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.

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).

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.

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.

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.

Different effect of serotonin on intracellular calcium ion dynamics in the smooth muscle cells between rat posterior ciliary artery and vorticose vein.

5-hydroxytriptamine (5-HT: serotonin) is an important transmitter that causes vessel constriction, although few studies have examined the effect of 5-HT on venous smooth muscles. The intracellular Ca(2+) concentration ([Ca(2+)]i) plays an essential role in stimulus-response coupling in numerous tissue/cells including vascular smooth muscle cells. The present study was performed to examine whether differences between arteries and veins in the response to 5-HT can be detected under confocal microscope with respect to [Ca(2+)]i dynamics. In posterior ciliary arteries of rats, 5-HT induced a [Ca(2+)]i increase. The 5-HT-induced responses were caused by both Ca(2+) influx and mobilization. Agonist and antagonist experiments revealed that arterial smooth muscles possess 5-HT1a, 1b, 2 (Gprotein-coupled type) and 5-HT3 (ion channel type) receptors, and that 5-HT2 in particular plays a major role in these responses. For vorticose veins, the 5-HT-induced responses were also caused by both Ca(2+) influx and mobilization. However, the cAMP dependent pathway (5-HT4-7) was found to be significant in vasocontraction with respect to 5-HT in these vessels. Thus, Ca(2+) mobilization was induced by 5-HT2 and 5-HT4-7 in a vessel-dependent manner, whereas Ca(2+) influx universally was induced by 5-HT3. These results indicate that the posterior ciliary arteries and vorticose veins in the same tissue might differ greatly in their responses to stimulus.