ASSTRACT
Cholinergic receptors controlling iridescence in the iridophores
of Loligo opalescens were found to be muscarinic. Selective agonists and
antagonists were applied, and responsiveness was measured by a change
in iridescence. Carbamulcholine and acetyl-beta-methgl-choline (ABHE)
are group A muscarinic agonists that elicited positive responses.
Effective group B agonists included carbamul-beta-methyl-choline
(CBMC) and oxotremorine. Atropine, scopolamine, and pirenzipine are
muscarinic antagonists that effectively hindered iridescence, while
tubocurarine, a nicotinic antagonist, did not block iridescence. Agonist
groupings are based on their ability to turn over phoshatidglinosito
(Phl). The results of these agonistsfantagonists combined with the
effect of Li and subsequent addition of myo-inositol, show that Phi
turnover mag be involved. Li' selectively inhibits a step in the Phl
pathwau. Lif was found to decrease iridescence and addition of
muo-inositol seemed to cause its reappearance. Ca seems to be
involved, since addition of an agonist in zero Ca' sea water did not
result in iridescence. Addition of forskolin and caffeine resulted in a
decrease in iridescence, impluing that high cAMP levels inhibit the
response. From this data four receptor models were constructed.
INTEODUCTION
Iridophores, specialized cells in the skin of squid produce
structural iridescence bu either diffraction or thin film interference
mechaniems (Schäfer, 1938, Brocco and Cloneg, 1980). Two types of
iridophore cells are recognized: iridocutes, cells that apparently
produce structural colors in a manner similar to a diffraction grating;
and reflector cells, which produce structural colors via thin-film
interference (Brocco and Cloneg, 1983). In Loliga, reflector cells are
located chiefly above the iris, on the ink sac and other internal organs.
The iridocutes are found in the skin over the whole body of the squid,
and are arranged in donut-shaped splotches in the third dermal lager of
the dorsal mantle. Iridescence of iridocytes seems to be caused by the
organization of iridosomal platelets of high refractive index located on
the periphery of the cell and surrounded by infoldings of the
plasmalemma (Brocco and Cloneg, 1980). These platelets are aligned on
edge, perpendicular to the integument of the squid (Schäfer, 1938,
Kawaguti and Ohgishi, 1962, Hirow, 1972).
Iridocutes differ from reflector cells not only in the mechanism
bu which they produce structural iridescence, but also in their ability to
change their iridescent properties. The neurotransmitter acetulcholine
(AChl causes a change in the strength of iridescence (Cooper and Hanlon,
1966), although no one has get successfully identified the innervation of
the iridocute dermal lager. This study was conducted to characterize the
nature of the receptor responsible for that effect, and the possible
signal transduction mechanisms that mag help mediate the structursl
changes involved in iridescence.
HATERIALS AND METHODS
ACh, atropine, tubocurarine, forskolin, caffeine, LiCl, EGTA,
HEPES and muo-inositol were obtained from Sigma Chemical.
Carbamulcholine, ABMC, CEHE, oxotremorine, pirenzipine,
quinuclidinul benzilate (ONB), and scopolamine were the kind gift of
Anthong Horielli. Loligo opalescens were collected from Montereg Bag
and maintained in round holding tanks 2 m in diameter. Photographs were
taken with Kodschrome 400 and Ektachrome 400 using an Olympus UM¬
Camera mounted to an Olumpus upright microscope. The 10k occular lens
was combined with either a 40x water immersion Zeiss lens, or a lox
American Optical lens, making overall viewing magnification either
400x or 100x. Fiber optic incident lighting was used as the single light
source.
Skin preparations were obtained by excising patches of the
iridophore dermal lauer from the dorsal mantle region and pinning them
in a 3 mm diemeter, Sulgard coated dish. The dish was filled with
artificial ses water (500 mM Nacl, 12 mMMgi, 10 mac, 10 m
KCI, 10 mM HEFES, and enough KOH to bring the pH to 7.8). The
sppropriate drugs were diluted in saline and spplied bu exchanging the
solution in the beth. The zero Ca solution was prepared by substituting
Ng for Ca and adding 2 mM EGTA.
