Quantitative measurement of melanophore responses to drugs and hormones
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Abstract
With the attention of many physiologists, pharmacologists, biochemists, and microbiologists focused upon the cell, the quantitation of the responses of single cell effectors is of current interest. This thesis is devoted to the consideration of methods of quantitating the melanophore responses of frogs and fish to drugs and hormones.
Color changes of animals have been studied since the time of Aristotle. For many years the melanophore responses of various species of animals were studied with respect to changes in environmental background. In the earlier part of this century the responses of melanophores to drugs and hormones, isolated from the endocrine glands, especially the pituitary and adrenal gland, were observed and quantitated, for the most part, subjectively. More recently the demonstration of the melanophore stimulating activity of preparations of ACTH has greatly increased interest in the responses of melanophores. A controversy of whether ACTH has intermedin activity or whether intermedin is a contaminant of ACTH preparations has increased the need for objective quantitative methods for measuring melanophore responses.
Demonstration of the melanophore stimulating principle in the blood of patients under stress, patients with Addison's disease, and in pregnancy increases the importance of quantitation of the melanophore responses. Similarity between the pigment cells of the retina of many animals and the pigment cells present in the skins, fascia, and mesenteries of animals has been another stimulus for this study.
Both morphology and the mechanism of melanin pigment migration are considered in the thesis. Photographs which confirm the theory that the melanophores of Fundulus heteroclitis are fixed stellate processed cells whose pigment migrates into and out of the processes and the body of the cell in response to epinephrine and an adrenergic blocking drug N-9-Fluorenyl-N-ethyl-2 Chlorethylamine HCl (SY-21) are presented.
The reason for the changes in color of animals with the migration of pigment within the melanophores was demonstrated by measuring the areas covered by single melanophores with their pigment in the dispersed and concentrated states. The melanophore with dispersed pigment was found to cover approximately six times more area than with pigment in the concentrated state.
A critical review of the previously reported methods of recording and quantitating melanophore responses is presented. Methods of quantitating melanophores are discussed under two large categories: subjective and objective. Subjective methods are those which use an arbitrary index by which the various stages of the melanophores are characterized by numbers. Together with the subjective eiement of such methods, the statistical treatment of such indices as measurement data is criticized on the ground that there is no mathematical reaction of the various subdivisions. The originators of the method expressed the fact that the indices were arbitrary designations. Criticisms of the objective quantitative methods developed up to the present time are discussed. It is concluded that only an objective quantitative method for measuring melanophore responses to which valid statistical procedures can be applied should be used for assay of the melanophore stimulating principle.
An objective quantitative method of measuring melanophore responses in the intact frog is presented. This method consists of series of measurements of the light reflected from the dorsal surface of intact light adapted frogs before and at various time intervals after the administration of an Armour ACTH preparation. An extensive analysis of the data is presented and discussed. A slight dependence of the peak and one-hour responses on the starting reflectances of the frogs was calculated by means of covariance analysis. No dependence of the responses on the vreight of the animals was found. The arbitrary practice of administering the hormone on the basis of animal weight is criticized on the grounds that previous workers did not establish the dependence of the response on the weight of the animal.
Dose-response regression lines are presented for the peak-response and the response one hour after administration of the ACTH. Analysis of these lines and comparison of the slopes, variances of error, and precision indices indicate the one-hour response has about the same degree of precision as the peakresponse. For purposes of assay, the one-hour-response is more convenient to use. This method fulfills the three criteria for quantitating responses set forth by Gaddum in that there is a linear relationship over a hundred fold range of doses; the standard deviation is independent of the response; and the precision index, lambda, has a small value.
The reliability of the photoelectric method for quantitating melanophore responses was demonstrated by means of the assay of unknown solutions of the standard Armour ACTH preparation. An average error of 14% for the unknowns was found.
This method was employed in the assay of the melanophore stimulating activity of anterior pituitary preparations provided by Dr. E. B. Astwood. Most of the melanophore stimulating activity of the preparations was found to be present in the acetic acid fraction produced by fractionation of Corticotrophin between butanol and acetic acid. We are unable to confirm the 400 fold purification of the ACTH (butanol fraction) reported by Astwood and coworkers. Results of the assay of an alkali-treated preparation of Corticotrophin indicate a potentiation reported previously by other workers.
Assay of the melanophore stimulating activity of posterior pituitary preparations indicated that almost all the intermedin activity is present in the Vasopressin fraction of posterior pituitary powder. Pitocin, or the oxytocic fraction of posterior pituitary powder, was found to have very little melanophore stimulating activity.
Comparison of the slopes, standard deviations and precision indices of the standard preparation over a period of seven months indicated a stability of the assay method. It is concluded, therefore, that the photoelectric method of measuring melanophore responses is reliable.
A study of the effects of a number of drugs on the melanophores of the intact frog and the response of the melanophores to a dose of 0.4 µg/frog was presented. Dimethylaminoethyl benzhydryl ether HCl (Benadryl), atropine sulfate, epinephrine in a dose of 0.1 mg/frog, methacholine chloride, and a drug produced by the condensation of an alkoxyphenylamine with formaldehyde (48/80) were found to disperse the pigment within the melanophores of the intact light adapted frogs. Histamine diphosphate, epinephrine - 1 mg/frog, and tetraethylammonium did not disperse the pigment within the melanophores at one half hour. None of the drugs studied blocked the responses to the standard dose of ACTH.
A method of quantitating the melanophore responses of isolated frog skins was described and compared with the in vivo method. When the results were expressed in the same way as that of the originators of the method, there was an increase in the variability of the responses and heterogeneity of the variances was revealed. The responses expressed as the light transmission measurement forty minutes after administration of the ACTH was variable. A log dose-response regression line was presented with its statistical analysis. The precision index of the in vitro photoelectric method was found to be greater than that of the in vivo photoelectric method. The sensitivity of the intact animal was greater than isolated pieces of frog skin.
An attempt to quantitate the melanophore responses of isolated Fundulus heteroclitis melanophores was presented. The method, similar to that reported by Smith consists of the measurement of changes in light transmission through the scales after the administration of piperidine methyl-3-benzodioxane (Benodaine), and in another series, epinephrine. Statistical analysis indicated variability of the responses and a precision index of 0.96. Because of the great variability of the responses no significant differences were produced by changing the temperature at which the experiments are conducted. The response to epinephrine was also variable and in this series no significant regression was found.
In conclusion, a method of quantitating melanophore responses of the intact frog has been developed. This method has the advantages of sensitivity, reliability and accuracy. The data permit the application of statistical procedures which give the results a known degree of confidence that is unobtainable with the in vivo methods that have been reported to date.
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Thesis (Ph.D.)--Boston University
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