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 osphradium. A–J: IF and CM analysis with R20 antibody; K–P: NADPHd assay. (A) Frontal section of the osphradium showing intense NOS staining in the lamellae (l) and a weaker immunolabeling in the central axis (ca) of the organ; (B) Higher magnification showing the weak and diffuse NOS staining in the central axis corresponding to the osphradial ganglion, and the intense staining in the lamellar axis (la). A weaker labeling is also seen in lamellar epithelium (ep); (C) IF image showing NOS-IR ganglion cells (gc) and nerve fibers (arrow) at the base of the lamella. The different intensity of NOS labeling in the central axis and ganglionic cells can be appreciated; (D) Detail of ganglion cells showing an intense dotted staining along the edge of the cells (arrow) and a less intense labeling in the cytoplasm. Nerve NOS-IR fibers contacting ganglionic cells are seen (arrowhead); (E) Frontal section of the lamella. Strongly labeled cells are visible in the central axis. An intense IF signal is also present in the apical portion of the epithelial cells (arrowheads). Among epithelial cells, NOS-IR fibers coursing from the lamellar axis toward the epithelium surface are seen (arrows); (F) Superimposition of NOS staining to DIC image showing the path of NOS-IR fibers (arrows) among epithelial cells. Near the surface of the epithelium a dotted NOS staining is present (arrowhead); (G) NOS-labeled cells beneath the epithelium can be observed (arrows); (H,I) Two focal planes at different Z positions of the lamellar axis reveal the presence of NOS-positive spots (red signal) around NOS-unlabeled cells (compare the cell pointed by the arrow in H and I). Nucleic acids are labeled by TOTO3 (blue signal). Due to the large number of immunoreactive spots, these cells appear as NOS-expressing cells in epifluorescence; (J) IF microphotograph showing NOS-IR processes contacting the lamellar surface (arrow points to the dotted staining); (K) NADPHd showing the intense labeling in the central axis of the osphradium (arrowhead). Clusters of NADPHd-positive ganglionic cells are present at the periphery of the osphradial neuropile; (L) Microphotography of ganglionic cells showing the intense NADPHd staining at the periphery of the cells and few positive spots (arrowheads) in the cell bodies. NADPHd-positive nerve fibers (arrows) can be observed. NADPHd staining is more intense in the regions where nerve fibers contact the cell body (asterisks); (M) NADPHd-positive cells at high magnification showing their bipolar morphology; (N) Frontal section of the lamella showing NADPHd-positive neurons (arrows) and nerve fibers (arrowhead) in the central axis. Scarce positive spots are seen in the epithelium (double arrowheads); (O) Detail of a NADPHd-positive cell in the lamellar axis showing intensely labeled spots on its surface (arrows); (P) Sagittal section of the lamellar tip showing NADPHd-positive spots on the epithelium surface. Bars: A = 500 μm; B–G, J, K, P = 25 μm; H–I, L, O = 10 μm; M = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f003: NOS distribution in the osphradium. A–J: IF and CM analysis with R20 antibody; K–P: NADPHd assay. (A) Frontal section of the osphradium showing intense NOS staining in the lamellae (l) and a weaker immunolabeling in the central axis (ca) of the organ; (B) Higher magnification showing the weak and diffuse NOS staining in the central axis corresponding to the osphradial ganglion, and the intense staining in the lamellar axis (la). A weaker labeling is also seen in lamellar epithelium (ep); (C) IF image showing NOS-IR ganglion cells (gc) and nerve fibers (arrow) at the base of the lamella. The different intensity of NOS labeling in the central axis and ganglionic cells can be appreciated; (D) Detail of ganglion cells showing an intense dotted staining along the edge of the cells (arrow) and a less intense labeling in the cytoplasm. Nerve NOS-IR fibers contacting ganglionic cells are seen (arrowhead); (E) Frontal section of the lamella. Strongly labeled cells are visible in the central axis. An intense IF signal is also present in the apical portion of the epithelial cells (arrowheads). Among epithelial cells, NOS-IR fibers coursing from the lamellar axis toward the epithelium surface are seen (arrows); (F) Superimposition of NOS staining to DIC image showing the path of NOS-IR fibers (arrows) among epithelial cells. Near the surface of the epithelium a dotted NOS staining is present (arrowhead); (G) NOS-labeled