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Back | Show only these items: | Intracellular localization of a long alkyl chain tetraphenylporphyrin and chloride channel activation in Psammobatis extenta electrocytes

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Intracellular localization of a long alkyl chain tetraphenylporphyrin and chloride channel activation in Psammobatis extenta electrocytes

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

The intracellular localization of a tetraphenylporphyrin bearing four long lipophilic alkyl chains in electrocytes from Psammobatis extenta (Rajidae) is described.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

In contrast to what is usually the case, this porphyrin derivative is localized in the electromotor nerves and the nuclear chromatin of the electrocytes.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

Both structures exhibited intense fluorescence, whereas, the mitochondria were only slightly fluorescent.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

These data are discussed in relation to electrocyte death in a weakly electric fish.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

Additionally, electron probe X-ray microanalysis suggests a migration of chloride and cationic ions, which might be implicated in chloride and cationic channel activation in the electrocyte.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

The development of new generation photosensitizers to improve the efficiency of photodynamic therapy (PDT) is an area of intensive research.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

In PDT, the activation of a photosensitizer by light generates singlet molecular oxygen (¹O₂), a highly active form of oxygen that reacts with many biomolecules, including lipids, proteins, and nucleic acids.¹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

The biomolecules are chemically modified by the action of ¹O₂ and, therefore, cannot accomplish their function, which leads to cell death.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

This therapy is applied for treatment of cancer, as well as for bacterial and viral eradication.^2–6

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

The fluorescence exhibited by photosensitizers means they can also be used for detection of tumors.⁷

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

The advantages of this method, as compared to other conventional cancer treatment modalities, are its low systemic toxicity and its ability to destroy tumors selectively.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

Nevertheless, the application of PDT remains restricted due to the limited penetration of light into tissues, the photosensitization of normal tissues, and the residual skin photosensitivity that is observed for several weeks following treatment.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

The lipophilicity of a particular photosensitizer determines its localization and, therefore, the site and nature of the damage it gives rise to in cells;^6,8,9 in general, lipophilic photosensitizers accumulate in the membrane of a cell and its organelles.⁵

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

On the other hand, hydrophilic, as well as aggregated states of photosensitizers, enter the cell by pynocitosis and are localized mainly in lysosomes and endosomes.¹⁰

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

There are many photosensitizers, mostly porphyrin or phthalocyanine derivatives, under clinical study that can be classified as lipophilic or hydrophilic.¹¹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

However, there are a few examples of neutral macrocycles with long alkyl chains which have been studied as potential photosensitizers for PDT.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

The efficiency and selectivity of tumor targeting has been shown to increase slightly upon increasing the length of the alkyl groups connected to a Zn(ii)–phthalocyanine complex.^12,13

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

In this work, we have investigated the uptake and intracellular localization of 5,10,15,20-tetrakis(4-n-dodecylphenyl)porphyrin (1), and the elemental composition and morphological changes in electrocytes of Psammobatis extenta.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

This species belongs to the Rajidae, one of three groups of weakly electric fish.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

We chose electrocytes for this study because they are large cells with very few organelles.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

This facilitates the study of the intracellular localization of the photosensitizer.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

In addition, electric ray electrocytes have myoproteins.^14,15

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

The electrocytes are highly polarized and multinuclear cells.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

They are semicircular in shape (Fig. 1) and have their concave face receiving innervations (IF) from electromotor neurons of the spinal cord.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

The other face, which is convex, is non-innervated (NIF) and shows a system of caveolae.¹⁴

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

The nuclei are localized at the posterior region of the cytoplasm.¹⁴

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

Electric organ cryostat sections were performed along the antero-posterior axis; sections were incubated with a solution of 1 in a mixture of xylene, EtOH, and imidazole buffer and observed with an epifluorescence microscope (Fig. 2).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

The phase-contrast micrograph [Fig. 2(a)] shows part of two electrocytes and the fluorescence micrograph [Fig. 2(b)] shows strong fluorescence in the nuclei, but only very slight fluorescence in the mitochondria.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

When the incubation was performed in CHCl₃ solution only, the intracellular localization of 1 is the same, but the micrograph shows better contrast (Fig. 3).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

The photosensitizer also shows affinity for the terminal nerves (TN).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

The affinity of this lipophilic photosensitizer for the nerves is a result of this tissue being rich in fatty acids.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac19

The nuclei show an intense red fluorescence and the shape of this fluorescence is similar to the semicircular distribution of the chromatin of these cells (Fig. 3), suggesting that 1 interacts with DNA.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

In contrast, the mitochondria show only slight fluorescence.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

This localization is unusual for other photosensitizers of similar polarity.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

For example, a lipophilic phthalocyanine has been found to localize on the lysosomes.¹⁶

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac20

Immediately after the penetration of 1 (1.2 × 10^–4 M in chloroform) into the electrocytes, they start to swell and the convex faces lose all their invaginations (Fig. 4).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

In order to understand the reason for the swelling of cells, microanalysis by energy-dispersive X-ray (EDAX) spectroscopy of the same region was carried out. After treatment of the electrocyte with 1, the relative semi-quantitative contents [K_α (wt%)] of oxygen, sodium, and chloride ions were 39, 17, and 15, respectively.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

Compared to the negative control, the peak for Na⁺ is five times larger, while for oxygen, the variation is not significant.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac21

Additionally, the Ca²⁺ peak is twice as large for the treated electrocyte compared to the negative control, but that for K⁺ is six times smaller.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac22

The simultaneous increase in the Na⁺ and Ca²⁺ concentrations and the decrease in the K⁺ concentration is good evidence for cationic channel activation by the porphyrin derivative.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

However, the most important change is the appearance of a large new peak corresponding to the Cl^– anion.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

Treatment of electrocytes with systems I and II [(I) 7.8 × 10^–5 M of 1 in CHCl₃–EtOH–imidazole buffer; (II) 3.9 × 10^–4 M of 1 in xylene–EtOH–imidazole buffer] produces similar behavior to that observed after treatment with the CHCl₃ solution of 1.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

In both cases, the EDAX patterns shows a new peak corresponding to the chloride anion (Fig. 5).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

The K_α values for this element are 47 (system I) and 33 wt% (system II), in spite of the higher concentration of 1 in system II (5 times higher) compared to system I. These results constitute suggestive evidence for chloride channel activation as a consequence of the penetration of 1 into the electrocytes.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

The massive intracellular accumulation of Cl^– and, particularly, the influx of Na⁺ lead to cell swelling, and eventually a necrotic response from the cells.^17,18

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac23

It should be pointed out that chloride channel activation is not usual for weakly electric fish.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac24

However, the presence of a voltage-gated chloride channel was demonstrated in the non-innervated plasma membrane of electrocytes of Torpedo, a strongly electric fish.¹⁹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac25

The activation of the chloride channel by 1 may constitute an alternative for the treatment of cystic fibrosis, which is related to a dysfunction of chloride ion transport.²⁰

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

In contrast, the increase in the Ca²⁺ concentration in the electrocytes suggests the participation of an apoptotic mechanism.¹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac26

Moreover, the interaction of 1 with DNA, and even the low amount of mitochondrial-bound porphyrin, may contribute to cell death by an apoptotic mechanism after excitation with light.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac27

The photosensitizer used in this work has liquid crystalline properties.^21,22

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac28

It would be very interesting to compare these results with those obtained using another photosensitizer with a similar chemical structure but no mesomorphic properties.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac29