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Goldfish Calmodulin: Molecular Cloning, Tissue
Distribution, and Regulation of Transcript Expression in
Goldfish Pituitary Cells
LONGFEI HUO, ERIC K. Y. LEE, P. C. LEUNG, AND
Department of Zoology, University of Hong Kong, Hong Kong, People’s Republic of China
Calmodulin (CaM) is a Ca2ⴙ-binding protein essential for bi-
ological functions mediated through Ca2ⴙ-dependent mecha-
nisms. In the goldfish, CaM is involved in the signaling events
mediating pituitary hormone secretion induced by hypotha-
lamic factors. However, the structural identity of goldfish
CaM has not been established, and the neuroendocrine mech-
anisms regulating CaM gene expression at the pituitary level
are still unknown. Here we cloned the goldfish CaM and tested
the hypothesis that pituitary expression of CaM transcripts
can be the target of modulation by hypothalamic factors.
Three goldfish CaM cDNAs, namely CaM-a, CaM-bS, and CaM-
bL, were isolated by library screening. These cDNAs carry a
450-bp open reading frame encoding the same 149-amino acid
CaM protein, the amino acid sequence of which is identical
with that of mammals, birds, and amphibians and is highly
homologous (>90%) to that in invertebrates. In goldfish pitu-
C ALMODULIN (CAM), a heat-stable acidic protein ex-
pressed in eukaryotes, serves as a major intracelluar
Ca2⫹ sensor in living cells. The functional roles of CaM in
regulating cell division and differentiation, gene expression,
programmed cell death, DNA replication and repairing, and
exocytosis of hormone/neurotransmitter are well docu-
mented (1–3). The molecular structure of CaM is character-
ized by the presence of four Ca2⫹-binding motifs known as
the helix-loop-helix EF-hands. Three-dimensional structural
analysis has revealed that CaM is dumbbell shaped with two
similar domains located at either end, each with two EF-
hands for Ca2⫹-binding. In the absence of Ca2⫹, these EF-
hands are in a closed conformation. This Ca2⫹-free form of
CaM (or ApoCaM) is not functional but can still bind to a
subset of target proteins, e.g. Nuf1p and Spc110p (4, 5). Upon
Ca2⫹ binding, CaM can shift to an open conformation. As a
result, two hydrophobic surfaces are exposed and allowed
for CaM interactions with Ca2⫹-sensitive target proteins (6,
7). CaM is known to be encoded by members of a multigene
family. At present, three CaM genes (CaM I, II, and III) in
mammals (8 –13) and two CaM genes (CaM I and II) in birds
Abbreviations: a.a., Amino acid; [Ca2⫹]i, intracellular Ca2⫹; CaM,
calmodulin; DIG, digoxigenin; DMSO, dimethylsulfoxide; GTH, gonad-
otropin; LSD, least significance difference; ORF, open reading frame;
PACAP, pituitary adenylate cyclase-activating polypeptide; PKC, pro-
tein kinase C; SDS, sodium dodecyl sulfate; SSC, saline sodium ci-
trate; TPA, 12-O-tetra-decanoyl-phorbol-13-acetate; UTR, untranslated
region.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
5056
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Endocrinology 145(11):5056 –5067
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0584
ANDERSON O. L. WONG
itary cells, activation of cAMP- or PKC-dependent pathways
increased CaM mRNA levels, whereas the opposite was true
for induction of Ca2ⴙ entry. Basal levels of CaM mRNA was
accentuated by GnRH and pituitary adenylate cyclase-acti-
vating polypeptide but suppressed by dopaminergic stimula-
tion. Pharmacological studies using D1 and D2 analogs re-
vealed that dopaminergic inhibition of CaM mRNA expression
was mediated through pituitary D2 receptors. At the pituitary
level, D2 activation was also effective in blocking GnRH- and
pituitary adenylate cyclase-activating polypeptide-stimu-
lated CaM mRNA expression. As a whole, the present study has
confirmed that the molecular structure of CaM is highly con-
served, and its mRNA expression at the pituitary level can be
regulated by interactions among hypothalamic factors. (En-
docrinology 145: 5056 –5067, 2004)
have been reported (14 –16). Although these CaM genes have
variations in their nucleotide sequences, all of them encode
the same CaM protein with identical a.a. sequence. When
compared with CaM molecules reported in lower verte-
brates, like the fish [e.g. electric eel (17)], only one amino acid
(a.a.) substitution in the primary sequence can be noted.