cells beneath the epithelium can be observed (arrows); (H,I) Two focal planes at different Z positions of the lamellar axis reveal the presence of NOS-positive spots (red signal) around NOS-unlabeled cells (compare the cell pointed by the arrow in H and I). Nucleic acids are labeled by TOTO3 (blue signal). Due to the large number of immunoreactive spots, these cells appear as NOS-expressing cells in epifluorescence; (J) IF microphotograph showing NOS-IR processes contacting the lamellar surface (arrow points to the dotted staining); (K) NADPHd showing the intense labeling in the central axis of the osphradium (arrowhead). Clusters of NADPHd-positive ganglionic cells are present at the periphery of the osphradial neuropile; (L) Microphotography of ganglionic cells showing the intense NADPHd staining at the periphery of the cells and few positive spots (arrowheads) in the cell bodies. NADPHd-positive nerve fibers (arrows) can be observed. NADPHd staining is more intense in the regions where nerve fibers contact the cell body (asterisks); (M) NADPHd-positive cells at high magnification showing their bipolar morphology; (N) Frontal section of the lamella showing NADPHd-positive neurons (arrows) and nerve fibers (arrowhead) in the central axis. Scarce positive spots are seen in the epithelium (double arrowheads); (O) Detail of a NADPHd-positive cell in the lamellar axis showing intensely labeled spots on its surface (arrows); (P) Sagittal section of the lamellar tip showing NADPHd-positive spots on the epithelium surface. Bars: A = 500 μm; B–G, J, K, P = 25 μm; H–I, L, O = 10 μm; M = 5 μm.

Mentions: Intense NOS staining was found in the osphradial lamellae compared to the central axis of the sensory organ containing the voluminous ganglion (Figure 3A). Higher magnifications showed that the ganglion neuropile was little reactive for NOS (Figure 3B). In contrast, peripheral ganglion cell bodies were strongly labeled (Figure 3C). Immunoreactive products were distributed on these cells in small spots located at the cell periphery (punctate staining) whereas neuronal cytoplasm was weakly reactive (Figure 3D). This distribution suggested that ShNOS is localized to axon terminals contacting ganglion neurons.


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 osphradium. A–J: IF and CM analysis with R20 antibody; K–P: NADPHd assay. (A) Frontal section of the osphradium showing intense NOS staining in the lamellae (l) and a weaker immunolabeling in the central axis (ca) of the organ; (B) Higher magnification showing the weak and diffuse NOS staining in the central axis corresponding to the osphradial ganglion, and the intense staining in the lamellar axis (la). A weaker labeling is also seen in lamellar epithelium (ep); (C) IF image showing NOS-IR ganglion cells (gc) and nerve fibers (arrow) at the base of the lamella. The different intensity of NOS labeling in the central axis and ganglionic cells can be appreciated; (D) Detail of ganglion cells showing an intense dotted staining along the edge of the cells (arrow) and a less intense labeling in the cytoplasm. Nerve NOS-IR fibers contacting ganglionic cells are seen (arrowhead); (E) Frontal section of the lamella. Strongly labeled cells are visible in the central axis. An intense IF signal is also present in the apical portion of the epithelial cells (arrowheads). Among epithelial cells, NOS-IR fibers coursing from the lamellar axis toward the epithelium surface are seen (arrows); (F) Superimposition of NOS staining to DIC image showing the path of NOS-IR fibers (arrows) among epithelial cells. Near the surface of the epithelium a dotted NOS staining is present (arrowhead); (G) NOS-labeled cells beneath the epithelium can be observed (arrows); (H,I) Two focal planes at different Z positions of the lamellar axis reveal the presence of NOS-positive spots (red signal) around NOS-unlabeled cells (compare the cell pointed by the arrow in H and I). Nucleic acids are labeled by TOTO3 (blue signal). Due to the large number of immunoreactive spots, these cells appear as NOS-expressing cells in epifluorescence; (J) IF microphotograph showing NOS-IR processes contacting the lamellar surface (arrow points to the dotted staining); (K) NADPHd showing the intense labeling in the central axis of the osphradium (arrowhead). Clusters of NADPHd-positive ganglionic cells are present at the periphery of the osphradial neuropile; (L) Microphotography of ganglionic cells showing the intense NADPHd staining at the periphery of the cells