Pharmacological evidence that alpha1.-adrenoceptors mediate metamorphosis of the pacific oyster, Crassostrea gigas

Abstract-Qyster larvae ~x?nbe inducc3dto metamorphose by exposure to the natural vertebmte ;adrenergic agonists, epiaephrine and norepi~~~~ne. The larval raEeptors mediating this induction were @arma* ~olo~~I]y char~~ct~rized by testing the ability of a v&%y of adrenergic ago&s and sefected structural analogs of epinephrine and norepinephrine to induce oyster metamorphosis, and by testing the ability of various adreaergic antagonists to black the induction of metamorphosis by epinephrine. oyster metamo~hos~s can be induced by vertebrate adrenergic agonists with relative potencies; drazoline r epithet > ~be~y~~~~e 3 no~~~~h~~ 3 atpha-metbyipb~~ > ~~prot~~o~ % me&= oxamin~ = ~~o~d~~e=C&her st~~~~ am4ogs of ~~~~b~ and no~~~~~ne~ in&ding dodgy and octupamine, were ineffective at inducing m~amo~~~s~s. fnduction 5fm~t~morptrosis by epinephrine can be blocked by vertebrate adrenergic antagonists with relative potencies: chlorpromazine Ir: prazosin > pbentolamine > WB4101 z propranolol z yohimbine :, metopralol, These data demonstrate that mceptcrrs similar to vurtebrate-type alpha,-adrenoceptors mediate oyster metamorphosis. This is the first evidence for a~p~~-ad~~~~to~ in mollusczl, and provides an ~~~~aut due to the cnntrof of the complex process ofm~~~ m~amor~ho~s and to the evob~tion of vertebra& adrenergic receptors.

~~~~~~~~sjs of many marine invert&rates involves a dramatic change from a swimming larval stage to 8 ~ttom*dwelling juvenile stage. Metamorphosis Is often a comp$ex process ~n~Iv~~g h&o&is of ~e~~~t~ve Iarvi t&Sues, histogen&s of transitory larval tissues and proliferation of adult anlagen, as well zcsmany biochr;mical and physiological changes, The mechanisms which control these many coneomitaut &anges are poor& ~nd~~t~. Rsx?&~ C&& ef db have shown that larvae of the P&Sc oyster3 Crmwmea gigas+ cm be induced to rn~t~o~hos~ by a brief exposure to the natural vertebrate a~~~~r~c agonists, ~pinep~~ne (EPX) and norepinephrine {NE). More than 50% of the larvae were induced to metamo~ho~ by exposures to 1W4 M EPI for f0min. ~etamo~hos~s in oysters is rapid+ with the major rn~o~~~~ &arxges, ~~~ud~ng ioss of major larval tissues, proliferation of the adult gill and secretion .of a new post-metamorphic shell structure, occurring within 24 h.6 Metamorphosis is norm Enally preceded by, and dependent upon, eompfex ~~~o~~ changes and jr~~~~ble argument to a substrate. Howeve;r, ~~neph~uaand norepinephrine&duced met;amorphoses are indmndent of these bhavioral changes and attachma~t~ the larvae, upon exposure to ~~~~eph~~~ or ~a~pineph~ne, sink to the bottom and metamorphose without attaching*6 The ability to separate m~~mo~h~s~s From these normai prerequisites provides a unique oppor*To whom correspondence should be addressed, Abbreviatiam: DA, dopamine; EPI, epinq?hrine; NE, aompinephrine.

tunity to study the m~han~sms which controI metamorphosis. The putative receptors rn~~at~~~ the ind~~on of ~amo~~o~s are sefective f&r &a ~t~~~y oceardng adrentxgie neuroactive compcsunds, EPI anii NE. A variety of other naturally occurring neuroactive substances, including dopamine, octopamine, scrotonin, ga~~~amiuobutyrate and a~ty~~hoI~n~~ do not induce m~tamo~ho~s.~ The specS&y of the receptors has led us to isolate the presezxe of vertebrate-type adrenoceptors in the oyster larvae. In the present study we have characterized the receptors on the basis of their ste~~h~~~~ specificity and their responses to known vertebrate adrenergic agonists and antagonists.

