Comp. Biochem. Physiol. Vol. 71C, pp. 15 to 19, 1982 0306-4492/82/010015-05503.00/0 Printed in Great Britain. All rights reserved Copyright © 1982 Pergamon Press Ltd

ADDITIONAL EVIDENCE FOR NOREPINEPHRINE AS A NEUROTRANSMITTER TRIGGERING RELEASE OF

MELANIN-DISPERSING HORMONE IN THE FIDDLER CRAB, UCA PUGILATOR: THE EFFECTS

OF ~l AND fl ADRENOCEPTOR BLOCKING DRUGS ON MELANIN MIGRATION*

MUKUND M. HANUMANTE and MILTON FINGERMAN Department of Biology, Tulane University, New Orleans, LA 70118, U.S.A.

Abstract--1. The effects of the ~ adrenoceptor blockers indoramin, azapetine and phenoxybenzamine and the fl adrenoceptor blockers propranolol and SK&F 92657 on the distribution and translocation of the pigment in the melanophores of the fiddler crab, Uca pugilator, were determined.

2. All of the ~q blockers (but none of the fl blockers) significantly inhibited the melanin dispersion which normally occurs when fiddler crabs are transferred from a white to a black background.

3. None of the drugs had a direct effect on the rate of pigment migration in the melanophores of isolated legs.

4. The results are consistent with the hypotheses that (a) norepinephrine produces melanin dispersion in Uca pugilator by triggering release of melanin-dispersing hormone (MDH) and (b) when fiddler crabs are transferred from a white to a black background cq adrenoceptor activation plays the dominant role in executing the MDH-release triggering action of norepineprhine.

INTRODUCTION

Translocation of the melanin within the melano- phores of the fiddler crab, Uca pugilator, is regulated by melanin-dispersing and melanin-concentrating hormones (Carlson, 1935; Sandeen, 1950; Fingerman, 1956). A series of experiments has led to the hypoth- esis that at least in this crab norepinephrine (NE) triggers the release of melanin-dispersing hormone (MDH) (Hanumante et al., 1980; Fingerman et al., 1981; Hanumante et al., 1981). Hanumante and Fingerman (1981) have postulated that ct 1 adreno- ceptors are involved in the M D H release-triggering effect of NE. However, in mammals postjunctional ct and fl adrenoceptors may both be involved in norad- renergic neurotransmission (Berthelsen & Pettinger, 1977), although it is well known that NE mainly acti- vates ~t 1 adrenoceptors (Maxwell, 1971; Jenkinson, 1973). Therefore, it is of interest for comparative phar- macological reasons to determine the nature of the adrenoceptors through whose excitation the release of M D H is triggered in the fiddler crab, Uca pugilator. This objective was carried out by administering ct 1 and fl adrenoceptor blockers and noting their effects on melanin migration in crabs kept either on a white or a black background throughout the experiment or in the crabs that underwent a background change from white to black or black to white at the time the drugs were administered.

MATERIALS AND METHODS

Adult male fiddler crabs, Uca pugilator, from the area of Panacea, FL, were used. The crabs were exposed to an illumination of 1200 lx at 24°C during the experiments.

* This investigation was supported by Grant PCM-7826094 from the National Science Foundation.

15

The melanophores were staged using the system of Hog- ben & Slome (1931). According to their scheme, stage 1.0 represents maximal pigment concentration, stage 5.0 maxi- mal dispersion, and stages 2.0, 3.0 and 4.0 the intermediate conditions. To facilitate comparison of the responses of the experimental and control crabs, the mean responses of both groups were used to calculate what we here refer to as "response indices." The response index is defined as the difference between the means of the control and experi- mental groups. For example, if the mean melanopbore stage of the control group is 2.8 and that of the experimen- tal group is 3.6 then the response index is +0.8, the " + " indicating the melanin of the experimental group is more dispersed than that of the control group. A " - ' ' will indi- cate that the melanin of the experimental group is less dispersed than that of the control group. The data presented below are the mean response indices for 10 crabs or 10 isolated legs. All of the data were analyzed by means of Student's t-test with significance set at the 95% confi- dence interval.

