Functional and Morphological Analyses of Rab Proteins and the Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) System in the Secretion of Pituitary Hormones

Matsuno, Akira; Itoh, Johbu; Takekoshi, Susumu; Nagashima, Tadashi; Osamura, R. Yoshiyuki

doi:10.1267/ahc.36.501

Cited by 6 publications

(11 citation statements)

References 39 publications

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“…To elucidate the role of rab3B with regard to SNARE mechanisms, we undertook experiments to elucidate the role of rab3B in GH secretion and the mutual relationships with SNARE proteins such as SNAP-25 and syntaxin [8,9]. CLSM of immunohistochemical double stainings for SNAP-25, syntaxin and rab3B demonstrated co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats.…”

Section: Resultsmentioning

confidence: 99%

“…2621, fax. : (+81) 436621357, e-mail: akirakun@med.teikyo-u.ac.jp ship in GH secretion [8,9]. In order to examine more specific relationship among molecules that play important roles in transportation and secretion of pituitary hormone, we developed an experimental pituitary cell line that has secretory granules of GH linked to enhanced yellow fluorescein protein (EYFP), which enabled us to visualize the real-time movement of the molecules in a living cell [10].…”

Section: Abstract: Gh Rab3b Porosome Eyfp Ecfpmentioning

confidence: 99%

“…T-SNAREs are located at the cell plasma membrane (present at the base of porosomes), and v-SNAREs are present at the secretory vesicle membrane. Previously, we investigated the modulations of the intracellular dynamics of growth hormone (GH), rab3B, SNARE proteins such as SNAP-25 and syntaxin, in rat pituitary cells, caused by growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) [8,9]. These proteins display a close relation-ship in GH secretion [8,9].…”

Section: Introductionmentioning

confidence: 99%

“…Previously, we investigated the modulations of the intracellular dynamics of growth hormone (GH), rab3B, SNARE proteins such as SNAP-25 and syntaxin, in rat pituitary cells, caused by growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) [8,9]. These proteins display a close relation-ship in GH secretion [8,9]. In order to examine more specific relationship among molecules that play important roles in transportation and secretion of pituitary hormone, we developed an experimental pituitary cell line that has secretory granules of GH linked to enhanced yellow fluorescein protein (EYFP), which enabled us to visualize the real-time movement of the molecules in a living cell [10].…”

Section: Introductionmentioning

confidence: 99%

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Co-transfection of EYFP-GH and ECFP-rab3B in an experimental pituitary GH3 cell: a role of rab3B in secretion of GH through porosome.

Matsuno

Itoh²,

Mizutani³

et al. 2009

Folia Histochem Cytobiol.