These findings indicate that CaM is highly conserved at the
protein level during vertebrate evolution.
In the goldfish, gonadotropin (GTH), and GH secretion are
regulated by a multitude of neuroendocrine factors (18, 19).
Among these regulators, most of them are hypothalamic
factors that can exert regulatory actions on GTH and GH
release simultaneously. For examples, GnRH and pituitary
adenylate cyclase-activating polypeptide (PACAP) are
known to stimulate GTH-II (or fish LH) and GH release
directly from goldfish pituitary cells (20 –23). Dopamine, on
the contrary, can exert opposite effects on the release of these
two hormones, being stimulatory to GH secretion via pitu-
itary D1 receptors (24, 25) and inhibitory to GTH-II release
through D2 receptors (26). Apparently, the availability of
extracellular Ca2⫹ and its entry through voltage-sensitive
Ca2⫹ channels are essential for both basal and stimulated
GTH-II and GH release in goldfish pituitary cells (27). Using
a pharmacological approach, it has been shown that CaM
antagonists and CaM kinase II inhibitors can block the reg-
ulatory effects on GTH-II and GH release by GnRH, PACAP,
and dopamine (28, 29), suggesting that CaM may be a key
component of the postreceptor signaling mechanisms for
these regulators. Although neuroendocrine regulation of
CaM expression at the pituitary level has not been previously
Huo et al. • CaM mRNA Expression in Pituitary Cells
examined, the findings in the goldfish have prompted us to
speculate that CaM gene expression at the pituitary level may
be the target of modulation by hypophysiotropic factors,
which may have direct consequences on pituitary hormone
synthesis and secretion.
In the present study, the structural identity of goldfish
CaM was established by molecular cloning using the stan-
dard techniques of cDNA library screening. The transcript
expression of CaM in various tissues and brain areas was
examined by Northern blot. Based on the nucleotide se-
quences of CaM cDNAs obtained, a slot blot system was set
up to quantify total CaM mRNA expression in primary cul-
tures of goldfish pituitary cells. Using this assay system, the
effects of hypophysiotropic factors in fish models, including
GnRH, PACAP, and dopamine, on CaM gene expression in
goldfish pituitary cells were investigated using a static in-
cubation approach. In parallel experiments, the effects of
activating cAMP-, protein kinase C (PKC)-, and Ca2⫹-depen-
dent pathways on CaM mRNA expression at the pituitary
level were also examined.
Materials and Methods
Animals
Goldfish (Carassius auratus) with body weight ranging from 35 to 50 g
were purchased from local pet stores and maintained in 200-liter aquaria
at 20 ⫾ 2 C for at least 7 d before pituitary cell preparation. During the
holding period, the fish were fed to satiation daily with commercial fish
feed. After the acclimation, pituitaries were collected for cell dispersion
from goldfish anesthetized in MS222 followed by spinosectomy accord-
ing to the regulations of animal use at the University of Hong Kong.
Reagents and test substances
TRIZOL, medium M199, and horse serum for cell culture were pur-
chased from Gibco Life Technologies (Grand Island, NY). PCR-digoxi-
genin (DIG) labeling kit, CDP-Star, and Anti-DIG-AP (Fab fragments)
were obtained from Roche Molecular Biochemicals (Mannheim, Ger-
many). Salmon GnRH and PACAP-38 were obtained from Peninsula
Laboratories Inc. (Belmont, CA). LY171555, (⫾) SKF38393, apomor-
phine, domperidone, SCH23390, forskolin, and 12-O-tetra-decanoyl-
phorbol-13-acetate (TPA) were purchased from RBI Sigma (St. Louis,
MO). A23187 was obtained from Calbiochem (La Jolla, CA). GnRH and
PACAP were dissolved in double-distilled deionized water to prepare
1-mm stock solutions, aliquoted in small volume, and stored frozen at
⫺80 C. Stock solutions of forskolin, TPA, and A23187 were prepared in
a similar manner except that they were dissolved in dimethylsulfoxide
(DMSO) to give a stock concentration at 10 mm. Ly171555, (⫾) SKF38393,
apomorphine, domperidone, and SCH23390 were freshly prepared on
the day of experiments. These pharmaceutical agents were first dis-
solved in DMSO to give a 10-mm stock solution, which was then diluted
with culture medium to appropriated concentrations 15 min before drug
treatment. The final concentration of DMSO in the medium was less than
0.1% and had no effect on CaM mRNA expression.
