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Prazosin decreases the aggregatory response caused by phenylephrine in scales from rainbow trount, Oncorhynchus mykiss

Jeevika Goyal*, Pakeezah Saadat1209306

*McMaster University, Department of Biology, 1280 Main Street West, Hamilton, ON Canada L8S 4K1

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Fish and other lower vertebrates such as fish and other amphibians protect themselves from predators by camouflaging in their environment. This defence mechanism of fishes is aided by the presence of dermal chromatophores, which allow for colour change. Melanosomes, a type of chromatophores, are present in scales of trout Oncorhynchus mykiss, with organelles called melanosomes that contain dark colour pigment granules. These melanosomes respond to hormonal or chemical stimuli by moving about the cell. In this study we observe this movement under a light microscope in trout scales of Oncorhynchus mykiss, to understand the effect of prazosin on these scales.1 We did this by comparing the average grade of melanophores in trout scales, in the presence of phenylephrine and ringer (control) to its presence in phenylephrine and prazosin. It was hypothesized, that chromatophores that interacted with prazosin will exhibit a higher grade, as it is an α 1 androgenic receptor antagonist and will inhibit the effects of phenylephrine; thereby, decreasing aggregation in scales. As anticipated, we found that average grade of melanophores was greater in control then in the presence of prazosin and phenylephrine. Therefore, the results demonstrate that prazosin decreases the rate of aggregation in trout scales. This study further opens doors for future experiment concerning other factors affecting melanophore movement, such as factors affecting microtubules.

As described earlier, melanosomes move about the cell, as a way to respond to stimuli.

This movement is described as aggregation or dispersion, and is the reason for colour change. Aggregation occurs when the pigment granules are aggregated in the centre of cell, giving the vertebrate a light colour. Contrarily, in dispersion, the pigment granules move away from the centre of the cell and the animal bearing such cells would appear darkly coloured. This movement is controlled by signal transduction pathways that control the direction of granule transport. Pigment dispersion is observed in the presence of cAMP in cells. In the presence of cAMP, cAMP dependent protein kinase (PKA) is activated, which phosphorylates many targets; thereby producing dispersion. Epinephrine on the other hand, binds to cell – surface receptors in scales, which then interacts with G – protein. Binding of epinephrine converts GTP into GDP on G - protein, which activates the G – protein. This activated G – protein, inhibits the enzyme adenylate cyclise, which is responsible for converting ATP into cAMP. This in turn, lowers the cAMP concentration, producing aggregation2.

In this study we sought to determine the effects of prazosin on chromatophores of trout scales from Oncorhynchus mykiss. Prazosin is an α -1 andregenic receptor antagonist; and is hypothesize to promote dispersion. We studied this effect by first placing scales in diginotin mixture to allow it fully aggregate. These scales were then, placed in ringer, as part of the control, for 7 minutes before they were placed in phenylephrine for 5 minutes and pictures were taken every 30 seconds for 5 minutes to account for any changes. This is was done in

several concentrations of phenylephrine. Similarly, to test the effects of prazosin, the scales were again placed in digintonin mixture for it fully aggregate. After these scales were placed in 10 -5 M concentration of prazosin, before placing them into phenyephrine for 5 minutes to take pictures every 30 seconds. Again this step was performed several times,

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using different concentrations of phenylephrine each time. The pictures taken at 30 seconds for 5 minutes, for both control and experiment, were graded from 1 to 5; with 1 being fully aggregated and 5 being fully dispersed. These average grades were then compared to examine the rate of aggregation in both sets of chromatophores (control and experiment)1.

We found that, in control, increasing the concentration of phenylephrine, increased the rate of aggregation. This can be seen by the difference in average grade of melanophores, presented by figure 1. It shows that 10-3M concentration of phenylephrine has the lowest average grade of melanophores; while 10-6M concentration of melanphores has the highest grade of melanophores. Figure 1 is a graph generated at 1 min; as in most of the cells had aggregated at this point. However, 10-3 Mcells were the first ones to be fully aggregated;while 10-6M, were last to be fully aggregated.

Figure 1: 10-3 M concentration of Phenylephrine has the lowest average grade at 1 min Pictures were taken using light microscope at every 30 seconds, for 5 minutes. These pictures were then analyzed to grade for melanophores, base d on the 1-5 grading scale provided. The graph generated is for the average grade of melanophores observed at 1minute. It exhibits the rate of melanophore aggregation in the presence of ringer and varying concentration of phenylephrine. It shows that 10-3 M concentration of phyenlephrine has the lowest average grade while the 10-6 M concentration of phenylephrine, has the highest average grade.

We observed a similar pattern in the chromatophores that were placed in prazosin and phenylephrine. We observed that, increasing the concentration of phenylephrine, increased the rate of aggregation. This was observed by difference in the average grade of melanophore depicted by figure 2. The graph shows that 10-4M had the lowest average grade melanophores, while 10-6M had the largest grade. This graph was produced by grading the melanosomes between 1 – 5 at 4 minutes. These chromatophores are best understood at 4 minutes, because after this point much of the cells remained same. Similar observation was made in Control (Fig1). The graph is generated for cells at 1 minute, no significant change was observed. However, we observed that rate of aggregation had significantly decreased in Fig 2 relative to control (Fig 1). This is because by 1 minute, in control (Figure 1), most of the cells can be see to aggregated through their lower average grade number while by 4 minutes, very few cells were aggrevated in the cells that interacted by prazosin; shown here by the differenece in average grade of melanophores. It can be seen that 10-3 in Figure 2 is at 1.6 while in figure 1 it is at 1.05; thereby, depicting decrease in aggrevatory response due to prazosin.3

Figure 2: 10-4M has the lowest average grade of melanophores. Pictures were taken using light microscope at every 30 seconds, for 5 minutes. These pictures were then analyzed to grade for melanophores, base d on the 1-5 grading scale provided. The graph generated is for the average grade of melanophores observed at 4minute. Graph exhibits the rate of melanophore aggregation in the presence of prazosin and varying concentration of Phenylephrine. It shows that 10-4 M concentration of phyenlephrine has the lowest average grade while the 10-6 M concentration of phenylephrine, has the highest average grade.