Assays were performed on intact crabs and isolated legs. When assays were performed on intact animals their mela- nophores were staged at the time of injection, 15 and 30 min thereafter, and subsequently at 30 rain intervals up to 120 rain. However, when assays were performed on iso- lated legs the melanophores were staged only at the time the legs were removed from the crabs (at which time the test solution or saline was injected into the legs), and 15, 30, 45 and 60 rain thereafter. The assays with isolated legs were performed according to the method of Fingerman (1956).

The volume of solution injected into each intact crab and isolated leg was 0.05 ml. In isolated legs the melano- phores remain responsive for at least 120 min (Herman & Dallman, 1975). When intact crabs were used, the melano- phores seen through the cuticle on the anteroventral sur- face of the second walking leg on the right side were staged. But when isolated legs were used, the second and third walking legs from both sides of the crab were re- moved, the legs from the left side serving as controls for the legs on the right side that received the test solution, and

16 MUKUND M. HANUMANTE and MILTON FINGERMAN

Table 1. Response indices of melanophores of crabs administered a drug and maintained throughout the experiment on a white (W) or a black (B) background

Time (min) Drug Background 0 15 30 60 90 120

Indoramin W 0.0 0.0 0.0 0.0 0.0 0,0 B 0.0 -0 .2 -0 .3 -0 .2 -0 .2 +0.1

Azapetine W 0.0 0.0 0.0 0.0 0.0 0.0 B 0.0 - 0.6* - 0.7* - 0.6* - 0.4 - 0.2

Phenoxybenzamine W 0.0 - 0.3 - 1.0" - 0.5 - 0.7* - 0.7* B 0.0 -0 .3* -0 .5* - 0 . 2 - 0 . 4 - 0 . 6

Propranolol W 0.0 0.0 0.0 0.0 0.0 0.0 B 0.0 -0.7* -0.7* -0.5* -0.6* -0.7*

SK&F 92657 W 0.0 0.0 0.0 0.0 0.0 0.0 B 0.0 -0 .3 -0 .4 -0 .5 -0 .2 +0.1

+, melanin more dispersed in experimentals than in controls; - , melanin less dispersed in experimentals than in controls. *Statistically significant, P ~< 0.05.

the melanophores on the anteroventral surface of these iso- lated legs were likewise observed for staging.

Four of the drugs used in the present investigation, indoramin HCI (Wyeth), azapetine HaPO, (Hoffmann- LaRoche), phenoxybenzamine HCI (Smith, Kline & French), and SK&F 92657 (DL-3-(2-(3-t-butylamino-2-hyd- roxypropoxy)phenyl)-6-hydrazinopyridazine dihydrochlor- ide hydrate) (Smith, Kline & French) were generous gifts. In addition, DL-propranolol HCI (Sigma) was used. The concentration used for each drug was 20/zg/dose of the free compound. All the drugs except phenoxybenzamine were dissolved in physiological saline (Pantin, 1934). The phe- noxybenzamine solution was prepared in 5% ethylene gly- col + 5% dextrose in distilled water (w/v). Control crabs received either physiological saline or the 5% ethylene gly- col + 5% dextrose solution.

RESULTS

Effects of ~q adrenoceptor blockers on melanin migra- tion

Indoramin (Waterbury & Dean, 1980), azapetine (Starke & Endo, 1976; Doxey et al., 1977) and phe- noxybenzamine (Weiner, 1980; Wate rbury & Dean, 1980) are antagonists of ~1 adrenoceptors. Indoramin and azapetine did not produce a significant response in the crabs main ta ined on a white background th roughout the experiment (Table 1). Their melanin

was initially fully aggregated. Phenoxybenzamine did, however, diminish the melanin dispersion which was evident in the control crabs tha t received the 5% ethylene glycol + 5~o dextrose vehicle and were then retained th roughou t the experiment on the white background along with the experimentals (Table 1). The strong melanin-dispersing response which occurred in these control crabs was presumably due to this vehicle for the phenoxybenzamine.

Among the crabs transferred from a white to a black background, a black background fostering melanin dispersion, significantly less melanin disper- sion was apparen t in the crabs tha t received the ct t blockers than in the corresponding controls (Table 2). The melanin of these crabs transferred to the black background was initially maximally concentrated.