Screening of goldfish pituitary cDNA library
A 32P-labeled probe prepared from bovine CaM cDNA was used for
the screening of a goldfish pituitary Zap Express cDNA library accord-
ing to the instructions by the manufacturer (Stratagene, La Jolla, CA).
Briefly, the 32P-labeled probe was allowed to hybridize with nylon
membranes lifted from agar plates with phage colonies of the goldfish
pituitary cDNA library. The areas corresponding to positive signals on
the membranes were identified on the original plates. The plaques in
these areas were cored out, extracted, and spread on agar plates for
secondary and tertiary screening until single colonies were isolated. As
a result of library screening, three positive clones of goldfish CaM,
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Endocrinology, November 2004, 145(11):5056 –5067 5057
namely CaM-a, CaM-bS, and CaM-bL, were isolated. The cDNA inserts
in these clones were sequenced using a BigDye terminator cycle se-
quencing kit (Applied Biosystems, Foster City, CA) in a 310 Genetic
analyzer (PerkinElmer, Boston, MA). Based on the sequences obtained,
a pair of primers (PU1, 5⬘-CAGATATGGCTGACCAACTCAC-3⬘ and
PD1, 5⬘-ACAGAAGAGCTTCACTTTGCCG-3⬘) were designed to cover
a common sequence of CaM-a, CaM-bS, and CaM-bL. After that, a
DIG-labeled cDNA probe that can recognize the mRNA transcripts
corresponding to these CaM clones was prepared for subsequent studies
using a PCR-DIG labeling kit (Roche Diagnostics, Mannheim, Germany).
Northern blot of CaM mRNA
Total RNA was extracted with TRIzol from selected tissues and brain
areas of the goldfish. After that, mRNA was purified from total RNA
using a PolyATract mRNA isolation system III (Promega, Madison, WI).
These mRNA samples were denatured, size fractionated in 1% agarose
gel with 0.22 m formaldehyde, and blotted onto a positively charged
nylon membrane (Roche) using a VacuGene vacuum blotting system
(Pharmacia Biotech, Piscataway, NJ). The membrane was UV cross-
linked using a Stratalinker 2400 (Stratagene), prehybridized for 3 h in
50% formamide-containing hybridization buffer, and incubated with the
DIG-labeled CaM cDNA probe overnight at 42 C. On the following day,
the membrane was washed two times at 68 C in 0.5⫻ saline sodium
citrate (SSC) with 0.1% sodium dodecyl sulfate (SDS) and hybridization
signals were visualized using a DIG luminescent detection kit (Roche)
with CPD-Star as the substrate. Unless stated otherwise, Northern blot
was conducted using the DIG-labeled probe common for CaM-a, CaM-
bS, and CaM-bL. In this study, Northern blot of ␤-actin was used as an
internal control.
Southern blot of genomic DNA
Genomic DNA was isolated from whole blood freshly collected from
the goldfish. Briefly, blood cells were collected from 0.5 ml whole blood
by centrifugation and washed three times with ice-cold PBS. After that,
blood cells was resuspended in 10 ml freshly prepared digestion buffer
[100 mm NaCl, 10 mm Tris-HCl, 25 mm EDTA, 0.5% SDS, and 0.1 mg/ml
proteinase K (pH 8.0)] and incubated at 50 C with gentle shaking for 18 h.
The final solution was extracted two times with equal volume of phenol
(pH 8.0) and one time with chloroform. After that, the genomic DNA in
the aqueous phase was precipitated with equal volume of isopropanol
and 1:3 volume of 3 m sodium acetate (pH 5.2). The DNA pellet was
harvested, soaked in 70% ethanol for 5 h, and dried in a fume hood. After
that, the genomic DNA obtained was dissolved in 1 ml TE buffer and
digested with restriction enzymes including PvuII, HindIII, PstI, and
HincII, respectively. The digested products were size fractionized in a
0.7% agarose gel and transferred onto a positively charged nylon mem-
brane (Roche) by vacuum blotting. After UV cross-linking, the mem-
brane was incubated in 6⫻ SSC with 1⫻ blocking reagent (Roche) for 3 h
followed by hybridization overnight with the DIG-labeled CaM cDNA
probe at 42 C. On the following day, the membrane was washed, and
signal development was carried out as described for Northern blot.