Larvae of the Pacific oyster, Cra.rsos~~~ gigs, which were competent to metamorphose were obtained and maintained 8s previously described*’ The larvae wera shipped by air &eilghP from the Coast Dyster Hatchery of QtikXm% Wasb~~to~~ and upan arrival were rn~ut~n~ in ftltered natural sea-water at 23°C on a mixed gigal diet. Experiments were conduits l-7 days after arrival Qf the iarvae. Experiments were $ouducted at 23°C in plastic tissm3 culture plates (24-well; Falcon No. 3047) using artificial (Marine Biological Laboratory) sea-watar.3 In each experimental repI&ate, 25-IQ0 Iarvae we= placed in a total volume of 1.5 mz of test so&ion. ~~~~~a~ experiments sEaowedthat resuits wem Bat affected by xarvaf de&&s up to 150 larvae/ml. Stock solutions were prepared in either &lass distilled water, or O.OOSN HCl, and diluted to 10 x in distilled water immediately before use. Stock solutions were added to Marine Biolo#Xd Laboratory sea-water contain-

1170

S. L. CORN and D. B.

B~NAR

Table 1. Relative ability of structural analogs of epinephrine and norepinephrine metamorphosis of oyster larvae

to induce

R2

0 +yi R3

RI 6

R4

Compound* Dihydroxyphenyiaianine Dopamine Norepinephrine Epinephrine Phcnyiephrine Isoproterenoi a-Methyinorepinephrine Epinine DL-Metanephrine DL-Normetanephrine Phenethyiamine Phenylethanoiamine Tyramine DL-Octopamine DL-Synephrine

R, -OH -OH -OH -OH -H 4H -OH --OH -OCH, -0CH, -H -llb”H --OH -OH

R, -OH --OH -OH --OH -OH --OH --OH -OH -OH -OH -H -H -H -H -H

R, -H -H -OH -OH -OH -OH -OH -H -OH -OH -H -OH -H -OH -OH

R, -COOH -H -H -H -H -H -CH, -H -H -H -H -H -H -H -H

RS

R, -H -H -H ---CH,

Relative activity?

--&$A,)* -H --CH, -CH, -H -H -H -H -H ---CH,

f : 0.43 1.00 0.38 0.05 0.07 _ _

*All compounds are L-isomers, unless otherwise specified. tReiative ability of compound to induce metamorphosis of oyster larvae compared to epinephrine. Based on EC, values as given in Table 2. SPresumabiy induce metamorphosis indirectly by inducing prerequisite behavioral changes.6

ing the larvae to achieve the desired final experimental concentration. The resulting slight decreases in pH and salinity did not affect the results. The larvae were not fed during the experimental period to eliminate extraneous factors contributed by the algae. In all experiments, larvae were periodically examined with a dissecting microscope to monitor behavior pattems or transKnt responses to the test solutions. At the end of each experiment, ail w&is were examined to determine the percentage of the total number of larvae which had metamorphosed (called spat). An oyster was categorized as metamorphosed if it had noticeably resorbed its vefum or showed new shell growth. Within 24 h after induction, morphogenesis progressed suiiiciently to pennit a clear distinction between metamorphosed spat and quiescent larvae. Therefore, results were determined after 24-48 h. Ail treatments in each experiment were conducted in triplicate. Results varied quantitatively between experiments and batches of larvae, but were quafitativeiy consistent. The results presented here are representative of experiments conducted on more than 25 batches of larvae. Determination of stereochemical specifkity The stereochemical specificity of the putative receptors was investigated by testing the ability of various structural analogs of EPI and NE to induct me&morPhoais. Structural analogs were chosen to test requimm@&ts for functional group substituents at five key positions as iadicpti in Table 1. In these experiments, larvae were exposed to a range of coBocntnisioMI (lQ-7-tO-3 M) of an&o&&r theduration of the apcrhnant (24-48 h). To normaliz se& ti#erenccs between batches of larvae, relative to )wasused as the control &cc ear&r expsfhesnts had shown this solution rates of in&c& ability of the vert&rate adrenergic agonists to induce

