curvature, suggesting that these nerve endings were one of the vagal mechanoreceptors. The present study investigated the morphological relationship between P2X3-immunoreactive nerve endings and serosal ganglia in the rat gastric antrum by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3-immunoreactive basket-like subserosal nerve endings with new morphology were distributed laterally to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into numerous nerve fibers with pleomorphic flattened structures to form basket-like nerve endings, and the parent axons were originated from large net-like structures of vagal afferent nerve endings. Basket-like nerve endings wrapped around the whole serosal ganglia, which were characterized by neurofilament 200 kDa-immunoreactive neurons with or without neuronal nitric oxide synthase immunoreactivity and S100B-immunoreactive glial cells. Furthermore, basket-like nerve endings were localized in close apposition to dopamine beta-hydroxylase-immunoreactive sympathetic nerve fibers immunoreactive for vesicular nucleotide transporter. These results suggest that P2X3-immunoreactive basket-like nerve endings associated with serosal ganglia are the specialized ending structures of vagal subserosal mechanoreceptors in order to increase the sensitivity during antral peristalsis, and are activated by ATP from sympathetic nerve fibers and/or serosal ganglia for the regulation of mechanoreceptor function.

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.

Distribution and morphology of P2X3-immunoreactive subserosal afferent nerve endings in the rat gastric antrum.

The present study investigated the morphological characteristics of subserosal afferent nerve endings with immunoreactivity for the P2X3 purinoceptor (P2X3) in the rat stomach by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3 immunoreactivity was observed in subserosal nerve endings proximal and lateral to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into several lamellar processes to form net-like complex structures that extended two-dimensionally in every direction on the surface of the longitudinal smooth muscle layer. The axon terminals in the periphery of P2X3-immunoreactive net-like structures were flat and looped or leaf-like in shape. Some net-like lamellar structures and their axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2. P2X3-immunoreactive nerve fibers forming net-like terminal structures were closely surrounded by S100B-immunoreactive terminal Schwann cells, whereas axon terminals twined around these cells and extended club-, knob-, or thread-like protrusions in the surrounding tissues. Furthermore, a retrograde tracing method using fast blue dye indicated that most of these nerve endings originated from the nodose ganglia of the vagus nerve. These results suggest that P2X3-immunoreactive subserosal nerve endings have morphological characteristics of mechanoreceptors and contribute to sensation of a mechanical deformation of the distal antral wall associated with antral peristalsis.

Morphological characterization of brush cells in the rat trachea.

Brush cells have recently been classified as solitary chemosensory cells. However, tracheal brush cells have not been morphologically and immunohistochemically characterized yet. In the present study, the morphological and immunohistochemical characteristics of tracheal brush cells were analyzed using immunohistochemistry and scanning, and transmission electron microscopies. Brush cells in the tracheal epithelium were barrel-like or columnar in shape and were immunoreactive for villin. Scanning and transmission electron microscopies revealed densely arranged thick microvilli on the apical surface of tracheal brush cells and tubular membranous elements and/or vesicular formations in the supranuclear region. A morphometrical analysis of tracheal whole-mount preparations showed that the density of brush cells was greater in the cranial third and the mucosa on the annular ligament. Double immunofluorescence revealed that the morphology of villin-immunoreactive brush cells was distinct from other non-ciliated cells in the tracheal epithelium, i.e., MUC5AC-immunoreactive mucous cells, SNAP25-immunoreactive neuroendocrine cells, and GNAT3-immunoreactive solitary chemosensory cells. On the other hand, tracheal brush cells were immunoreactive for the marker proteins for intestinal brush cells, CK18, DCLK1, and Cox1; however, these antibodies also recognized cells other than brush cells. Furthermore, immunoreactivity for PKD2L1, a cation channel subunit, was detected in brush cells. The present results demonstrated that tracheal brush cells are independent cell types. These brush cells may be activated by acid and the secretion of prostaglandins. In conclusion, the present study revealed that tracheal brush cells are independent cell types based on the morphological and immunohistochemical characteristics.

Vesicular nucleotide transporter-immunoreactive type I cells associated with P2X3-immunoreactive nerve endings in the rat carotid body.

ATP is the major excitatory transmitter from chemoreceptor type I cells to sensory nerve endings in the carotid body, and has been suggested to be released by exocytosis from these cells. We investigated the mRNA expression and immunohistochemical localization of vesicular nucleotide transporter (VNUT) in the rat carotid body. RT-PCR detected mRNA expression of VNUT in extracts of the tissue. Immunoreactivity for VNUT was localized in a part of type I cells immunoreactive for synaptophysin (SYN), but not in glial-like type II cells immunoreactive for S100 and S100B. Among SYN-immunoreactive type I cells, VNUT immunoreactivity was selectively localized in the sub-population of tyrosine hydroxylase (TH)-immunorective type I cells associated with nerve endings immunoreactive for the P2X3 purinoceptor; however, it was not detected in the sub-population of type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for VNUT, P2X3, and Bassoon revealed that Bassoon-immunoreactive products were localized in type I cells with VNUT immunoreactivity, and accumulated on the contact side of P2X3-immunoreactive nerve endings. These results revealed the selective localization of VNUT in the subpopulation of TH-immunoreactive type I cells attached to sensory nerve endings and suggested that these cells release ATP by exocytosis for chemosensory transmission in the carotid body.

Different immunohistochemical localization for TMEM16A and CFTR in acinar and ductal cells of rat major salivary glands and exocrine pancreas.