Preparations were placed under the microscope, and a single
iridophore donut was observed. A baseline level of iridescence was
obtained bu adjusting the light to produce a maximum amount of
iridescence (incident light made an angle of approximately 30° with the
horizontal). A photograph was then taken to objectively evaluate this
level of iridescence. Iridescence was judged by three factors: 1) Area:
the number of cells that were iridescent in the donut vs those that were
not, 2) Intensity: intensity of iridescence, and 3) Color: the color of
the light seems to be related to the strength of the response. White
corresponds to completely off, Blues and Greens represent a
semi-iridescent stage, and Reds, Golds, and Oranges indicate a more
fullu iridescent form of the iridophore. The various drugs were added
topicallu to the bath, and changes in levels of iridescence were noted.
Photographs were taken at times of 1, 5, and 10 minutes after addition
of each chemical. Photographs were taken at later times depending on
the response to the drug.
RESULTS
Characterization of the Receptor
Various cholinergic agonists and antagonists were tested at a
final concentration of .1 mM. Agonists were judged to induce iridescence
if application to the bath resulted in an increase in iridescence. Theg
were judged ineffective if addition to the bath caused no change, and
subsequent washing followed by application of ACh resulted in a positive
change in iridescence. Antagonists were considered ineffective if the
combined application of the antagonist and ACh resulted in a change in
iridescence that could not be enhanced by subsequent washing and
application of ACh alone. The antagonists were judged to be successful
blockers if iridescence could be enhanced by washing and addition of ACh.
See Table 1
Cholinergic receptors can be divided into two tupes: muscarinig
and niocotinic. The non-specific cholinergic agonist carbamylcholine
uielded a positive result, as would be expected in either a muscarinic or
nicotinic system. Specific muscarinic agonists, ABMC, CBME, and
oxotremorine, gave positive responses to varging degrees. Host of the
specific muscarinic antagonists that were tested, atropine,
scopolamine, and pirenzipine effectively competed with the agonist and
hindered iridescence. From these results, it appears that a muscarinic
system is present in the iridocytes of Loligo.
Muscarinic agonists have been divided into two groups depending
on their effect on Phl turnover (Fisher et al, 1983). Group A agonists
trigger Phl turnover to a large degree, while Group B agonists trigger it
to a somewhat lesser extent. Oftentimes, the degree of response to the
various muscarinic agonists can indicate whether or not Phi turnover is
involved in the system. Solely on the basis of agonist binding
experiments, the results of this study are too preliminaru to assess the
role of Phl turnover in iridescence. Oxotremorine, a Group B agonist, did
elicit a less dramatic response than ang of the Group A agonists that
were applied, but CBMC, also a Group B agonist, gave just as strong a
response. Other methods of determining Phl involvement were required.
Effect of Li', myo-inositol
The cation, Li', provides one of the few ways to selectivelu
inhibit the cycle of Phl turnover, thereby providing a method for testing
the role of Phl involvement (Drummond et al, 1986). LiCl (lOmM) was
added to a bath containing a semi-iridescent preparation. After 30
minutes, no change in the level of iridescence was observed. After 45
minutes, iridescence began to fade, and after an hour, there was a
substantial decrease in iridescence. 10 m myo-inositol was then added
to the bath, and iridescence returned within 30 seconds to a minute.
This procedure was repeated twice. The first repeat showed no fading of
virtually full iridescence after an hour, and addition of myo-inositol did
not increase it. The second repeat resulted in fading of a
semi-iridescent preparation after 30 minutes, but addition of
myo-inositol did not have ang effect.
Effect of 2ero Ca Ses Water
There is some evidence that Ca' is required for iridescence
(Cooper and Hanlon, 1986), and muscarin has been shown to affect Ca'
fluxes in some sustems (Parod et al, 1980). After bathing the iridophore
in zero Ca' sea water for 50 minutes, addition of ACh produced no
change in iridescence. The prep was then washed with regular artificial
ses water, and subsequent addition of the agonist resulted in a
significant increase in iridescence. This procedure was repeated on a
different preparation and the results were similar, with slightlg less
iridescence after washing and application of ALh.
