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

Foot: histology, NOS distribution and NADPHd staining. A–E: Histological analysis. F–N: NOS distribution analyzed by R20 antibody. O: NADPHd staining. (A,B) Sagittal section of the foot stained with hematoxylin/eosin. The ciliated columnar epithelium contains two types of gland cells with a different affinity for hematoxylin (arrows and arrowheads). Asterisks indicate pigment granules. The wavy basal lamina (double arrowhead) separates the epithelium from the underlying tissues; (C) Picture showing gland cells pouring their secretion on the foot surface. Slender cell processes are seen between epithelial elements (arrowhead); (D) The superimposition of TOTO3 staining to DIC allows a better appreciation of the cell morphology. Gland (arrows) and epithelial (asterisk) cells are visible. Beneath the epithelium, bipolar neurons are clearly seen (arrowhead); (E) DIC image at high magnification showing the presence of thin cell processes among epithelial elements (arrow). Cell processes contact the epithelial surface (arrowhead); (F) Sagittal section showing the distribution of NOS-like proteins beneath the epithelium; (G) Enlargement of the small boxed area in F. NOS-IR cells (arrow) and fibers (arrowheads) are seen; (H) Detail of the large dot-boxed area in F. NOS staining is intense at the base of the epithelium where NOS-IR cells and fibers are in close contact with epithelial cells (arrows). The epithelium exhibits a much weaker staining that is more intense in the apical portion; (I) IF image showing NOS-IR neurons (arrows) with processes penetrating the epithelium (arrowheads); (J) Detail showing intensely labeled NOS-IR cells in the subepithelial layers (arrows). Aligned NOS-positive spots toward the surface are visible (arrowheads) among epithelial cells, suggesting the presence of NOS-IR varicose fibers; (K,L) CM images of the same section confirming the wide distribution of NOS-like proteins beneath the epithelium. NOS-IR cells (arrows) and fibers are present. In the epithelium, a diffuse NOS labeling was observed in gland (arrowheads) and epithelial cells. The apical portion of epithelial cells shows an intense NOS-immunoreactivity. In L triple labeling with NOS antibodies (green), phalloidin (red) and TOTO3 (blue) is shown to better evaluate the distribution of NOS-positive staining; (M) Detail of the epithelium where the presence of NOS-IR thin processes (arrowheads) directed to the epithelium surface can be glimpsed among epithelial cells; (N,O) The same sagittal section of the foot stained with NOS-antibodies (N) and NADPHd assays (O). The two stainings overlapped in both cells (arrow) and cell processes (arrowheads). The number of NOS-positive elements was higher in IF than in NADPHd images. The detection of NADPHd reactivity in the epithelium was difficult due to the presence of natural pigments that obscure NADPHd staining. Bars: A, C–E, G–O = 25 μm; B, M = 10 μm; F = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f006: Foot: histology, NOS distribution and NADPHd staining. A–E: Histological analysis. F–N: NOS distribution analyzed by R20 antibody. O: NADPHd staining. (A,B) Sagittal section of the foot stained with hematoxylin/eosin. The ciliated columnar epithelium contains two types of gland cells with a different affinity for hematoxylin (arrows and arrowheads). Asterisks indicate pigment granules. The wavy basal lamina (double arrowhead) separates the epithelium from the underlying tissues; (C) Picture showing gland cells pouring their secretion on the foot surface. Slender cell processes are seen between epithelial elements (arrowhead); (D) The superimposition of TOTO3 staining to DIC allows a better appreciation of the cell morphology. Gland (arrows) and epithelial (asterisk) cells are visible. Beneath the epithelium, bipolar neurons are clearly seen (arrowhead); (E) DIC image at high magnification showing the presence of thin cell processes among epithelial elements (arrow). Cell processes contact the epithelial surface (arrowhead); (F) Sagittal section showing the distribution of NOS-like proteins beneath the epithelium; (G) Enlargement of the small boxed area in F. NOS-IR cells (arrow) and fibers (arrowheads) are seen; (H) Detail of the large dot-boxed area in F. NOS staining is intense at the base of the epithelium where NOS-IR cells and fibers are in close contact with epithelial cells (arrows). The epithelium exhibits a much weaker staining that is more intense in the apical portion; (I) IF image showing NOS-IR neurons (arrows) with processes penetrating the epithelium (arrowheads); (J) Detail showing intensely labeled NOS-IR cells in the subepithelial layers (arrows). Aligned NOS-positive spots toward the surface are visible (arrowheads) among epithelial cells, suggesting the presence of NOS-IR varicose fibers; (K,L) CM images of the same section confirming the wide distribution of NOS-like proteins beneath the epithelium. NOS-IR cells (arrows) and fibers are present. In the epithelium, a diffuse NOS labeling was observed in gland (arrowheads) and epithelial cells. The apical portion of epithelial cells shows an intense NOS-immunoreactivity. In L triple labeling with NOS antibodies (green), phalloidin (red) and TOTO3 (blue) is shown to better evaluate the distribution of NOS-positive staining; (M) Detail of the epithelium where the presence of NOS-IR thin processes (arrowheads) directed to the epithelium surface can be glimpsed among epithelial cells; (N,O) The same sagittal section of the foot stained with NOS-antibodies (N) and NADPHd assays (O). The two stainings overlapped in both cells (arrow) and cell processes (arrowheads). The number of NOS-positive elements was higher in IF than in NADPHd images. The detection of NADPHd reactivity in the epithelium was difficult due to the presence of natural pigments that obscure NADPHd staining. Bars: A, C–E, G–O = 25 μm; B, M = 10 μm; F = 50 μm.

