Limits...
Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress.

Toni M, De Angelis F, di Patti MC, Cioni C - Mar Drugs (2015)

Bottom Line: The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located.Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception.The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Charles Darwin", Sapienza University, 00161 Rome, Italy. mattia.toni@uniroma1.it.

ABSTRACT
Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca(2+)/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

Show MeSH

Related in: MedlinePlus

NOS distribution in the tentacle. (A) Sagittal section showing that NOS staining is more intense in the region beneath the epithelium (ep, arrows), in proximity of the eye (arrowhead) and in nerve fibers (nf); (B,C) CM double-staining with NOS antibodies (green) and phalloidin (red). Weakly NOS-labeled cells are seen in the epithelium whereas a dense network of NOS-IR nerve fibers are interspersed with phalloidin-positive actin filaments in the subepithelial layers. In the epithelium both gland (arrow in C) and epithelial cells are weakly labeled by NOS antibody; (D) CM double-staining with NOS antibodies (green) and the nucleic acid stain TOTO 3 (blue) to identify cell bodies. Dotted NOS staining are seen at the base of epithelial and gland cells (arrowhead) and along nerve fibers (arrows); (E) Superimposition of NOS/TOTO3 staining to DIC of the boxed area in D, showing NOS-IR fibers contacting the base of epithelial cells (arrowhead); (F) Superimposition of NOS staining (red) to DIC image clearly shows the presence of NOS-IR spots around unlabeled cells (arrow); (G) Detail of a nerve fascicle showing NOS-positive spots; (H) DIC image of panel G; (I) NOS/DIC superimposition showing the absence of NOS staining in muscle cells (m). Among muscle cells, NOS-positive spots are present; (J) Magnification of the boxed area in I. NOS-positive neuromuscular junctions can be observed (arrow); (K) NOS-IR cells (arrows) and nerve fibers (arrowheads) in the eye. (L) DIC image of the boxed area in J in which BRN, PR, and RL layers are recognizable; (M–O) Three focal planes taken at different z positions of the boxed area in K showing NOS-IR neurons (arrows) and nerve fibers (arrowhead) in the BRN. NOS staining is also detected in the RL and PR. A–O: R20 antibody. Bars: A = 200 μm; B, C = 50 μm; D, G, H = 25 μm; E, F, I, K = 10 μm; J = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663546&req=5

marinedrugs-13-06636-f005: NOS distribution in the tentacle. (A) Sagittal section showing that NOS staining is more intense in the region beneath the epithelium (ep, arrows), in proximity of the eye (arrowhead) and in nerve fibers (nf); (B,C) CM double-staining with NOS antibodies (green) and phalloidin (red). Weakly NOS-labeled cells are seen in the epithelium whereas a dense network of NOS-IR nerve fibers are interspersed with phalloidin-positive actin filaments in the subepithelial layers. In the epithelium both gland (arrow in C) and epithelial cells are weakly labeled by NOS antibody; (D) CM double-staining with NOS antibodies (green) and the nucleic acid stain TOTO 3 (blue) to identify cell bodies. Dotted NOS staining are seen at the base of epithelial and gland cells (arrowhead) and along nerve fibers (arrows); (E) Superimposition of NOS/TOTO3 staining to DIC of the boxed area in D, showing NOS-IR fibers contacting the base of epithelial cells (arrowhead); (F) Superimposition of NOS staining (red) to DIC image clearly shows the presence of NOS-IR spots around unlabeled cells (arrow); (G) Detail of a nerve fascicle showing NOS-positive spots; (H) DIC image of panel G; (I) NOS/DIC superimposition showing the absence of NOS staining in muscle cells (m). Among muscle cells, NOS-positive spots are present; (J) Magnification of the boxed area in I. NOS-positive neuromuscular junctions can be observed (arrow); (K) NOS-IR cells (arrows) and nerve fibers (arrowheads) in the eye. (L) DIC image of the boxed area in J in which BRN, PR, and RL layers are recognizable; (M–O) Three focal planes taken at different z positions of the boxed area in K showing NOS-IR neurons (arrows) and nerve fibers (arrowhead) in the BRN. NOS staining is also detected in the RL and PR. A–O: R20 antibody. Bars: A = 200 μm; B, C = 50 μm; D, G, H = 25 μm; E, F, I, K = 10 μm; J = 5 μm.

Mentions: A diffuse NOS staining was observed in the tentacle with a more intense labeling in the subepithelial region (Figure 5A). Triple CM staining by using NOS antibodies, phalloidin (for actin filaments) and TOTO3 (nucleic acid stain) showed that the subepithelial labeling was due to a dense network of varicose NOS-IR nerve fibers and phalloidin-positive fascicles of actin filaments (Figure 5B,C). Conversely, epithelial cells were weakly reactive for NOS (Figure 5B,C). NOS-IR varicosities were observed in close contact to the basal surface of epithelial cells (Figure 5D,E). The overlapping of NOS staining with DIC bright field clearly showed the presence of abundant NOS-positive spots around unstained cells (Figure 5F). The granular distribution of NOS immunostaining was also evident in large nerve fascicles (Figure 5G) and the varicose aspect of the nerve fibers was confirmed in the DIC pictures (Figure 5H).


Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress.

Toni M, De Angelis F, di Patti MC, Cioni C - Mar Drugs (2015)

NOS distribution in the tentacle. (A) Sagittal section showing that NOS staining is more intense in the region beneath the epithelium (ep, arrows), in proximity of the eye (arrowhead) and in nerve fibers (nf); (B,C) CM double-staining with NOS antibodies (green) and phalloidin (red). Weakly NOS-labeled cells are seen in the epithelium whereas a dense network of NOS-IR nerve fibers are interspersed with phalloidin-positive actin filaments in the subepithelial layers. In the epithelium both gland (arrow in C) and epithelial cells are weakly labeled by NOS antibody; (D) CM double-staining with NOS antibodies (green) and the nucleic acid stain TOTO 3 (blue) to identify cell bodies. Dotted NOS staining are seen at the base of epithelial and gland cells (arrowhead) and along nerve fibers (arrows); (E) Superimposition of NOS/TOTO3 staining to DIC of the boxed area in D, showing NOS-IR fibers contacting the base of epithelial cells (arrowhead); (F) Superimposition of NOS staining (red) to DIC image clearly shows the presence of NOS-IR spots around unlabeled cells (arrow); (G) Detail of a nerve fascicle showing NOS-positive spots; (H) DIC image of panel G; (I) NOS/DIC superimposition showing the absence of NOS staining in muscle cells (m). Among muscle cells, NOS-positive spots are present; (J) Magnification of the boxed area in I. NOS-positive neuromuscular junctions can be observed (arrow); (K) NOS-IR cells (arrows) and nerve fibers (arrowheads) in the eye. (L) DIC image of the boxed area in J in which BRN, PR, and RL layers are recognizable; (M–O) Three focal planes taken at different z positions of the boxed area in K showing NOS-IR neurons (arrows) and nerve fibers (arrowhead) in the BRN. NOS staining is also detected in the RL and PR. A–O: R20 antibody. Bars: A = 200 μm; B, C = 50 μm; D, G, H = 25 μm; E, F, I, K = 10 μm; J = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663546&req=5

marinedrugs-13-06636-f005: NOS distribution in the tentacle. (A) Sagittal section showing that NOS staining is more intense in the region beneath the epithelium (ep, arrows), in proximity of the eye (arrowhead) and in nerve fibers (nf); (B,C) CM double-staining with NOS antibodies (green) and phalloidin (red). Weakly NOS-labeled cells are seen in the epithelium whereas a dense network of NOS-IR nerve fibers are interspersed with phalloidin-positive actin filaments in the subepithelial layers. In the epithelium both gland (arrow in C) and epithelial cells are weakly labeled by NOS antibody; (D) CM double-staining with NOS antibodies (green) and the nucleic acid stain TOTO 3 (blue) to identify cell bodies. Dotted NOS staining are seen at the base of epithelial and gland cells (arrowhead) and along nerve fibers (arrows); (E) Superimposition of NOS/TOTO3 staining to DIC of the boxed area in D, showing NOS-IR fibers contacting the base of epithelial cells (arrowhead); (F) Superimposition of NOS staining (red) to DIC image clearly shows the presence of NOS-IR spots around unlabeled cells (arrow); (G) Detail of a nerve fascicle showing NOS-positive spots; (H) DIC image of panel G; (I) NOS/DIC superimposition showing the absence of NOS staining in muscle cells (m). Among muscle cells, NOS-positive spots are present; (J) Magnification of the boxed area in I. NOS-positive neuromuscular junctions can be observed (arrow); (K) NOS-IR cells (arrows) and nerve fibers (arrowheads) in the eye. (L) DIC image of the boxed area in J in which BRN, PR, and RL layers are recognizable; (M–O) Three focal planes taken at different z positions of the boxed area in K showing NOS-IR neurons (arrows) and nerve fibers (arrowhead) in the BRN. NOS staining is also detected in the RL and PR. A–O: R20 antibody. Bars: A = 200 μm; B, C = 50 μm; D, G, H = 25 μm; E, F, I, K = 10 μm; J = 5 μm.
Mentions: A diffuse NOS staining was observed in the tentacle with a more intense labeling in the subepithelial region (Figure 5A). Triple CM staining by using NOS antibodies, phalloidin (for actin filaments) and TOTO3 (nucleic acid stain) showed that the subepithelial labeling was due to a dense network of varicose NOS-IR nerve fibers and phalloidin-positive fascicles of actin filaments (Figure 5B,C). Conversely, epithelial cells were weakly reactive for NOS (Figure 5B,C). NOS-IR varicosities were observed in close contact to the basal surface of epithelial cells (Figure 5D,E). The overlapping of NOS staining with DIC bright field clearly showed the presence of abundant NOS-positive spots around unstained cells (Figure 5F). The granular distribution of NOS immunostaining was also evident in large nerve fascicles (Figure 5G) and the varicose aspect of the nerve fibers was confirmed in the DIC pictures (Figure 5H).

Bottom Line: The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located.Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception.The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Charles Darwin", Sapienza University, 00161 Rome, Italy. mattia.toni@uniroma1.it.

ABSTRACT
Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca(2+)/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

Show MeSH
Related in: MedlinePlus