metamorphosis was calculated as EC= values by using a probit transformation of the normalized &ta.‘* Determination of adrenergic agonist potency The selectivity of the putative receptors for ve*ate adrenergic agonists was mined by tcstinp; the &i&y of selected agonists to +tidtree tMt@orp&o&.. Larvae Were exposed to various concentrations oF the agonists-f& the duration of the exparimeRts (24-48 hf. EC% crab& were calculated as desc&ed above. Determination of adrenergic antagonist potency The selectivity of the putative receptors for vertebrate adrenergic antagonists was dstwrminsd by tit&g &t&&&y of selected antagonists to i&M t&e im&%ion of 8*l#IIMsrphosis by Efl~. Larvae were prci%&t&l in var@s carapantrations of the antagonists for 15 dth prior to * of EPI (1O-4 M finai concentration). Larvae were &&Med in the antagonist p&s East s&&on for 1h, than I+&&& rinsed and placed in fresh MBL sea-water, A 1h rcxp48sne to io-*M EPI was used b@&lse it had shown to induce conaiatentiy m metamorphose,6 w@e mini&i&n@

tranjFo~tion

of-the ti&&e&data.‘*

Materials

MD). Methoxamjne

HCl and cirazo&e

Alpha,-ad~n~to~

1171

mediate oyster me~mo~hosis

o ClRAZOLINE~a,)

80 -

l EPINEPHRINE (a, d1 0 N~~PINEPHRIN~ (cx , r0f o PHENYLEPHRlNE(~,)

60 -

A a-METHYLNOREPiNEPHRlNE (a,

40 -

A CLONlDlNE (at)

m ISOPROTERENOL (6) 0 METHOXAMINE (a,

1

20 -

O-

m I

I -8

-7

-6 AGONIST

-4

-5

CO~ENTRATION

-3

(LOG MI

Fig. 1. Percentage of larvae induced to metamorphose in response to continuous exposure to increasing concentrations of vertebrate adrenergic agonists. All points represent the mean of triplicate determinations; error bars represent the S.E.M.

Dr IX Kiein (National Institutes of Health, Bethesda, MD) who obtained them from Burrou~s-Wellcome (Research Triangle Park, NC) and Synthelabo (Paris, France), respectively. All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO). RESULTS The

stereochemical specificity of the putative reis systematically demonstrated in Table 1. These data show the receptors to he specific in their

ceptors

requirement for selected functional group substitutions. Epinephrine was the most potent of the structural analogs tested. Removal of the R, hydroxyl group (phenyleph~ne) or the R, methyl group (NE) reduced the activity of the compounds slightly. Alternatively, increasing the size of the functional groups at R, (metanephrine and normetanephrine) or RS (isoproterenol) sharply decreased the inductive activity. The importance of the hydroxyl group at R, is demonstrated by the inability of octopamine and synephrine to induce metamorphosis. Likewise, the lack of effect of epinine demonstrates the importance of the hydroxyl group at R,. A preference for a hydrogen at R4 is suggested by the decrease in activity of alpha-methylnorep~neph~ne relative to norepinephrine. This stereochemical specificity is indicative of vertebrate a1pha,-adrenoceptors.23 Figure 1 shows the ability of selected vertebrate adrenergic agonists to induce metamorphosis of oyster larvae as a function of agonist concentration. Table 2 lists the demonstrated vertebrate adrenergic selectivity of each compound along with their ECm values based on the data shown in Fig. 1. Cirazoline was the most effective agonist, being about 40 times more potent than EPI. Norepinephrine and phenylephrine were about half as potent as EPI. Alpha-methylnorepinephrine and isoproterenol

were 15-20-fold less potent than EPI, and clonidine and methoxamine showed no activity at any concentration. These data, with the exception of methoxamine, also indicate that vertebrate-type alpha,-adrenoceptors mediate metamorphosis.2,23,26 Methoxamine, while considered to be selective for alpha,-adren~pto~, has been shown to exhibit only low, or partial, agonist activity at alpha,-adrenoceptors in several vertebrate preparations.‘6.24*26Thus the lack of effect of methoxamine in this study is not considered problematic for the demonstration of alpha,-adrenoceptors in iight of the other evidence. Figure 2 shows the ability of selected vertebrate antagonists to block the induction of metamorphosis by EPI as a function of antagonist concentration. Table 3 lists the demonstrated vertebrate adrenergic selectivity of each compound along with their ICY values based on the data shown in Fig. 2. Chlorpromazine and prazosin were approximately equipotent as antagonists of EPI induction. Phentolamine and WB4101 were about 5 and 10 times less effective than prazosin, respectively. Propranolol and

Table 2. Ability of adrenergic agonists to induce metamorphosis in oyster larvae Agonist

Selectivity

Cirazoline Epinephrine Phenytephrine Nompin~h~ne Alpha-methylnorepineph~ne Isoproterenol Methoxamine Clonidine

alpha, alpha, beta alpha, alpha, beta beta beta alpha, alpha,

*No activity at any concentration

tested.