Static incubation of goldfish pituitary cells
Primary cultures of pituitary cells were prepared from the goldfish
as described previously (30). Briefly, pituitaries were excised from gold-
fish and washed three times in washing medium [Medium M199 with
Hanks’ salts, 25 mm HEPES, 26 mm NaHCO3, 100 U/ml penicillin, 100
␮g/ml streptomycin, 250 ␮g/ml Fungizone, and 0.3% BSA (pH 7.2)] to
remove blood clots. After that, pituitaries were diced into 0.5-mm frag-
ments using a McILwain tissue chopper (Mickle Laboratory Engineer-
ing, Gomshall, UK) and exposed to trypsin (40 mg/10 ml, Sigma) for 30
min at 28 C. After the incubation, trypsin digestion was terminated by
adding soybean trypsin inhibitor (25 mg/10 ml, Sigma) and pituitary
fragments were rinsed with DNase II (0.1 mg/10 ml, Sigma) followed
by a two-step washing with EDTA (2 mm and 1 mm, respectively) in
Ca2⫹-free medium [Medium M199 with Hanks’ salts without CaCl2,
supplemented with 25 mm HEPES, 26 mm NaHCO3, 100 U/ml peni-
cillin, 100 ␮g/ml streptomycin, and 0.3% BSA (pH 7.2)]. Pituitary frag-
ments were then dispersed in Ca2⫹-free medium with DNase II (0.1
mg/50 ml) by gentle trituration using a DPTP transfer pipette (Bio-Rad
5058 Endocrinology, November 2004, 145(11):5056 –5067 Huo et al. • CaM mRNA Expression in Pituitary Cells

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Huo et al. • CaM mRNA Expression in Pituitary Cells Endocrinology, November 2004, 145(11):5056 –5067 5059

FIG. 2. Alignment of goldfish CaM with the corresponding a.a. sequences reported in other species. Sequence alignment was conducted using
Clustal W with MacVector 6.5.3 program and the conserved a.a. residues in these protein sequences were shaded for identification. The four
EF-hands (i.e. EF-I, EF-II, EF-III, and EF-IV) were marked by arrows, and the Ca2⫹-binding loop in the helix-loop-helix structure of individual
EF-hands was boxed. The a.a. sequences of CaM in representative species were downloaded from the GenBank.

Laboratories, Richmond, CA). Pituitary cells were harvested by filtration
through a nylon mesh (⬃30 ␮m pore size) followed by centrifugation at
200 ⫻ g for 10 min. The cell pellet obtained was resuspended in 5 ml
Ca2⫹-free medium, and the cell yield and percentage viability were
estimated by cell counting in the presence of trypan blue. The average
cell yield was 0.5– 0.7 million cells per pituitary and cell viability was
always 94% or greater. After cell counting, pituitary cells were pelleted
by centrifugation and resuspended in plating medium [Medium M199
with Earle’s salts, 25 mm HEPES, 26 mm NaHCO3, 100 U/ml penicillin,
and100 ␮g/ml streptomycin (pH 7.2)] to give a concentration of ap-
proximately 3 million cells/ml. The cell suspension was then dispensed
into 24-well culture plates (0.8 ml/well) precoated with poly-d-lysine
(Sigma). After 3 hr incubation at 28 C, 200 ␮l of 5% horse serum in plating
medium was added to individual wells, and pituitary cells were then
incubated overnight at 28 C under 5% CO2 and saturated humidity. On
the following day, test substances including GnRH, PACAP, and do-
pamine D1/D2 analogs were prepared in testing medium [Medium
M199 with 25 mm HEPES, 26 mm NaHCO3, 100 U/ml penicillin, 100
␮g/ml streptomycin, and 0.1% BSA (pH 7.2)] at appropriate concen-
trations and gently added onto pituitary cells after removing the plating
medium. Based on the results of our initial validation, the optimal
duration of drug treatment was routinely fixed at 48 h. After drug
treatment, total RNA was extracted from individual wells using TRIzol
according to the instructions of the manufacturer.