Mentions: The foot is the main locomotory organ of gastropods. It is coated by a ciliated columnar epithelium constituted by narrow and elongated cells (about 28 μm high) with brown granules at their apex (Figure 6A–C). A wavy basal lamina separates the epithelium from underlying tissues (Figure 6B). Two types of gland cells were interspersed among epithelial cells similar to those observed in other epithelia (Figure 6B,C). Among epithelial cells, slender processes of cells located in deeper layers are present (Figure 6C). Small bipolar neurons were identified by DIC in subepithelial layers innervating the base of the epithelium (Figure 6D). In DIC images, cell processes interspersed with epithelial cells are clearly seen (Figure 6D,E).


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)

Foot: histology, NOS distribution and NADPHd staining. A–E: Histological analysis. F–N: NOS distribution analyzed by R20 antibody. O: NADPHd staining. (A,B) Sagittal section of the foot stained with hematoxylin/eosin. The ciliated columnar epithelium contains two types of gland cells with a different affinity for hematoxylin (arrows and arrowheads). Asterisks indicate pigment granules. The wavy basal lamina (double arrowhead) separates the epithelium from the underlying tissues; (C) Picture showing gland cells pouring their secretion on the foot surface. Slender cell processes are seen between epithelial elements (arrowhead); (D) The superimposition of TOTO3 staining to DIC allows a better appreciation of the cell morphology. Gland (arrows) and epithelial (asterisk) cells are visible. Beneath the epithelium, bipolar neurons are clearly seen (arrowhead); (E) DIC image at high magnification showing the presence of thin cell processes among epithelial elements (arrow). Cell processes contact the epithelial surface (arrowhead); (F) Sagittal section showing the distribution of NOS-like proteins beneath the epithelium; (G) Enlargement of the small boxed area in F. NOS-IR cells (arrow) and fibers (arrowheads) are seen; (H) Detail of the large dot-boxed area in F. NOS staining is intense at the base of the epithelium where NOS-IR cells and fibers are in close contact with epithelial cells (arrows). The epithelium exhibits a much weaker staining that is more intense in the apical portion; (I) IF image showing NOS-IR neurons (arrows) with processes penetrating the epithelium (arrowheads); (J) Detail showing intensely labeled NOS-IR cells in the subepithelial layers (arrows). Aligned NOS-positive spots toward the surface are visible (arrowheads) among epithelial cells, suggesting the presence of NOS-IR varicose fibers; (K,L) CM images of the same section confirming the wide distribution of NOS-like proteins beneath the epithelium. NOS-IR cells (arrows) and fibers are present. In the epithelium, a diffuse NOS labeling was observed in gland (arrowheads) and epithelial cells. The apical portion of epithelial cells shows an intense NOS-immunoreactivity. In L triple labeling with NOS antibodies (green), phalloidin (red) and TOTO3 (blue) is shown to better evaluate the distribution of NOS-positive staining; (M) Detail of the epithelium where the presence of NOS-IR thin processes (arrowheads) directed to the epithelium surface can be glimpsed among epithelial cells; (N,O) The same sagittal section of the foot stained with NOS-antibodies (N) and NADPHd assays (O). The two stainings overlapped in both cells (arrow) and cell processes (arrowheads). The number of NOS-positive elements was higher in IF than in NADPHd images. The detection of NADPHd reactivity in the epithelium was difficult due to the presence of natural pigments that obscure NADPHd staining. Bars: A, C–E, G–O = 25 μm; B, M = 10 μm; F = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f006: Foot: histology, NOS distribution and NADPHd staining. A–E: Histological analysis. F–N: NOS distribution analyzed by R20 antibody. O: NADPHd staining. (A,B) Sagittal section of the foot stained with hematoxylin/eosin. The ciliated columnar epithelium contains two types of gland cells with a different affinity for hematoxylin (arrows and arrowheads). Asterisks indicate pigment granules. The wavy basal lamina (double arrowhead) separates the epithelium from the underlying tissues; (C) Picture showing gland cells pouring their secretion on the foot surface. Slender cell processes are seen between epithelial elements (arrowhead); (D) The superimposition of TOTO3 staining to DIC allows a better appreciation of the cell morphology. Gland (arrows) and epithelial (asterisk) cells are visible. Beneath the epithelium, bipolar neurons are clearly seen (arrowhead); (E) DIC image at high magnification showing the presence of thin cell processes among epithelial elements (arrow). Cell processes contact the epithelial surface (arrowhead); (F) Sagittal section showing the distribution of NOS-like proteins beneath the epithelium; (G) Enlargement of the small boxed area in F. NOS-IR cells (arrow) and fibers (arrowheads) are seen; (H) Detail of the large dot-boxed area in F. NOS staining is intense at the base of the epithelium where NOS-IR cells and fibers are in close contact with epithelial cells (arrows). The epithelium exhibits a much weaker staining that is more intense in the apical portion; (I) IF image showing NOS-IR neurons (arrows) with processes penetrating the epithelium (arrowheads); (J) Detail showing intensely labeled NOS-IR cells in the subepithelial layers (arrows). Aligned NOS-positive spots toward the surface are visible (arrowheads) among epithelial cells, suggesting the presence of NOS-IR varicose fibers; (K,L) CM images of the same section confirming the wide distribution of NOS-like proteins beneath the epithelium. NOS-IR cells (arrows) and fibers are present. In the epithelium, a diffuse NOS labeling was observed in gland (arrowheads) and epithelial cells. The apical portion of epithelial cells shows an intense NOS-immunoreactivity. In L triple labeling with NOS antibodies (green), phalloidin (red) and TOTO3 (blue) is shown to better evaluate the distribution of NOS-positive staining; (M) Detail of the epithelium where the presence of NOS-IR thin processes (arrowheads) directed to the epithelium surface can be glimpsed among epithelial cells; (N,O) The same sagittal section of the foot stained with NOS-antibodies (N) and NADPHd assays (O). The two stainings overlapped in both cells (arrow) and cell processes (arrowheads). The number of NOS-positive elements was higher in IF than in NADPHd images. The detection of NADPHd reactivity in the epithelium was difficult due to the presence of natural pigments that obscure NADPHd staining. Bars: A, C–E, G–O = 25 μm; B, M = 10 μm; F = 50 μm.
Mentions: The foot is the main locomotory organ of gastropods. It is coated by a ciliated columnar epithelium constituted by narrow and elongated cells (about 28 μm high) with brown granules at their apex (Figure 6A–C). A wavy basal lamina separates the epithelium from underlying tissues (Figure 6B). Two types of gland cells were interspersed among epithelial cells similar to those observed in other epithelia (Figure 6B,C). Among epithelial cells, slender processes of cells located in deeper layers are present (Figure 6C). Small bipolar neurons were identified by DIC in subepithelial layers innervating the base of the epithelium (Figure 6D). In DIC images, cell processes interspersed with epithelial cells are clearly seen (Figure 6D,E).

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