EC&M) 0.24 ; 26 140 206 NA’ NA*

S. L. COONand D. B. BONAR

1172

Fig. 2. Percentage of larvae induced to metamorphose by a l-h exposure to lo-’ M epinephrine (EPI) in the presence of increasing concentrations of selected vertebrate adrenergic antagonists. All points represent the mean of triplicate

determinations; error bars represent the S.E.M. (A) Compounds which were relatively ineffective antagonists of

EPI induction. (B) Compounds which were relatively potent antagonists of EPI induction. None of the antagonists induced metamorphosis

in the absence of WI.

yohimbine were more than lOO-fold less effective than prazosin while metoprolol demonstrated little antagonist activity at any concentration. These data probably overestimate much of propranulol’s antagonist activity since exposure to high concentrations of propranolol caused many of the larvae to withdraw tightly within their shells, thereby severely limiting their exposure to EPI. This was not observed for any other antagonist tested. These data are consistent with vertebrate-type alpha,-adrenoceptor mediation of EPI-induced metamorphosis.2*” Also of interest is the enhancement of EPI-induced metamorphosis at low concentrations of WMIOl, although the significana of this phenomenon is unknown. None of the antagonists, including WB4101, demonstrated any agonist activity in the absence of EPI (data not shown). DI!XXJSSION

The results of this investigation provide pharmacological evidence that vertebrate-type alpha,adrenoceptors mediate the induction of metamorphosis in the oyster, C. gigas. This is the lint evidena for alpha,-adrenoceptors in a mollusc. Usually dopamine (DA) and octopamine are considered as the primary catecholaminergic neuroactive compounds in the molluscs. I9 In most instances where

receptors with alpha-adrenergic properties have been described these receptors were primarily either dopaminergic or octopaminergic.‘a~2’~2nReceptors similar to vertebrate D, and D, dopaminergic receptors have been demonstrated to control the release of growth hormone in Lymnaea but NE was ineffective at these receptors. 25 Glaizner’,” identified a neuron in the snail, Helix, that responded to NE but not to DA. However, this response could be blocked by both alpha- and beta-adrenergic antagonists. The finding that DA, octopamine, and epinine (a molluscan DA agonist) did not directly induce metamorphosis in oysters indicates the receptors described herein are distinct from DA and octopamine receptors.’ ‘I Phenethylamine, phenylethanolamine and tyramine are also considered putative neuroactive compounds in molluscs,17,27 but they have no effect on oyster metamorphosis. Therefore, the putative alpha,adrenoceptors studied here are different from any known molluscan receptor. Evidence for alpha,-adrenoceptors in oyster larvae suggests that EPI or NE are functional in vivo in mediating metamorphosis. Norcpinephrine is normally found in molluscs in relatively small amounts and, with the exception of a study by Osborne,” EPI is considered to be absent from the mo11uscs.‘7~19~29 We have found NE in C. gigus larvae which are competent to metamorphose, although the presence of EPI is still equivocal.’ The presence of NE in these larvae along with putative alpha,-adrenoceptors constitute the first evidence of a functional role for NE in a mollusc. Receptors with alpha,-adrenergic properties have been reported for only one other invertebrate.” Melanin-dispersing hormone which, along with melanin-concentrating hormone, controls the melanin translocation within the melanophores of the fiddler crab, Uca pugilaator, is released by the action of NE on receptors identified pharmacologically as alpha,-adrenoceptors.“*” The presence of alpha,-adrenoceptors in both the arthropods and the molluscs may indicate that these receptors appeared early in evolutionary history and may be present in other invertebrate phyla despite the generally low levels of EPI and NE in invertebrates.‘7*‘9

Table 3. Ability of adrenergic antagonists to block the induction of metamorphosis by eoinenhrine . Antagonist Chtorpromazine Prazosin Phentolamine wB4101 Propranolol Yohimbine Metoprolol

Wectivity

~c,(tiM)

alpha, alpha, alpha alpha, beta atvha, beta -

0.69 0.86 4.5 7.1 862 93 NAt

_I..