Fig. 1. Nucleotide and deduced a.a. sequences of goldfish CaM. The nucleotide sequences of three goldfish CaM cDNAs, namely CaM-a, CaM-bS,
and CaM-bL, were aligned using Clustal W with MacVector 6.5.3. Conserved nucleotides in these three clones were shaded for identification.
The a.a. sequences of goldfish CaM deduced from the ORFs of these CaM cDNAs were found to be identical. Sequence analysis has revealed
that CaM-bS is a truncated form of CaM-bL, probably caused by differential use of polyadenylation signals in 3⬘UTR. The nucleotide
substitutions in the ORF of CaM-a are all silence mutations and do not alter the a.a. sequence of goldfish CaM. The putative polyadenylation
signals (AATAAA or AACAAA) are underlined and an asterisk (*) represents the stop codon at the end of the coding sequence.

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5060 Endocrinology, November 2004, 145(11):5056 –5067
Measurement of total CaM mRNA by slot blot
Total RNA samples were extracted from goldfish pituitary cells, heat
denatured at 70 C for 15 min, and vacuum blotted onto nylon membrane
using a Bio-Dot SF microfiltration unit (Bio-Rad Laboratories). The mem-
brane was UV cross-linked, incubated for 3 h at 42 C in 50% formamide-
containing 5⫻ SSC with 1⫻ blocking reagent (Roche), and hybridized
overnight (18 h or longer) under the same conditions with the DIG-
labeled CaM cDNA probe. After hybridization, the membrane was sub-
jected to two washes in 2⫻ SSC with 0.1% SDS at room temperature
followed by two washes in 0.5⫻ SSC with 0.1% SDS at 68 C. After that,
the membrane was washed two times in maleic acid buffer [0.1 m maleic
acid, 0.1 m NaCl, and 0.03% Tween 20 (pH 7.5)] and incubated for at least
5 min in Tris detection buffer [100 mm Tris-HCl, 100 mm NaCl (pH 9.5)].
Diluted solution (1:100) of CPD-Star was then added and hybridization
signals were quantified using a 440 image station (Kodak Digital Science,
Rochester, NY). In these experiments, slot blots of ␤-actin mRNA or 18S
rRNA were used as the internal control.
Data transformation and statistical analysis
For quantitation of CaM gene expression in goldfish pituitary cells,
CaM mRNA levels were measured in terms of arbitrary density units
and normalized against ␤-actin mRNA or 18S rRNA data of the same
sample. These ratio data were then transformed into a percentage of the
mean value in the control group without drug treatment for statistical
analysis (as percent control). This data transformation was performed to
allow for pooling of data from separate experiments together without
increasing the overall variability of the data in individual groups. In this
study, data were analyzed using Student’s t test or ANOVA followed by
Fisher’s least significance difference (LSD) test. Differences were con-
sidered significant at P ⬍ 0.05.
FIG. 3. Phylogenetic analysis of CaM cDNAs from the goldfish and
other animals. The cDNA sequences of representative species were
downloaded from the GenBank and unrooted analysis was conducted
using the neighbor-joining method after 100 bootstraps. The guide
tree was constructed with PHYLIP and TreeView V.32 programs. The
stippled oval indicates the branch of CAM I gene subfamily, whereas
the scale bar represents the genetic distance for evolution.
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Huo et al. • CaM mRNA Expression in Pituitary Cells
FIG. 4. Interdomain comparison of EF-hands in goldfish CaM. A,
Homology analysis based on Clustal W alignment of a.a. sequences of
EF-I, EF-II, EF-III, and EF-IV domains. Identical a.a. residues were
marked by dark gray color and conserved substitutions were shaded
by light gray. B, Comparison of the nucleotide sequences of EF-hands.
Percentage homology was calculated using the Haggin & Sharp al-
gorithm with MacDNAsis 3.1 program.