Effect of Forskolin, Caffeine
Muscarinic receptor mechanisms, in some cases, have been
shown to act by decreasing the sunthesis of cAMP (Birdsall et al, 1980).
Forskolin (.1 mt1) and caffeine were added to a bath containing a
semi-iridescent preparation. After 5 minutes, iridescence decreased
dramaticallu. After 30 minutes, iridescence came back slightly but was
still far from the original level. Addition of carbamylcholine to the bath
at this stage resulted in a sharp increase in iridescence. This procedure
wos repeated twice on different preparations, a fully iridescent
preparation and another semi-iridescent preparation, each time
resulting in a virtually complete loss of iridescence, that was reversed
bu application ofImM carbamglcholine.
DISCUSSION
The iridescence of iridocutes seems to be controlled by a
muscarinic-tupe receptor system that involves Phi turnover, a rise in
intracellular Ca' levels, and a decrease in cAMP levels. The responses
to a number of muscarinic agonists and the block of the response by
Specific muscarinic antagonists indicates that a muscarinic receptor is
involved. Muscarinic receptors have been proposed to exist in two
subtipes, MI and M2, each able to express more than one conformational
state (Fisher and Bartus, 1985). pirenzipine specificallu blocks the Mi
subtupe (Fisher and Bartus, 1985), and on the basis of this result, it
seems that iridocytes possess at least the MI tupe of muscarinic
receptor.
The distinction between the two muscarinic receptor subtupes
based on the effects on Phl turnover is still vaque (Fisher and Bartus.
1985). Comparisons of the effectiveness of Group A and B agonists also
gield inconclusive evidence whether Phl turnover is involved in the
induction of iridescence. A role for Phl turnover is suggested in
iridescence on the basis of the experiment involving Li and
myo-inositol. Addition of Li' to cells has been shown to induce a sharp
decrease in myo-inositol concentrations, while causing a drastic
increase in myo-inositol 1-phosphate levels, apparently by inhibiting
the enzyme myo-inositol 1-phosphatase (Allison et al, 1976). Ph
breskdown products that are known secondarg messengers, IP- and DAG
occur well before the myo-inositol 1-phosphatase step in the pathwad,
and Li' action has been hupothesized to occur by depleting the Fhi pool of
mgo-inositol, therebg rendering it unable to regenerate Phl, and placing
the turnover mechaniem into check (Berridge et al, 1982). The cell can
either absort myo-inositol through the plasma mernbrane or sunthesize
it from glucose (Drummond et al, 1986). The fading of iridescence upon
addition of small concentrations of Li' can be correlated with a
depletion of myo-inositol from the Phl pool, since addition of
myo-inositol seems to restore iridescence, at least to some degree, The
preparation that did not fade after an hour perhaps had an inordinatelu
high Phl pool that was not get depleted.
The Fhl breakdown product, IFz, may act by releasing Cat from
intracellular stores. Roger Hanlon and Kag Cooper have been
investigating the role of Ca“ in iridocyte iridescence of L. Brevis.
Addition of the Ca ionophore 423187 to a bath solution containing Ca"
caused an increase in iridescence. Verapamil, a Ca channel blocker,
inhibited agonist-induced iridescence to a great degree. Addition of the
agonist ACh to a zero Ca bath solution resulted in a brief increase in
iridescence that could not be maintained (Hanlon and Cooper, 1986). The
zero Ca sea water experiment executed in this study implied that Ca"
was required in the external medium to induce iridescence. However.
the 50 minute bathing period might be ample time for intracellular
stores to become depleted; therefore, Hanlon's experiment with zerg
Car sea water mag indeed support the theory of releasing Ca from
intracellular stores. Agonist binding resulted in a brief increase in
iridescence, perhaps associated with this intracellular Ca release.
