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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.

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NOS distribution in the pedal ganglion. (A) Low magnification showing the gross distribution of NOS staining in the pedal ganglion. Both round neuron cell bodies (arrows) and nerve fibers (arrowhead) are labeled by NOS antibodies. (B) In this CM microphotography, the weak NOS staining in the cytoplasm (cyt) can be compared to the intense spotted staining on the neuronal surface (arrows); (C) Superimposition of NOS staining in B to the DIC image showing the position of NOS-positive spots in relation to cell morphology; (D,E) NOS/DIC image showing NOS-IR axons (arrows). NOS labeling appears particularly intense in the region where axons contact the neuron cell soma (arrowheads); (F) CM image showing the intense NOS staining around unstained neuron cell bodies (arrows); (G) Superimposition of NOS staining to DIC image allows us to better appreciate the intense NOS staining at the periphery of neuron cell bodies (arrowheads) and NOS-IR nerve fibers (arrows); (H) Detail of NOS staining in a single neuron: intense and defined NOS-labeled spots are present at the periphery of the cell (arrows); (I) IF microphotography showing NOS-positive neuronal processes (arrow) contacting one NOS-IR neuron. Note that NOS staining is diffuse and weak in the cell cytoplasm. NOS-stained neuronal processes have a basket-like distribution on the neuron cell soma (arrowheads). (J) NADPHd staining of the consecutive section to that in I containing the same neurons. A good overlapping of NOS and NADPHd staining can be observed; (K–L) Double-CM immunostainings of the pedal ganglion for NOS (K) and ChAT (L); (M) Overlay of fluorescence signals shown in K and L show that NOS and ChAT are co-localized in several neurons; the numbers “1” and “2” refer to the same neurons. A–E, K: M_R20 antibody; F–H: H299 antibody. A= 200 μm; B–D, F, G, K–M = 25 μm; E, H–J = 10 μm.
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marinedrugs-13-06636-f007: NOS distribution in the pedal ganglion. (A) Low magnification showing the gross distribution of NOS staining in the pedal ganglion. Both round neuron cell bodies (arrows) and nerve fibers (arrowhead) are labeled by NOS antibodies. (B) In this CM microphotography, the weak NOS staining in the cytoplasm (cyt) can be compared to the intense spotted staining on the neuronal surface (arrows); (C) Superimposition of NOS staining in B to the DIC image showing the position of NOS-positive spots in relation to cell morphology; (D,E) NOS/DIC image showing NOS-IR axons (arrows). NOS labeling appears particularly intense in the region where axons contact the neuron cell soma (arrowheads); (F) CM image showing the intense NOS staining around unstained neuron cell bodies (arrows); (G) Superimposition of NOS staining to DIC image allows us to better appreciate the intense NOS staining at the periphery of neuron cell bodies (arrowheads) and NOS-IR nerve fibers (arrows); (H) Detail of NOS staining in a single neuron: intense and defined NOS-labeled spots are present at the periphery of the cell (arrows); (I) IF microphotography showing NOS-positive neuronal processes (arrow) contacting one NOS-IR neuron. Note that NOS staining is diffuse and weak in the cell cytoplasm. NOS-stained neuronal processes have a basket-like distribution on the neuron cell soma (arrowheads). (J) NADPHd staining of the consecutive section to that in I containing the same neurons. A good overlapping of NOS and NADPHd staining can be observed; (K–L) Double-CM immunostainings of the pedal ganglion for NOS (K) and ChAT (L); (M) Overlay of fluorescence signals shown in K and L show that NOS and ChAT are co-localized in several neurons; the numbers “1” and “2” refer to the same neurons. A–E, K: M_R20 antibody; F–H: H299 antibody. A= 200 μm; B–D, F, G, K–M = 25 μm; E, H–J = 10 μm.

Mentions: Both neuronal somata of pedal neurons and axosomatic terminals were stained for NOS (Figure 7A,B). CM of individual focal planes clearly showed weaker diffuse intracellular labeling compared to the intense punctate staining at the cell periphery, where immunoreactive spots were arranged in a “basket-like” fashion (Figure 7B,C). A similar distribution was described for the synaptic boutons on glandular cells of Helix pomatia [43], the salivary glands and cerebral ganglia of Aplysia [44], and the buccal ganglia of Tritonia diomedea [47].


