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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 expression in S. haemastoma organs. Western blot analysis of NOS expression levels in the nerve ring (NR), osphradium (Os), tentacle (Te) and foot (F) of S. haemastoma acclimated at 15 °C for 40 days. A: The NOS immunodetection was performed using three different NOS polyclonal antibodies (R20, H299 and K20) purchased from Santa Cruz Biotechnology, USA. Rat brain homogenate (R) was used as positive control. Anti-GAPDH and anti-actin antibodies were used on the same samples in order to normalize NOS expression. Standard molecular weights are indicated on the left. Each lane was loaded with 75 μg of protein homogenate. B: Band quantification of NOS expression levels detected by R20 antibody normalized on GAPDH levels.
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marinedrugs-13-06636-f001: NOS expression in S. haemastoma organs. Western blot analysis of NOS expression levels in the nerve ring (NR), osphradium (Os), tentacle (Te) and foot (F) of S. haemastoma acclimated at 15 °C for 40 days. A: The NOS immunodetection was performed using three different NOS polyclonal antibodies (R20, H299 and K20) purchased from Santa Cruz Biotechnology, USA. Rat brain homogenate (R) was used as positive control. Anti-GAPDH and anti-actin antibodies were used on the same samples in order to normalize NOS expression. Standard molecular weights are indicated on the left. Each lane was loaded with 75 μg of protein homogenate. B: Band quantification of NOS expression levels detected by R20 antibody normalized on GAPDH levels.

Mentions: Before analyzing NOS distribution, the crossreactivity of commercial anti-NOS antibodies against ShNOS was tested by Western blot (WB). Three different polyclonal antibodies (R20, H299 and K20, Santa Cruz Biotechnology, USA) were tested on protein homogenates of S. haemastoma tissues and then used to assess levels of NOS-like protein expression in the foot, nerve ring, osphradium and tentacle (Figure 1). The use of antibodies directed against mammalian nNOS on molluskan tissues was justified by the moderate sequence homology between ShNOS/rat nNOS (49.3%) and ShNOS/human nNOS (48.3%). The specificity of the antibodies was evaluated on the basis of (a) molecular weight (MW), (b) intensity of immunolabeled bands and (c) level of background labeling. Among the three antibodies tested, both R20 and H299 intensely labeled a band at about 170 kDa that corresponds to the predicted ShNOS molecular weight (169 kDa). However, R20 produced better results than H299 which revealed the presence of more intense background labeling (Figure 1A). Finally, K20 was not able to label defined bands in S. haemastoma tissues. For this reason, K20 was excluded and only R20 and H299 antibodies were used in IF experiments.


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 expression in S. haemastoma organs. Western blot analysis of NOS expression levels in the nerve ring (NR), osphradium (Os), tentacle (Te) and foot (F) of S. haemastoma acclimated at 15 °C for 40 days. A: The NOS immunodetection was performed using three different NOS polyclonal antibodies (R20, H299 and K20) purchased from Santa Cruz Biotechnology, USA. Rat brain homogenate (R) was used as positive control. Anti-GAPDH and anti-actin antibodies were used on the same samples in order to normalize NOS expression. Standard molecular weights are indicated on the left. Each lane was loaded with 75 μg of protein homogenate. B: Band quantification of NOS expression levels detected by R20 antibody normalized on GAPDH levels.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06636-f001: NOS expression in S. haemastoma organs. Western blot analysis of NOS expression levels in the nerve ring (NR), osphradium (Os), tentacle (Te) and foot (F) of S. haemastoma acclimated at 15 °C for 40 days. A: The NOS immunodetection was performed using three different NOS polyclonal antibodies (R20, H299 and K20) purchased from Santa Cruz Biotechnology, USA. Rat brain homogenate (R) was used as positive control. Anti-GAPDH and anti-actin antibodies were used on the same samples in order to normalize NOS expression. Standard molecular weights are indicated on the left. Each lane was loaded with 75 μg of protein homogenate. B: Band quantification of NOS expression levels detected by R20 antibody normalized on GAPDH levels.
Mentions: Before analyzing NOS distribution, the crossreactivity of commercial anti-NOS antibodies against ShNOS was tested by Western blot (WB). Three different polyclonal antibodies (R20, H299 and K20, Santa Cruz Biotechnology, USA) were tested on protein homogenates of S. haemastoma tissues and then used to assess levels of NOS-like protein expression in the foot, nerve ring, osphradium and tentacle (Figure 1). The use of antibodies directed against mammalian nNOS on molluskan tissues was justified by the moderate sequence homology between ShNOS/rat nNOS (49.3%) and ShNOS/human nNOS (48.3%). The specificity of the antibodies was evaluated on the basis of (a) molecular weight (MW), (b) intensity of immunolabeled bands and (c) level of background labeling. Among the three antibodies tested, both R20 and H299 intensely labeled a band at about 170 kDa that corresponds to the predicted ShNOS molecular weight (169 kDa). However, R20 produced better results than H299 which revealed the presence of more intense background labeling (Figure 1A). Finally, K20 was not able to label defined bands in S. haemastoma tissues. For this reason, K20 was excluded and only R20 and H299 antibodies were used in IF experiments.

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