Localization of endothelin A receptors in the rat pituitary TSH cells: light- and electron-microscopic immunohistochemical studies

Abstract

Endothelins modulate hormonal secretion in the pituitary gland. Intense signaling of endothelin A receptors (ET(A)R) has been detected by in situ hybridization, binding assay and receptor autoradiography. We used light- and electron-microscopic immunohistochemistry of ET(A)R with polyclonal antibody against a synthetic peptide corresponding to the carboxyl terminus (403-427) of human ET(A)R. Immunoreactivity was observed in 6-8% of anterior pituitary cells, which were rather large polygonal or stellate cells. These cells were often clustered. Double-staining immunofluorescence showed that the ET(A)R-positive cells immunoreacted with antibody against the beta-subunit of thyroid-stimulating hormone (TSH), but not adrenocorticotropic hormone (ACTH) or lutenizing hormone beta (LHbeta). Pre- and postembedding electron-microscopic immunohistochemistry showed that ET(A)R-positive cells had vacuolated or parallel-lined rough endoplasmic reticulum (rER) and numerous round granules in their periphery and the elongated processes. By pre-embedding immunohistochemistry, diaminobenzidine tetrahydrochloride (DAB) products were shown to be mostly located around the granules and occasionally underneath the plasma membrane. By postembedding immunohistochemistry, granules in the ET(A)R-positive cells were 90-150 nm in diameter, and colloidal gold particles due to ET(A)R were associated with about 10% of these granules. These results indicate that ET(A) receptors are associated mostly with the secretory granules of TSH cells.

Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum

“…Preparation of antipeptide antibodies specific for ET A and ET B receptor As described previously (Sasaki et al 1998;Tanaka et al 1998;Furuya et al 2000), polyclonal antipeptide antibodies specific for ET A R and ET B R were prepared. Briefly, a peptide (TSIQWKNHDQNNHNTDRSSHKDSMN) corresponding to the carboxyl terminus (403-427) of human ET A R (Hosoda et al 1991) or a peptide (CLKFKANDHGYDNFRSSNKYSSS) corresponding to the carboxyl terminus (420-442) of human ET B R (Sakamoto et al 1991) was coupled to keyhole limpet hemocyanin (KLH).…”

“…After fixation, cerebella were washed with phosphate-buffered saline (PBS) four times, immersed in 10%, 20%, and then 30% sucrose in PBS for 4 h to overnight, and then frozen as described previously (Furuya et al 2000). Midsagittal sections of 10 µm thickness were cut by a cryostat (Yung Frigocut 2800E) and mounted on albumin-coated glass slides.…”

“…Recently, we prepared antipeptide antibodies specific for ET A and ET B receptors (Sasaki et al 1998;Tanaka et al 1998). These antibodies were designed to recognize the intracellular carboxyl terminus of ET receptors and are very useful for lightmicroscopic (LM) and electron-microscopic (EM) immunohistochemistry (Furuya et al 2000).…”

Distribution of aquaporin 1 in the rat pancreatic duct system examined with light- and electron-microscopic immunohistochemistry

“…Therefore, it is necessary to view a large area to search various parts of the duct system. In the brain, we usually make 60-to 70-µm sections by vibratome for EM immunohistochemistry (Furuya et al 2000(Furuya et al , 2001. However, pancreas is too soft to make sections by vibratome.…”

“…After fixation, the pancreas was cut into small pieces and washed with phosphate-buffered saline (PBS) 4 times, immersed in 10%, 20% and then 30% sucrose in PBS at 4°C for 4 h to overnight, and then frozen as described previously (Furuya et al 2000(Furuya et al , 2001. For EM immunohistochemistry, the tissues were immersed in 10% and 20% sucrose in PBS for 4 h to overnight and then in 20% sucrose-5% glycerin for 1 h at 4°C before being frozen with liquid N 2 .…”