We investigated the mRNA expression and immunohistochemical localization of Cl- channels, transmembrane member 16A (TMEM16A or anoctamin 1), and cystic fibrosis transmembrane conductance regulator (CFTR) in rat major salivary glands and exocrine pancreas. RT-PCR detected mRNA expression of TMEM16A and CFTR in the extracts of the parotid gland (PG), submandibular gland (SMG), sublingual gland (SLG), and pancreas. Immunoreactivity for TMEM16A was localized in the apical membrane of serous acinar and intercalated ductal cells in the PG and SMG as well as mucous acinar cells in the SLG; however, it was not detected in striated ductal cells of these tissues. Although striated ductal cells in the PG, SMG and SLG, and granular ductal cells in the SMG, were immunoreactive for CFTR in the luminal side, serous, mucous acinar, and intercalated ductal cells were not immunoreactive for CFTR in any of the major salivary glands. In the exocrine pancreas, immunoreactivity for TMEM16A was localized in the apical membrane of acinar cells, while immunoreactivity for CFTR was localized in the luminal side of intercalated ductal cells. These results suggest that different localization of TMEM16A and CFTR immunoreactivities reflects the respective functions of acinar and ductal cells in major salivary glands and exocrine pancreas.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart.

Inhibitory role of Munc13-1 in antigen-induced mast cell degranulation.

Secretory granules (SGs) of mast cells are lysosome-related organelles that contain various inflammatory molecules such as histamine, which are stored in the cytoplasm. Mast cell degranulation is the regulated exocytosis of SGs in response to external stimuli, such as the antigen-mediated cross-linking of the high-affinity IgE receptor, FcεRI. Upon stimulation, SGs undergo priming to become fusion-competent prior to fusing with the plasma membrane, which is mediated by Munc13-4, one of the five members of the vesicle-priming Munc13 protein family. Although Munc13-4 is shown to be crucial for mast cell degranulation, the functional involvement of other Munc13 isoform(s) remains unknown. Herein, this was investigated using the RBL-2H3 mast cell line. We found that Munc13-1 and Munc13-4 are the only Munc13 isoforms that are expressed in the RBL-2H3 cells, and Munc13-1 is distributed in the cytoplasm, but highly concentrated on the late endosome and/or lysosome. Unexpectedly, antigen-induced degranulation was considerably increased by Munc13-1 knockdown, but decreased by its overexpression. Further, we found that the hypersecretion phenotype of the Munc13-1-knockdown cells was attenuated by simultaneous Munc13-4 knockdown. These results suggested that Munc13-1 has an inhibitory role in antigen-induced mast cell degranulation, which is performed in a Munc13-4-dependent manner.

Role of pituitary adenylyl cyclase-activating polypeptide in intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Serotonin-mediated modulation of acetylcholine-induced intracellular calcium responses in chromaffin cells isolated from the rat adrenal medulla.

We examined serotonin (5-HT)-mediated modulation of acetylcholine (ACh)-induced intracellular Ca2+ ([Ca2+]i) responses in rat adrenal chromaffin cells using calcium imaging. 5-HT did not induce any [Ca2+]i response in clustered chromaffin cells. However, the magnitude of ACh-induced [Ca2+]i increases in the same specimens was inhibited in the presence of 5-HT. ACh-induced [Ca2+]i increases in chromaffin cells were also inhibited by the 5-HT1A receptor agonist, 8-hydroxy-2-(dipropylamino) tetralin hydrobromide, but were not changed by the 5-HT1B, 5-HT2, or 5-HT3 receptor agonists, CP93129, α-methyl-5-HT, or 1-(m-chlorophenyl) biguanide, respectively. RT-PCR analysis detected the expression of all 5-HT receptor subtype mRNAs, except for 5-HT5 receptors, in extracts of the adrenal medulla. Immunohistochemistry revealed that immunoreactivity for 5-HT1A receptor was located in the chromaffin cells immunoreactive for the biosynthetic enzyme for noradrenaline, dopamine ß-hydroxylase. These results suggest that 5-HT inhibits ACh-induced excitability in adrenal chromaffin cells via the 5-HT1A receptor in order to reduce catecholamine release during preganglionic sympathetic stimuli.

Topographic distribution of serotonin-immunoreactive urethral endocrine cells and their relationship with calcitonin gene-related peptide-immunoreactive nerves in male rats.

We investigated the topographic distribution and morphology of serotonin (5-HT)-immunoreactive endocrine cells in the urethra of male rats, and focused on their relationship with peptidergic nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP). Urethral endocrine cells immunoreactive for 5-HT were densely distributed in the epithelial layers of the prostatic part, but were sparsely distributed in the membranous and spongy parts of urethra. Distribution of urethral endocrine cells with 5-HT immunoreactivity in the prostatic part was restricted from the internal urethral orifice to the region of seminal colliculus. 5-HT-immunoreactive endocrine cells were also observed in the ductal epithelial layers of coagulating glands, prostatic glands, and seminal vesicles. 5-HT-immunoreactive endocrine cells were triangular or flask in shape and possessed an apical projection extending toward the urethral lumen, and basal or lateral protrusions intruding between other epithelial cells were also detected in some cells. Double immunolabeling for 5-HT and CGRP revealed that CGRP-immunoreactive nerve fibers attached to urethral endocrine cells with 5-HT immunoreactivity in the prostatic part. These results suggest that urethral endocrine cells may release 5-HT in response to luminal stimuli, and that these cells and CGRP-immunoreactive nerves may regulate each other by an axon reflex mechanism.