Results and Discussion
After three rounds of library screening, three positive
clones, namely CaM-a, CaM-bS, and CaM-bL, were isolated
from the goldfish ␭-phage pituitary cDNA library. The in-
serts in these positive clones were sequenced and found to
be 722, 690, and 1530 bp in size for CaM-a, CaM-bS, and
CaM-bL, respectively (Fig. 1). The open reading frames
(ORFs) of these inserts encode a 149 a.a. CaM protein with
identical a.a. sequence. Alignment of nucleotide sequences of
these three positive clones has revealed that CaM-bS is a
truncated form of CaM-bL, probably caused by differential
use of polyadenylation signals in 3⬘ untranslated region
(UTR). There are 32 mismatches in the nucleotide sequence
of CaM-a, compared with that of CaM-bS and CaM-bL. These
mismatches are not clustered together in a particular region
but randomly distributed throughout the nucleotide se-
quence of CaM-a, indicating that the transcripts of CaM-a
and CaM-b (S and L forms) may be originated from two
separate genes. When compared with the coding sequence of
CaM-b, the 14-bp substitutions found in the ORF of CaM-a
are all silent mutations and do not alter the primary structure
of goldfish CaM.
Sequence alignment at the a.a. level (Fig. 2) has revealed
that the primary structure of goldfish CaM is identical with
that of mammals (e.g. human, bovine, mouse, and rat), birds
(e.g. chicken and duck), amphibians (e.g. xenopus), and mod-
ern bony fish (e.g. perch and medaka) and highly homolo-
gous to that reported in early evolved bony fish (e.g. eel),
cyclostome (e.g. hagfish), mollusca (e.g. aplysia), cephalo-
chordate (e.g. brachiostoma), urochordate (e.g. ciona), nem-
atode (e.g. Caenorhabditis elegans), porifera (e.g. sponge), and
protozoa (e.g. trypanosome). Except for a single a.a. substi-
tution in the hagfish and eel CaMs, the primary structure of
CaM is conserved in vertebrates. The a.a. sequences of CaMs
in invertebrates, however, are more diverse. When compared
with the mammalian counterpart, multiple substitutions
(3–15 a.a.) could be noted in the CaMs of brachiostoma,
Huo et al. • CaM mRNA Expression in Pituitary Cells Endocrinology, November 2004, 145(11):5056 –5067 5061

aplysia, ciona, sponge, and trypanosoma, respectively. These
results, as a whole, indicate that the molecular structure of
CaM is highly conserved during the course of evolution. This
structural conservation may also reflect a high level of evo-
lutionary constraints/selection pressure imposed by the es-
sential functions of CaM in eukaryotic cells.
Although the protein structure of CaM is highly con-
served, the genes coding for CaM tend to vary quite a bit
among different species or even within the same species.
Multiple copies of CaM genes encoding the same CaM pro-
tein have been reported in the human (8, 9, 31), rat (13, 32),
chicken (15, 16), and frog (33), respectively. Based on se-
quence comparison, these nonallelic CaM genes can be di-
vided into CaM I, CaM II, and CaM III subfamilies (8 –13),
which form the basis of the multiple genes, single protein
model for CaM evolution (9, 34). This genetic redundancy is
commonly accepted to be the result of gene duplication oc-
curred 1400 million years ago (35, 36). Using phylogenetic
analysis based on nucleotide sequence comparison (Fig. 3),
the goldfish CaM-a and CaM-b cDNAs were grouped with
perch CaM and found to be related to the branch of vertebrate
CaM I gene but less related to CaM II and CaM III genes.
Furthermore, sequence analysis of the helix-loop-helix EF-
hands in goldfish CaM has confirmed that these Ca2⫹-bind-
ings domains are highly conserved at the protein level (Fig.
4A). Parallel comparison of nucleotide sequences of these
EF-hands (Fig. 4B) also revealed that the sequence homology
between EF-I and EF-III (55.17%) and EF-II and EF-IV
(44.82%) are in general higher than that between other EF
repeats (31.03% to 34.48%). These findings are consistent
with the idea that CaM was evolved from an ancestral gene
with a single EF-hand followed by two rounds of tandem
duplication and divergence into the common ancestor of
CaM gene family with four EF hands (34, 37, 38). Because a
high degree of sequence homology (ⱖ 95%) was noted be-
tween CaM-a and CaM-b cDNAs, it raises the possibility that
these two genes may be the products of a more recent gene
duplication event, e.g. tetraploidization in the Cyprinid lin-
eage (39).