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 pedal ganglion. (A) Low magnification showing the gross distribution of NOS staining in the pedal ganglion. Both round neuron cell bodies (arrows) and nerve fibers (arrowhead) are labeled by NOS antibodies. (B) In this CM microphotography, the weak NOS staining in the cytoplasm (cyt) can be compared to the intense spotted staining on the neuronal surface (arrows); (C) Superimposition of NOS staining in B to the DIC image showing the position of NOS-positive spots in relation to cell morphology; (D,E) NOS/DIC image showing NOS-IR axons (arrows). NOS labeling appears particularly intense in the region where axons contact the neuron cell soma (arrowheads); (F) CM image showing the intense NOS staining around unstained neuron cell bodies (arrows); (G) Superimposition of NOS staining to DIC image allows us to better appreciate the intense NOS staining at the periphery of neuron cell bodies (arrowheads) and NOS-IR nerve fibers (arrows); (H) Detail of NOS staining in a single neuron: intense and defined NOS-labeled spots are present at the periphery of the cell (arrows); (I) IF microphotography showing NOS-positive neuronal processes (arrow) contacting one NOS-IR neuron. Note that NOS staining is diffuse and weak in the cell cytoplasm. NOS-stained neuronal processes have a basket-like distribution on the neuron cell soma (arrowheads). (J) NADPHd staining of the consecutive section to that in I containing the same neurons. A good overlapping of NOS and NADPHd staining can be observed; (K–L) Double-CM immunostainings of the pedal ganglion for NOS (K) and ChAT (L); (M) Overlay of fluorescence signals shown in K and L show that NOS and ChAT are co-localized in several neurons; the numbers “1” and “2” refer to the same neurons. A–E, K: M_R20 antibody; F–H: H299 antibody. A= 200 μm; B–D, F, G, K–M = 25 μm; E, H–J = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f007: NOS distribution in the pedal ganglion. (A) Low magnification showing the gross distribution of NOS staining in the pedal ganglion. Both round neuron cell bodies (arrows) and nerve fibers (arrowhead) are labeled by NOS antibodies. (B) In this CM microphotography, the weak NOS staining in the cytoplasm (cyt) can be compared to the intense spotted staining on the neuronal surface (arrows); (C) Superimposition of NOS staining in B to the DIC image showing the position of NOS-positive spots in relation to cell morphology; (D,E) NOS/DIC image showing NOS-IR axons (arrows). NOS labeling appears particularly intense in the region where axons contact the neuron cell soma (arrowheads); (F) CM image showing the intense NOS staining around unstained neuron cell bodies (arrows); (G) Superimposition of NOS staining to DIC image allows us to better appreciate the intense NOS staining at the periphery of neuron cell bodies (arrowheads) and NOS-IR nerve fibers (arrows); (H) Detail of NOS staining in a single neuron: intense and defined NOS-labeled spots are present at the periphery of the cell (arrows); (I) IF microphotography showing NOS-positive neuronal processes (arrow) contacting one NOS-IR neuron. Note that NOS staining is diffuse and weak in the cell cytoplasm. NOS-stained neuronal processes have a basket-like distribution on the neuron cell soma (arrowheads). (J) NADPHd staining of the consecutive section to that in I containing the same neurons. A good overlapping of NOS and NADPHd staining can be observed; (K–L) Double-CM immunostainings of the pedal ganglion for NOS (K) and ChAT (L); (M) Overlay of fluorescence signals shown in K and L show that NOS and ChAT are co-localized in several neurons; the numbers “1” and “2” refer to the same neurons. A–E, K: M_R20 antibody; F–H: H299 antibody. A= 200 μm; B–D, F, G, K–M = 25 μm; E, H–J = 10 μm.
Mentions: Both neuronal somata of pedal neurons and axosomatic terminals were stained for NOS (Figure 7A,B). CM of individual focal planes clearly showed weaker diffuse intracellular labeling compared to the intense punctate staining at the cell periphery, where immunoreactive spots were arranged in a “basket-like” fashion (Figure 7B,C). A similar distribution was described for the synaptic boutons on glandular cells of Helix pomatia [43], the salivary glands and cerebral ganglia of Aplysia [44], and the buccal ganglia of Tritonia diomedea [47].

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