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Abstract:Recently, in order to elucidate the role of rab3B in porosome, we have observed the incorporation of rab3B in the secretion of GH through porosome under confocal laser scanning microscopy (CLSM). Transfected cells with GH-EYFP fusion protein and rab3B-ECFP fusion protein were observed under CLSM, which showed the colocalization of EYFP-GH and ECFP-rab3B in the budding configuration of secretory process. These structural and functional images of rab3B imply the incorporation of rab3B in the secretion of GH through porosome. Key words: GH, rab3B, porosome, EYFP, ECFPCorrespondence: A. Matsuno, Dept. of Neurosurgery, Teikyo University Chiba Medical Center, 3426-3 Anesaki, Ichihara City, Chiba 299-0111, Japan; tel.: (+81) 436621211 ext. 2621, fax.: (+81) 436621357, e-mail: akirakun@med.teikyo-u.ac.jp ship in GH secretion [8,9]. In order to examine more specific relationship among molecules that play important roles in transportation and secretion of pituitary hormone, we developed an experimental pituitary cell line that has secretory granules of GH linked to enhanced yellow fluorescein protein (EYFP), which enabled us to visualize the real-time movement of the molecules in a living cell [10]. Recently, in order to elucidate the role of rab3B in porosome, we have observed the incorporation of rab3B in the secretion of GH through porosome under confocal laser scanning microscopy (CLSM). In this paper, we describe the synergistic dynamics of rab3B and GH in porosome with illustrative fluorescein imaging of rab3B and GH in an experimental cell line. Materials and methodsConstruction of rat EYFP-GH-1 plasmid for transfection of GH3 cell. The rat GH cDNA clone pRGH-1 and the EYFP-expression construct pEYFP-N1 were obtained from the American Type Culture Collection (Manassas, VA, USA) and Clontech Laboratories, Inc., (Palo Alto, CA, USA), respectively. The GH-EYFP fusion construct pCMV-Sig-EYFP-GH-1 was derived from pEYFP-N1 and contained a sequence encoding the rat GH signal peptide (1 to 26 in the rat GH amino-acid sequence) and the EYFPcoding segment, followed by another rat GH coding sequence (27 to 217 in the rat GH amino-acid sequence).Construction of rat rab3B-enhanced cian fluorescein protein (ECFP) plasmid for transfection of GH3 cell. prab3B-ECFP was constructed as follows. A cDNA fragment encoding rat rab3B was amplified by RT-PCR and cloned in-frame into the EcoRI/BamHI sites of pECFP-N1 (Clontech Laboratories, Inc.), which expresses rab3B-ECFP fusion proteins in the mammalian cell. Plasmids were purified using Qiagen midi-prep kit (Qiagen GmbH, Hilden, Germany), and were verified by nucleotide sequencing.GH3 cell culture. GH3 cells were maintained at 37°C in a 5% CO 2 in DMEM/Ham's F-12 medium supplemented with 2.5% heatinactivated fetal bovine serum (FBS), 10% horse serum (HS), 100U/ml of penicillin and 100 μg/ml of streptomycin at 70% confluency on poly-L-lysin coated 35-mm glass base dishes.Transfection. Transfection of GH3 cells was performed using lipofectamine 2000 (Invitrogen Corp., Carlsba...

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Section: Resultsmentioning

confidence: 99%

Section: Abstract: Gh Rab3b Porosome Eyfp Ecfpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Co-transfection of EYFP-GH and ECFP-rab3B in an experimental pituitary GH3 cell: a role of rab3B in secretion of GH through porosome.

Matsuno

Itoh²,

Mizutani³

et al. 2009

Folia Histochem Cytobiol.

Self Cite

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“…Another important issue is the intracellular transport and secretion of pituitary hormone. We have investigated the modulations of the intracellular dynamics of growth hormone (GH), rab3B, soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins such as synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin in rat pituitary cells, caused by growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) [ 34 , 35 ]. In order to examine the relationships among molecules that play important roles in the transport and secretion of pituitary hormone, the real-time observation in a living cell is essential.…”

Section: Introductionmentioning

confidence: 99%

Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging

et al. 2011

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In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca2+ influx or Ca2+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.

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Functional molecular morphology of anterior pituitary cells, from hormone production to intracellular transport and secretion

et al. 2011

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Combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH & IHC) has sufficient ultrastructural resolution to provide two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots; Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH & IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin revealed that both rab3B and SNARE system proteins play an important role and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. An experimental pituitary cell line, the GH3 cell, in which growth hormone (GH) is linked to enhanced yellow fluorescein protein (EYFP), has been developed. This stable GH3 cell secretes GH linked to EYFP upon being stimulated by Ca(2+) influx or Ca(2+) release from storage. This GH3 cell is useful for real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the processes of transcription, translation, transport, and secretion of pituitary hormone.

show abstract

Functional and Morphological Analyses of Rab Proteins and the Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) System in the Secretion of Pituitary Hormones

Cited by 6 publications

References 39 publications

Co-transfection of EYFP-GH and ECFP-rab3B in an experimental pituitary GH3 cell: a role of rab3B in secretion of GH through porosome.

Co-transfection of EYFP-GH and ECFP-rab3B in an experimental pituitary GH3 cell: a role of rab3B in secretion of GH through porosome.

Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging

Functional molecular morphology of anterior pituitary cells, from hormone production to intracellular transport and secretion

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