The result from the study concludes two main things. Firstly, increasing the concentration of phenylephrine in both, control and chromatophores in prazosin and phenylephrine, results in increase in the rate of aggregation; which can be observed by the decrease in average grade of melanophores (Fig 1, Fig2). Secondly, prazosin decreases the rate of aggregation, which can also be seen through the elevated average grade values in figure 2 as compare to figure 1. Increasing the concentration increased the rate of aggregation, as it increased the total number of interactions between the phenylephrine and the receptors; which further increased the G- protein mediated response to increase the rate of aggregation. Increase in phenylephrine means that cells will be able to aggregate faster, which is supported by the decreased the grade scale values. As described earlier, this occurs because increase in phenylephrine (epinephrine) decreases the concentration of cAMP, which decreases the PKA, thereby producing aggregation, rather than dispersion. It was hypothesized that prazosin will decrease the rate of aggregation caused by phenylephrine. Our results support our hypothesis, as Figure 2 suggests a decrease in aggregatory response compare to Figure 1. Figure 2 shows a decrease in aggregation in cells because at every point on the graph, the average grade is higher than the points on Fig1. Additionally, the cells that were placed in prazosin, took longer to aggregate. This is can be seen from Figure2, as this graph was produced for chromatophores at 4 minutes while Figure 1 was produced for chromatophores at 1 minute. Despite the three minute difference, chromatophore in control

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(Fig1), are more aggregated; thereby exhibiting faster rate of aggregation, This is because prazosin is an α1 androgenic receptor antagonist; therefore, it will bind to the androgenic receptor, blocking the phenylephrine from binding to it.2 Since, less phenylephrine will be able to bind to androgenic receptor, due to the presence of prazosin, less G – protein mediated response will be activated; which in turn decreases the rate of aggregation and promotes dispersion. This shows that prazosin behaves similar to cAMP, as it promotes dispersion.4 Comparatively, phenylephrine on chromatophores in control (Fig1), face no such competition regarding binding to receptors on the scales; which is why the chromatophores in control exhibit an accelerated melanosome aggregation.

As stated earlier, figure 2 is a graph created using the average grade of melanophores at 4 minutes. In this 10-3M is higher in average grade than lower. This could be due to the potential source of errors during the study. One of the errors is that digitonin has a life of about 45 minutes to an hour; therefore, it may have expired towards the end of this experiment. This would affect the results, as not all the melansomes will be fully aggregated prior to the experiment. This would affect the pictures, as it will be harder to distinguish between melanosomes with the dispersion caused by the phenlephrine and pre – existing dispersion. Secondly, fish trout scales were dying towards the end, and did not respond to drugs very effectively. Lastly, each manipulation was made on a different scale. Different scales may respond differently to changes and have different number of melanosomes, which will affect the average grade scale. Also, the average grading of melanophores is biased; as difference in opinion regarding the grade being assigned to a melanosome may exist.

Therefore, we conclude that prazosin decreases the aggregatory response in trout scales, as it is an alpha 1 androgenic receptor antagonist, which interferes the binding of phenylephrine to the receptor; thereby decreasing the G – protein mediated aggregation.

MethodsControl: A 12- well plate was obtained and the following solutions are prepared, digitonin solution, by mixing 5μl of digitonin with 2mL of perfusion buffer; ringer solution of 10-5 M concenration; and serial dilutions of phenylephrine, from 10 -3 M to 10 -6 M concentration. First scales were allowed to sit in digitonin mixture for 3 minutes and a picture was taken.

After, all the digitonin was removed and ringer solution was added; in which they were allowed to sit for another 7 minutes. Lastly, the scales were place in 10-3M concentration of phenylephrine and pictures were taken at every 30 seconds, for five minutes. This process was repeatd with several different concentrations of phenylephrine.

Manipulation:A12- well plate was obtained and the following solutions are prepared, digitonin solution, by mixing 5μl of digitonin with 2mL of perfusion buffer; prazosin of 10-5 M concenration; and serial dilutions of phenylephrine, from 10 -3 M to 10 -6 M concentration. First scales were allowed to sit in digitonin mixture for 3 minutes and a picture was taken. After, all the digitonin was removed and prazosin solution was added; in which they were allowed to sit for another 7 minutes. Lastly, the scales were place in 10-3M concentration of phenylephrine and pictures were taken at every 30 seconds, for five minutes. This process was repeatd with several different concentrations of phenylephrine.

AckowledgementsA special thanks to Dr. Daniel for their assistance in deciphering the results and on theory/ conceptual aspect of the experiment. We would also like to acknowledge Britney Boroweic and Sinah Lee for their assistance during the experiment.

1 Bio 2 L03 Plant Bio Lab, Dr. Daniel2 Lodish H. Molecular Cell Biology 6th Edition. Macmillan;

2008.3Oshima N, Nakamaru N, Araki S, Sugimoto M.

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores. Comp Biochem Physiol C Toxicol Pharmacol. 2001;129(2):75-84.

4Brosnan CF, Goldmuntz EA, Cammer W, Factor SM, Bloom BR, Norton WT. Prazosin, an alpha 1-adrenergic receptor antagonist, suppresses experimental autoimmune encephalomyelitis in the Lewis rat. Proc Natl Acad Sci USA. 1985;82(17):5915-9.

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