and few positive spots (arrowheads) in the cell bodies. NADPHd-positive nerve fibers (arrows) can be observed. NADPHd staining is more intense in the regions where nerve fibers contact the cell body (asterisks); (M) NADPHd-positive cells at high magnification showing their bipolar morphology; (N) Frontal section of the lamella showing NADPHd-positive neurons (arrows) and nerve fibers (arrowhead) in the central axis. Scarce positive spots are seen in the epithelium (double arrowheads); (O) Detail of a NADPHd-positive cell in the lamellar axis showing intensely labeled spots on its surface (arrows); (P) Sagittal section of the lamellar tip showing NADPHd-positive spots on the epithelium surface. Bars: A = 500 μm; B–G, J, K, P = 25 μm; H–I, L, O = 10 μm; M = 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f003: NOS distribution in the osphradium. A–J: IF and CM analysis with R20 antibody; K–P: NADPHd assay. (A) Frontal section of the osphradium showing intense NOS staining in the lamellae (l) and a weaker immunolabeling in the central axis (ca) of the organ; (B) Higher magnification showing the weak and diffuse NOS staining in the central axis corresponding to the osphradial ganglion, and the intense staining in the lamellar axis (la). A weaker labeling is also seen in lamellar epithelium (ep); (C) IF image showing NOS-IR ganglion cells (gc) and nerve fibers (arrow) at the base of the lamella. The different intensity of NOS labeling in the central axis and ganglionic cells can be appreciated; (D) Detail of ganglion cells showing an intense dotted staining along the edge of the cells (arrow) and a less intense labeling in the cytoplasm. Nerve NOS-IR fibers contacting ganglionic cells are seen (arrowhead); (E) Frontal section of the lamella. Strongly labeled cells are visible in the central axis. An intense IF signal is also present in the apical portion of the epithelial cells (arrowheads). Among epithelial cells, NOS-IR fibers coursing from the lamellar axis toward the epithelium surface are seen (arrows); (F) Superimposition of NOS staining to DIC image showing the path of NOS-IR fibers (arrows) among epithelial cells. Near the surface of the epithelium a dotted NOS staining is present (arrowhead); (G) NOS-labeled cells beneath the epithelium can be observed (arrows); (H,I) Two focal planes at different Z positions of the lamellar axis reveal the presence of NOS-positive spots (red signal) around NOS-unlabeled cells (compare the cell pointed by the arrow in H and I). Nucleic acids are labeled by TOTO3 (blue signal). Due to the large number of immunoreactive spots, these cells appear as NOS-expressing cells in epifluorescence; (J) IF microphotograph showing NOS-IR processes contacting the lamellar surface (arrow points to the dotted staining); (K) NADPHd showing the intense labeling in the central axis of the osphradium (arrowhead). Clusters of NADPHd-positive ganglionic cells are present at the periphery of the osphradial neuropile; (L) Microphotography of ganglionic cells showing the intense NADPHd staining at the periphery of the cells and few positive spots (arrowheads) in the cell bodies. NADPHd-positive nerve fibers (arrows) can be observed. NADPHd staining is more intense in the regions where nerve fibers contact the cell body (asterisks); (M) NADPHd-positive cells at high magnification showing their bipolar morphology; (N) Frontal section of the lamella showing NADPHd-positive neurons (arrows) and nerve fibers (arrowhead) in the central axis. Scarce positive spots are seen in the epithelium (double arrowheads); (O) Detail of a NADPHd-positive cell in the lamellar axis showing intensely labeled spots on its surface (arrows); (P) Sagittal section of the lamellar tip showing NADPHd-positive spots on the epithelium surface. Bars: A = 500 μm; B–G, J, K, P = 25 μm; H–I, L, O = 10 μm; M = 5 μm.
Mentions: Intense NOS staining was found in the osphradial lamellae compared to the central axis of the sensory organ containing the voluminous ganglion (Figure 3A). Higher magnifications showed that the ganglion neuropile was little reactive for NOS (Figure 3B). In contrast, peripheral ganglion cell bodies were strongly labeled (Figure 3C). Immunoreactive products were distributed on these cells in small spots located at the cell periphery (punctate staining) whereas neuronal cytoplasm was weakly reactive (Figure 3D). This distribution suggested that ShNOS is localized to axon terminals contacting ganglion neurons.

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