*Apparent value; see text for explanation. tNo activity at any concentration tested.

Alpha,-adren~ptom

mediate oyster me~mo~hosis

It is not (yet) clear whether the putative alpha,adrenoceptors are directly or indirectly involved in the control of metamorphosis of C. gigas at the tissue level. That is, the receptors could mediate the release of a factor, which is actually active at the tissue level, from some secretory or neurohumoral site (centralized receptor theory). In this case, the receptors might be located on a limited number of cells,

analogous to control of neurosecretory release of growth hormone in Ly~~uea.***~ Alternatively, the receptors could be located on the target tissues and receive the adrenergic stimulus (NE) which would be released as a hormone into the circulatory system of the larva (peripheral receptor theory). Whether the receptors are located centrally or peripherally, they are not restricted to hormonal control, but could also mediate metamorphic control acting through direct neural stimulation. At present there is ins~cient evidence to conclude the relative contributions of

1173

hormonal, neural or other mechanisms to the control of metamorphosis in any mollusc.‘*4 Whatever the mechanism of metamorphosis in oysters, it must

account for the multiplicity of changes which occur, including histolysis, prolif~ation, bistogenesis and biochemical and physiological changes. Norepinephrine is present and sufficient to initiate this sequence of events and alpha,-adrenoceptors are present which mediate this process. Further research is required to more fully characterize the putative alpha,-adren~pto~ and to assess the actual role of the receptors and the nervous system in mediating metamorphosis. Acknowledgements-The

authors wish to thank the Coast

Oyster Hatchery of Quilcene. Washington, for providing us with quality larvae. This work was supported by the National Science Foundation grant No. PCM 831678 and the University of Maryland Sea Grant No. NOAA-NA83lOAA-D-00040.

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S. L. CWN and~D.

B. BONAR

25. Stoof J. C., Vlieger T. A. de and Lodder J. C. (1984) Opposing roles for D-i and D-2 dopamine receptors in regulating the excitability of growth hormone-producing cells in the snail Lymnaea sfagnalis. Eur. J. Phurmac. 106, 431-435. P. B. M. W. M. and Zwieten P. A. van (1981) Selectivity of some alpha 26. Van Meel J: C. A., Jonge A. de, Timmermans adrenoceptor agonists for peripheral alpha-l and alpha-2 adrenoceptorsin the normotensive rat. J. Pharmac. e.xp. 7’her. 219, 760-767. noradrenaline, dopamine, octopamine. tymmine, 27. Walker R. J. and Kerkut G. A. (1978) The first family (adrenaline, phenylethanolamine and phenylethylamine). Camp. Biochem. Physiol. 61C, 261-266. G. N., Glaizner B., Sedden C. B. and Kerkut G. A. (1968) The pharmacology of Helik 28. Walker R. J., Woodruff dopamine receptor of specific neurones in the snail, Helix uspersu. Comp. Biochem. Physiol. 24, 455-469. in the invertebrates. In Cutecholumines (eds Blaschko H. and Muscholl E.). pp. 29. Welsh J. H. (1972) Catecholamines 79-109. Springer, New York. (Accepted

23 February

1987)