Internal Ca pumps would then have removed Ca' from the cell where
it would be chelated in the external medium.
cAMP levels may also have an important modulating effect on the
iridescence of squid iridocutes. Huscarinic agonist binding has been
shown, in some cases, to decrease sunthesis of CAMP (Birdsall et al,
1983). The results of the forskolin and caffeine experiment may indicate
that cAMP levels cannot be too high if the iridocute is to maintain
iridescence. This does not necessarily imply that agonist binding in
iridocytes decresses cAMP levels, but it does seem to show that high
levels of intracellular CAMF produce some kind of inhibitorg effect on
iridescence. In general, muscarinic agonists affect cAMP levels by
linking to the G, protein that inhibits adenglate cyclase, therebg
decressing sunthesis of CAMP. A useful follow up experiment would then
be to test the effect of cholers and pertussis tokins on iridocyte
iridescence, since theg are known to affect cAMP sunthesis by acting on
the G protein.
Consideration of these aspects of intracellular mediation of the
receptor signal, has led to four receptor models that attempt to link the
receptor to Phl turnover and to inhibition of cAMP sunthesis. (See
Figures 1 to 4.) Theg assume that the Car rise is under control of IPz,
one of the products in the Phl cycle.
EECEPTOR MODEL 1: The receptor is linked directlg to
Phospholipase C, the enzyme involved in the initial stages of Fhl
turnover. Activation of this enzyme sets this pathwag into motion. The
products of Fhl turnover then either stimulate the G; protein to inhibit
adenglate cuclase and decrease overall cAMP synthesis, or theg inhibit
adenglate cyclase directly. IP causes Ca- release to turn on
iridescence.
EECEETOP MODEL 2: Two receptors are used: one directig linked
to Fhospholipase C, and the other to the G, protein. The two receptors
may have different binding affinities, affecting each process to varging
degrees. There is some evidence that muscarinic receptors mag each
interconvert to high and low affinity forms, adding another aspect to
requlation and allowing more flexibilty in the system.
FECEPTOR HODEL3 There is one receptor directlg linked to both
Phospholipase C and the G;. Agonist binding would cause a joint effect,
triggering both aspects of the system at once.
PECEPTOR HODEL 4: Both Fhospholipsse C and the G, protein are
under control of a single receptor, but the receptor is linked to only one
of them at ang given time. The receptor interconverts between high and
low affinitu forms and floats in the membrane of the cell, showing
preference for one integral membrane protein or the other depending on
the receptor's state.
These models deal only with the earliest parts of the signal. It
seems likelu that the external signal is transduced into an enzymatic
process. The muscarinic signal mag utilize cyclic quanine phosphate
products to either inhibit cAMP production or stimulate a specific
enzume. DAG, one of the products of Phl turnover, effects change
through protein kinase C, allowing for phosphorglation of various
enzumes, and switching them to an active state. Ca effects usuallg
occur through some sort of Ca* binding protein, like calmodulin. No
one has conclusively proven how the external signal elicits an
ultrastructural change in the iridosomal platelets. Hicrotubule and
microfilament mechaniems have been proposed, but further
experimentation is needed.
In conclusion, muscarinic receptors are involved in the
iridescence of iridocytes, and they appear to act through the cycle of Phl
turnover, rise in internal Ca' levels, and by hindering the synthesis of
CAMP.
would like to thank Stuart Thompson for his support and
quidance, Anthonu Morielli for his time and drugs in the characterization
of the receptor, Chris Patton for his technical expertise, Bruce Hopkins
for providing all the squid I could possibly kill, and mang hearty thanks
to Roger Hanlon for his helpful advice and the use of his unpublished data.
Lastlu, I would like to thank the entire class of Sio 175H for one helluva
good time.
REFERENCES
Allison, J. H., Blisner, M. E., Holland, W. H., Hipps, P. P. and W. R.
Sherman. 1976. Biachem. Biophus. Res. Commun 71,
664-670.
Berridge, M.J., Downes, C. P. and M. R. Hanleg. 1982. Biachem
206, 587-595
Birdsall, M. J.M., Berrie, C.P., Burgen A. S. V., and E. C. Hulme. 1980. in
Feceptors for Neuratrensmitters and Feptide Hormenes
(Pepeu, G., Kuhar, M. J., and J. S. Enna, eds.), pp. 107-116, Raven
Press, Hew Tork.
Brocco, S.L. and R. A. Cloneg. 1980. Reflector Cells in the skin of
Octopus dofleim. Cell Tissue Res. 205: 167-136.