Indoramin had no significant effect on the extent of melanin dispersion in those crabs tha t initially had maximally dispersed melanin and were mainta ined on a black background th roughou t the experiment (Table 1). In contrast , azapetine and phenoxyben- zamine evoked a small, but significant concentra t ion of the melanin in the crabs kept on a black back- ground th roughout the experiment (Table 1). But in the crabs that were transferred from a black to a white background (Table 2), none of these blockers of at adrenoceptors significantly affected the rate of cen-

Table 2. Response indices of melanophores of crabs administered a drug and then changed from a white to a black background (W ~ B) or from a black to a white background (B ---* W)

Background Time (min) Drug change 0 15 30 60 90 120

Indoramin W --~ B 0.0 - 1.1" - 1.6" - 1,3" - 1.6" - 1.6" B ---*W 0.0 -0 .5 -0 .2 +0.1 +0.1 -0 .2

Azapetine W---* B 0.0 -0.6* -0.9* -0.8* -1 .2" -1 .1" B ---,W 0.0 -0 .6 -0 .6 -0 .4 -0 .3 -0 .4

Phenoxybenzamine W ---, B 0.0 - 1.1" - 1.0" - 1.4" - 1.7" - 1.3" B ---~W 0.0 -0 .3 -0.3 -0 .4 -0 .3 -0 .6

Propranolol W ~ B 0.0 - 0.2 - 0.5 - 0.3 - 0.3 - 0.3 B --*W 0.0 -0 .3 -0 .5 -0 .6 -0 .6 -0 .8

SK&F 92657 W ~ B 0.0 -0 .3 -0.3 -0 .2 -0 .2 -0 .3 B ---*W 0.0 -0 .2 -0 .6 -1 .2" -1.1" -1 .2"

+, melanin more dispersed in experimentals than in controls; - , melanin less dispersed in experimentals than in controls. *Statistically significant, P ~ 0.05.

Color change hormone release 17

Table 3. Response indices of melanophores in isolated legs administered a drug. None of the responses was statistically significant

Initial state Time (min) Drug of pigment 0 15 30 45 60

Indoramin Maximally concentrated 0.0 - 0.2 + 0.2 0.0 0.0 Maximally dispersed 0.0 + 0.1 + 0.! 0.0 0.0

Azapetine Maximally concentrated 0.0 - 0.1 + 0.1 + 0.1 + 0.3 Maximally dispersed 0.0 + 0.1 + 0.1 + 0.1 + 0.1

Phenoxybenzamine Maximally concentrated 0.0 + 0.1 0.0 - 0.2 0.0 Maximally dispersed 0.0 +0.2 +0.1 0.0 +0.1

Propranolol Maximally concentrated 0.0 +0.1 -0.1 -0.1 -0.2 Maximally dispersed 0.0 +0.2 -0.1 0.0 +0.1

SK&F 92657 Maximally concentrated 0.0 +0.3 -0.1 +0.1 0.0 Maximally dispersed 0.0 - 0.2 - 0.2 - 0.1 0.0

+, melanin more dispersed in experimentals than in controls; - , melanin less dispersed in experimentals than in controls.

tripetal migration of the melanin, the white back- ground fostering melanin concentration.

As is evident from Table 3, none of the ~ adreno- ceptor blockers affected melanin migration when injected into isolated legs whether the melanin was initially maximally concentrated or maximally dis- persed. The melanin in isolated legs ultimately assumes an approximately intermediate degree of dis- persion regardless of the initial state.

Effects o f fl adrenoceptor blockers on melanin migration

Propranolol (Ratner et al., 1980) and SK&F 92657 (Taylor et al., 1979) block both fll and f12 adreno- ceptors. When these drugs were injected into crabs whose melanin was initially fully aggregated, and that were kept on a white background throughout the ex- periment, no melanin-dispersing response was ob- served (Table 1). Furthermore, when these drugs were injected into crabs with maximally concentrated melanin that were then transferred from a white to a black background both drugs had a slight inhibitory effect on melanin dispersion but the differences between the experimentals and controls were not stat- istically significant (Table 2).