A DIG-labeled probe covering the common sequence of
CaM-a and CaM-b was used to examine the tissue distribu-
tion of CaM gene expression in the goldfish (Fig. 5A). Except
for the testes, a 1.6-kb transcript was predominantly ex-
pressed in all the tissues examined, including the pituitary,
brain, liver, gill, intestine, kidney, spleen, muscle, and ovary.
The highest levels of CaM mRNA expression were found in

FIG. 5. Tissue distribution of CaM transcripts in

the goldfish. A, Northern blot of CaM mRNA in
selected tissues of the goldfish, including the
brain, pituitary, liver, gill, intestine, kidney,
spleen, muscle, ovary, and testes. B, Northern blot
of CaM mRNA in the ovary and testes of the gold-
fish using a DIG-labeled probe flanking the distal
region of 3⬘UTR in CaM-bL, which is absent in
CaM-a and CaM-bS. C, Northern blot of CaM
mRNA in selected brain areas of the goldfish, in-
cluding the telencephalon, olfactory bulbs, hypo-
thalamus, cerebellum, optic tectum, medulla ob-
longata, and spinal cord. Unless stated otherwise,
Northern blot was conducted using a DIG-labeled
probe covering a common region in the ORF of
CaM-a, CaM-bS, and CaM-bL. In these experi-
ments, parallel blotting of ␤-actin mRNA was used
as an internal control.

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5062 Endocrinology, November 2004, 145(11):5056 –5067 Huo et al. • CaM mRNA Expression in Pituitary Cells

the brain and liver; to a lesser extent in the pituitary, gills, of neurotransmitters (46 – 49). Besides, CaM is also known to
kidney, muscle, and ovary; and to a lower level in the in- increase nitric oxide production in various neuronal path-
testine, spleen, and testes. An additional transcript of 0.8 kb ways by activating neuronal NO synthase coupled to Ca2⫹
in size was also detected in the gonads. In the case of the influx through N-methyl-d-aspartate receptors (50). This
testes, the dominant transcript for CaM was the one with 0.8 functional coupling can be modulated by CaM kinase II-
kb but not 1.6 kb in size, which was quite the opposite as in
the case of the ovary. Although the physiological relevance
of this phenomenon is unclear, these results suggest that the
expression levels of different CaM genes (e.g. CaM-a and
CaM-b) and/or posttranscriptional processing of CaM tran-
scripts (e.g. by differential use of polyadenylation signals)
may be tissue-specific in fish models. Using a DIG-labeled
probe covering the distal region of 3⬘UTR of CaM-bL (which
is absent in CaM-a and CaM-bS), only the 1.6-kb transcript
could be detected in the gonads (Fig. 5B), confirming that this
transcript was originated from CaM-bL. In parallel studies,
the probe was also effective in detecting the 1.6-kb transcript
in other tissues previously examined (data not shown), in-
dicating that, except in the testes, CaM-bL mRNA is pre-
dominantly expressed in the goldfish.
In mammals, CaM is highly expressed in the central ner-
vous system (40, 41) and accounts for up to 0.5% of brain
proteins (42, 43). In the goldfish, the brain is among the
tissues with the highest expression of CaM mRNA. To ex-
amine the brain distribution of CaM mRNA, Northern blot
was performed in different brain areas of the goldfish, in-
cluding the olfactory bulbs, optic tectum, telencephalon, hy-
pothalamus, cerebellum, medulla oblongata, and spinal cord
(Fig. 5C). In this case, the 1.6-kb transcript was found to be
ubiquitously expressed in all the brain areas, with the highest
levels in the hypothalamus, optic tectum, and medulla ob-
longata and to a lesser extent in the telencephalon, cerebel-
lum, olfactory bulbs, and spinal cord. These findings are in
agreement with the previous reports that CaM is involved in
neuronal excitability (44, 45) and synthesis and/or secretion

FIG. 7. Stimulation of CaM mRNA expression in goldfish pituitary

FIG. 6. Southern blot of goldfish genomic DNA for CaM genes.
