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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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Effect of thermal stress on NOS gene expression and SOD and GPX activity in S. haemastoma organs. A–D: Semi-quantitative RT-PCR analysis of NOS gene expression normalized on actin performed in the osphradium (A), tentacle (B), foot (C) and nerve ring (D) samples of specimens placed at 28 °C for 2–24 h. C1 refers to control samples acclimated for 40 days at 15 °C and C2 refers to control samples transferred from the “home tank” at 15 °C to the “experimental tank” at the same temperature of 15 °C for 2 h. Band quantification was performed by Image J software; (E,F) SOD activity expressed as percentage of inhibition and GPX activity expressed as U/mL evaluated in the nerve ring (black bars) and osphradium (grey bars) subjected to thermal stress. The graph shows the average ± s.d. of three different experiments, performed under identical conditions. Asterisks indicate statistically significant difference (* = p < 0.05; ** = p < 0.02) between each sample and C2.
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marinedrugs-13-06636-f008: Effect of thermal stress on NOS gene expression and SOD and GPX activity in S. haemastoma organs. A–D: Semi-quantitative RT-PCR analysis of NOS gene expression normalized on actin performed in the osphradium (A), tentacle (B), foot (C) and nerve ring (D) samples of specimens placed at 28 °C for 2–24 h. C1 refers to control samples acclimated for 40 days at 15 °C and C2 refers to control samples transferred from the “home tank” at 15 °C to the “experimental tank” at the same temperature of 15 °C for 2 h. Band quantification was performed by Image J software; (E,F) SOD activity expressed as percentage of inhibition and GPX activity expressed as U/mL evaluated in the nerve ring (black bars) and osphradium (grey bars) subjected to thermal stress. The graph shows the average ± s.d. of three different experiments, performed under identical conditions. Asterisks indicate statistically significant difference (* = p < 0.05; ** = p < 0.02) between each sample and C2.

Mentions: Levels of NOS gene expression were assessed by semi-quantitative RT-PCR by normalizing results on actin expression (Figure 8). Comparison between expression levels detected in C1 and C2 showed small variations in NOS gene expression in all samples that were statistically significant in only tentacle and foot (Figure 8B,C). Evidently, despite all precautions taken, mollusks are mildly affected by the transfer between tanks. In order to obtain information only related to temperature, NOS expression in thermally stressed samples was only compared with C2 controls.


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)

Effect of thermal stress on NOS gene expression and SOD and GPX activity in S. haemastoma organs. A–D: Semi-quantitative RT-PCR analysis of NOS gene expression normalized on actin performed in the osphradium (A), tentacle (B), foot (C) and nerve ring (D) samples of specimens placed at 28 °C for 2–24 h. C1 refers to control samples acclimated for 40 days at 15 °C and C2 refers to control samples transferred from the “home tank” at 15 °C to the “experimental tank” at the same temperature of 15 °C for 2 h. Band quantification was performed by Image J software; (E,F) SOD activity expressed as percentage of inhibition and GPX activity expressed as U/mL evaluated in the nerve ring (black bars) and osphradium (grey bars) subjected to thermal stress. The graph shows the average ± s.d. of three different experiments, performed under identical conditions. Asterisks indicate statistically significant difference (* = p < 0.05; ** = p < 0.02) between each sample and C2.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f008: Effect of thermal stress on NOS gene expression and SOD and GPX activity in S. haemastoma organs. A–D: Semi-quantitative RT-PCR analysis of NOS gene expression normalized on actin performed in the osphradium (A), tentacle (B), foot (C) and nerve ring (D) samples of specimens placed at 28 °C for 2–24 h. C1 refers to control samples acclimated for 40 days at 15 °C and C2 refers to control samples transferred from the “home tank” at 15 °C to the “experimental tank” at the same temperature of 15 °C for 2 h. Band quantification was performed by Image J software; (E,F) SOD activity expressed as percentage of inhibition and GPX activity expressed as U/mL evaluated in the nerve ring (black bars) and osphradium (grey bars) subjected to thermal stress. The graph shows the average ± s.d. of three different experiments, performed under identical conditions. Asterisks indicate statistically significant difference (* = p < 0.05; ** = p < 0.02) between each sample and C2.
Mentions: Levels of NOS gene expression were assessed by semi-quantitative RT-PCR by normalizing results on actin expression (Figure 8). Comparison between expression levels detected in C1 and C2 showed small variations in NOS gene expression in all samples that were statistically significant in only tentacle and foot (Figure 8B,C). Evidently, despite all precautions taken, mollusks are mildly affected by the transfer between tanks. In order to obtain information only related to temperature, NOS expression in thermally stressed samples was only compared with C2 controls.

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