In crabs maintained on a black background throughout the experiment, their melanin having been initially maximally dispersed, propranolol induced a significant degree of melanin concentration (Table 1). SK&F 92657 also produced some melanin concen- tration in crabs kept on a black background through- out the experiment, but this response was not statisti- cally significant. Enhancement in the rate of melanin concentration was observed following the administra- tion of both propranolol and SK&F 92657 into crabs that were shifted from a black to a white background at the time of drug injection and whose melanin was initially fully dispersed (Table 2). However, only with the SK&F 92657 was this response statistically signifi- cant. Like the cta blockers, propranolol and SK&F 92657 also did not directly affect melanin migration when tested on isolated legs (Table 3).

D I S C U S S I O N

In light of the established pharmacological actions in mammalian systems of the drugs used herein and

the presence of NE (0.51 ___ 0.08SEMpg/g) (our unpublished data) in supraesophageal ganglia re- moved from male fiddler crabs between noon and 1300 hr, the sex and species being the same as used in the present investigation, the data presented above strongly support our hypothesis that NE serves as a neurotransmitter in Uca pugilator to trigger release of MDH (Hanumante et al., 1980; Fingerman et al., 1981; Hanumante et al., 1981; Hanumante & Finger- man, 1981). The data presented herein are consistent with the evidence of Hanumante & Fingerman (1981) that postsynaptic ~1 adrenoceptors are involved in this MDH release-stimulating action of NE in Uca pugilator.

The lack of any melanin-dispersing response to indoramin and azapetine in the crabs kept through- out the experiment on a white background where their melanin was fully aggregated initially (Table 1) is consistent with the ability of a white background to foster melanin concentration and the blocking action of these drugs on ~1 (postsynaptic) adrenoceptors (Starke & Endo, 1976; Doxey et al., 1977; Waterbury & Dean, 1980).

Phenoxybenzamine significantly reduced the mela- nin dispersion that was produced in the crabs kept on a white background throughout the experiment by the 5% ethylene glycol + 5% dextrose vehicle (Table 1), presumably by blocking postjunctional cq adreno- ceptors. Such a blockade would probably result in decreased release of NE-triggered MDH into the hemolymph than in the controls receiving the vehicle in which the phenoxybenzamine was dissolved. The vehicle itself must have evoked MDH release through an unknown mechanism, because the vehicle had no effect on melanin migration in isolated legs (Table 3).

In the crabs that underwent a background change from white to black all three cq adrenoceptor blockers significantly inhibited the melanin dispersion fostered by the black background (Table 2). However, none of them, like prazosin (Hanumante & Finger- man, 1981) which is another highly selective ct 1 blocker (Cavero & Roach, 1980), completely blocked the melanin dispersion in these crabs that had been transferred to the black background. At least there are three possible explanations for this observed inabi- lity of these cq adrenoceptor blockers to inhibit corn-

c.B.P. 71 / Ic - -B

18 MUKUND M. HANUMANTE and MILTON FINGERMAN

pletely melanin dispersion in crabs which underwent a background change from white to black. One, instead of NE triggering MDH release by activating ~1 adrenoceptors exclusively, in crabs transferred from a white to a black background NE also triggers release of some MDH by activating another type of postsynaptic adrenoceptor. At least in mammals, in addition to ct 1 adrenoceptors, ct 2 and fl adrenoceptors may also be involved in the postjunctional effects of NE neurotransmission (Berthelsen & Pettinger, 1977). The second possibility is that only ct 1 adrenoceptors are involved in mediating NE-induced release of MDH and the injected quantity (20#g/crab) of each drug was insufficient to block completely all of the postsynaptic ~1 adrenoceptors. Third, NE does not trigger the release of all the available MDH, another as yet unidentified neurotransmitter also triggering MDH release independently of NE.