Cloneu, R. A. and S. L. Brocco. 1983. Chromatophore organs, reflector
cells, iridocutes and leucophores in Cephalopods. Amer. 2ool.
23. 581-592.
K. H. and R.T. Hanlon. 1986. Correlation of iridescence with
Cooper
changes in iridophore platelet ultrastructure in the squid
Lolliguncula brevis. J Exg. Bicl Commin.
Drummond, A. H., Joels, L. A. and P. H. Hughes. 1986. The interaction of
lithium ions with inositol lipid signalling sustems. Biochem.
Soc. Transact. 15:32-35.
Fisher, S. K., Klinger, P. D and B. W. Agranoff. 1983. Muscarinic agonist
binding and phosholipid turnover in brain. J. Biol. Chem. 258,
7350-7363
Fisher, S. K. and R. T. Bartus. 1985. Regional differences in the coupling
of muscarinic receptors to inositol phospholipid hudrolisis in
quinea pig brain. J. Neurochem. 45, 1085-1095.
Hanlon, R. T. 1982. The functional organization of chromatophores and
iridescent cells in the bodg patterning in the body patterning of
Loligo Flei (Cephalopoda. Huopsida). Halacologia 23(1:
S9-119.
Kawaguti, S. and S. Ohgishi. 1962. Electron microscopic study of the
iridophores of a cuddlefish, Segia esculente. Biol. J. Okamaga
University 8: 115-129.
Mirow, S. 1972. Skin color in the squids Laliga Fealiiand Laliga
Cralescens. II. Iridophores. 2. Zellforsch. 125: 176-190.
Parod, R. J., Leslie, B. A. and J. W. Putneg. 1980. Am d Physiol 239,
699-105
Schäfer, W. 1938. Bau Entwicklung und Farbenentstehung bei den
Flitterzellen von Sepis officinslis. 2. Zellforsch. 27:

222-245.
EIGURE LEGEND
Eigure 1: Single link model. Single receptor linked to
Phospholipase C. Phospholipase C activation indirectly decreases cAMP
levels.
Eigure 2: Dual receptor model. Two receptors, each linked to
either Phosholipase Cor the G, inhibitorg protein.
Eigure 3: Double link model. Single receptor directlg linked to
both Phosholipase C and the G; inhibitorg protein.
Eigure 4: Floating receptor model. Single receptor links to
either Fhosholipase C or the G, inhibitorg protein, depending on the
receptors conformational state.
Plate 1: Lateral view of an iridophore donut in the off position.
Some Blues and Greens are visible.
Plate 2: 30 seconds after addition of carbarnglcholine. The donut
shows semi-iridescence. More Blues and Greens are visible.
Elate 3: Five minutes after addition of carbamglcholine. The
donut now shows a ruch greater degree of iridescence. Many more
iridocutes are on, and they show predominantly Reds and Oranges. Some
Blues and Greens are still visible.
ABLE
IRIDESCENCE RESPONSE TO VARIOUS AGONISTS/ANTAGONISTS
IRIDESCENCE EESC
HUSCAPINIC AGONIST
GROUP
***
cetyl Cholne
***
Carbamyl Cholne
***
Acetyl eta ethyl holne (O
Groue 8
***
Carbamyl Beta Methyl holne ()
Oxotremorne
ANTAGONISTS
HUSCARINIC
trone
Scopolamne
nucyl enate (INB)
Pirenzipire
NICCTINIC
fubcurare
* : INDUCED IRIDEECENCE
- : BLOCKED IRIDESCENCE
: NO ESFECT
aost
a aso
RECEPTOR MODEL
AGONIST

——
Gi
PHOSPHOLIPASE C

ADENYLATE CYCLASE
— dire link
- - indire link
IGURE
RECEPTOR MODEL 2
AGONIST
PHOSPHOLIPASE C
FIGURE 2
AGONIST
RECEPTOR MODEL 3
AGONIST

PHOSPHOLIPASE C
Gi
GURE 3
RECEPTOR MOOEL 4
AGONIST


PHOSPHOLIPASEC
P floating link
FIGURE 4