The fact that ct 1 and fl adrenoceptor blocking drugs produced, at least at some time during the experi- ment, some melanin concentration in the crabs kept on the black background (Table 1) is consistent with the hypothesis that adrenoceptors mediate MDH release, insufficient MDH having been released to maintain the melanin fully dispersed. However, the fact that among the crabs transferred from the black to the white background (Table 2) the amount of melanin concentration, as compared with the corre- sponding controls, was greater among those that received the fl blockers, particularly the SK&F 92657, unlike the crabs injected with cq-blocking drugs, remains for future experiments to explain. Perhaps the ~t and fl adrenoceptors in the fiddler crab undergo reciprocal changes in reactivity such as are known for a variety of vertebrate hormones and neurotrans- mitters. They modulate their expression through modification of the reactivity of their cellular recep- tors (Kahn, 1976; Tohmeh & Cryer, 1980). In particu- lar, there are many examples from vertebrates of re- ciprocal changes in end-organ responses of catechol- amines from an ct receptor mechanism to a fl type, and vice versa, in about 30 min, (Kunos, 1980). In crabs maintained on a black background for at least 24 hr prior to use in an experiment, such as those that were either kept on a black background throughout the experiment or were transferred from the black to the white background, MDH release would certainly be occurring at a greater rate during this acclimation period because the black background fosters melanin dispersion than in crabs kept on a white background. Presumably the greater role of MDH secretion is achieved by the higher NE output during this accli- mation period which in turn induces at least a partial change in the reactivity of the Ctl adrenoceptors to the fl type. Such a change in reactivity would not only account for the relative responses of the crabs trans- ferred from the black to the white background but also for the lack of a significant response to indor- amin in the crabs kept on the black background throughout the experiment (Table 1). The small but significant amount of melanin aggregation that occurred in the crabs that received azapetine or phe- noxybenzamine and were maintained on a black background throughout the experiment (Table 1) is presumably because of the occupation by azapetine and phenoxybenzamine of some of the cq adreno-

ceptors which did not undergo a change in their reac- tivity and which implement the MDH-releasing effect of NE, NE release being presumably continuously fostered by a black background. A partial change of reactivity of the ~tl adrenoceptors would account for the significant melanin concentration that occurred in the crabs that were kept on a black background and which received the propranolol as well as the mela- nin-concentrating response produced by the SK&F 92657 which, although not statistically significant was larger than that produced by indoramin.

Although, at least in vertebrates, ct and fl adreno- ceptors usually mediate antagonistic physiological effects (Kordon et al., 1980), in some tissues their acti- vation can lead to the same biological end response (Kunos, 1980). Propranolol and SK&F 92657, in keeping with their abilities in mammals to block both fll and f12 adrenoceptors (Taylor et al., 1979; Dreyer & Offermeier, 1980; Ratner et al., 1980), would presum- ably occupy both presynaptically and postsynapti- cally situated beta adrenoceptors, if present. Presyn- aptic fl adrenoceptors, at least in mammals, modulate a positive feedback mechanism (see Westfall, 1980) of action potential-induced NE release; agonists of pre- synaptic beta adrenoceptors increasing and antagon- ists producing a decrease in NE secretion. Similarly, the blockade of postjunctional beta adrenoceptors prevents NE from executing its end-organ effect. If in the fiddler crab presynaptic and/or postsynaptic beta adrenoceptors are involved in MDH release, propra- nolol and SK&F 92657 should have some inhibitory effect on melanin dispersion. But, since neither pro- pranolol nor SK&F 92657 produced a statistically significant amount of inhibition of melanin dispersion in the crabs transferred from the white to the black background, in contrast to the ~tl adrenoceptor an- tagonists (Table 2 and Hanumante & Fingerman, 1981), it appears, therefore, that NE executes its MDH release-triggering effect mainly by activating ~1 adrenoceptors. Also consistent with the interpretation that ~l adrenoceptors are the dominant ones in NE- mediated MDH release is the observation of Finger- man et al. (1981) that isoproterenol, the classic fl ad- renoceptor agonist, was much less effective in promot- ing melanin dispersion in crabs on a white back- ground than was NE. From the comparative view- point it is interesting that in the fiddler crab ~q ad- renoceptors have at least the dominant role in trigger- ing MDH release whereas in the pituitary gland of the rat f12 adrenoceptors play an important role in regu- lating the release of what is in ectothermic vertebrates a color change hormone, ct-melanocyte-stimulating hormone (Cote et al., 1980).

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