Genomic DNA prepared from the goldfish was digested with restric-
tion enzymes including PvuII, HindIII, PstI, and HincII. After size
fractionation in 0.7% agarose gel and transblotting onto a nylon mem-
brane, positive signals for goldfish CaM genes were detected by hy-
bridization with a DIG-labeled CaM cDNA probe.
cells by the adenylate cyclase activator forskolin and PKC activator
TPA. Pituitary cells were exposed for 48 h with increasing doses of
forskolin (0.1–30 ␮M, A) and TPA (10 –300 nM, B). In these experi-
ments, parallel blotting of 18S rRNA was used as an internal control.
Data presented are expressed as mean ⫾ SEM (n ⫽ 9) and are the
pooled results of three separate experiments. Individual groups de-
noted by the same letter represent a similar magnitude of CaM mRNA
expression (P ⬎ 0.05, ANOVA followed by Fisher’s LSD test).

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Huo et al. • CaM mRNA Expression in Pituitary Cells
induced phosphorylation of neuronal NO synthase associ-
ated with N-methyl-D-aspartate receptors via the postsyn-
aptic density protein PSD-95 (51). To shed light on the copy
number of CaM gene(s) in the goldfish, Southern blot was
conducted using genomic DNA digested with restriction
enzymes including PvuII, HindIII, PstI, and HincII, respec-
tively (Fig. 6). In the samples examined, multiple bands (five
to seven bands) were consistently detected, implying that
there are multiple copies of CaM genes in the goldfish. In
mammals, CaM genes, including CaM I, CaM II, and CaM III,
are consisted of six exons interrupted by introns of varying
sizes (52), and multiple bands are commonly observed in the
results of Southern blot (13, 53). As mentioned in the pre-
ceding section, the scattering pattern of nucleotide substitu-
tions observed in the cDNAs of CaM-a and CaM-b argues for
the presence of at least two CaM genes in the goldfish. Given
that the CaM-a and CaM-b genes have not been cloned in this
study, a detailed analysis of exon-intron structures related to
the interpretation of the results in Southern blot was not
performed.
In this study, using goldfish pituitary cells as a cell model,
signal transduction mechanisms regulating CaM gene ex-
pression at the pituitary level were examined using a phar-
macological approach. In this case, pituitary cells were ex-
posed to increasing doses of the adenylate cyclase activator
forskolin (0.1–30 ␮m, Fig. 7A), PKC-activator TPA (10 –300
nm, Fig. 7B) and Ca2⫹ ionophore A23187 (1 nm, 10 nm, and
10 ␮m, Fig. 8). Treatment with forskolin and TPA dose-
dependently increased CaM mRNA levels in goldfish pitu-
itary cells, suggesting that cAMP- and PKC-dependent path-
ways can activate pituitary expression of CaM gene in fish
species. In mammalian cell models, e.g. PC12 cells, cAMP
analogs can up-regulate CaM I and CaM II mRNA levels
without affecting CaM III transcripts (54). In NRK-44F cells,
CaM mRNA levels can be elevated by TPA-induced PKC
activation (55). Our findings in the goldfish may indicate that
the involvement of cAMP- and PKC-dependent mechanisms
in CaM gene expression is highly conserved in vertebrates.
FIG. 8. Inhibition of CaM mRNA expression in goldfish
pituitary cells by the Ca2⫹ ionophore A23187. Pituitary
cells were incubated for 48 h with nanomolar doses (1–10
nM, A) or micromolar dose of A23187 (10 ␮M, B). In these
experiments, parallel blotting of 18S rRNA was used as
an internal control. Data presented are expressed as
mean ⫾ SEM (n ⫽ 9) and are the pooled results of three
separate experiments. Individual groups denoted by the
same letter represent a similar magnitude of CaM mRNA
expression (P ⬎ 0.05, ANOVA followed by Fisher’s LSD
test).
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Endocrinology, November 2004, 145(11):5056 –5067 5063
Unlike forskolin and TPA, A23187 was effective in reducing
CaM mRNA expression in the nanomolar dose range (Fig.
8A). When a high dose of A23187 (10 ␮m) was used, basal
levels of CaM mRNA were almost undetectable (Fig. 8B).
These results, however, are at variance with the stimulatory
effects of Ca2⫹ ionophores on CaM expression reported in
mammalian cells, e.g. in NRK-44F cells (55). Although a pro-
longed elevation of intracellular Ca2⫹ ([Ca2⫹]i) caused by
Ca2⫹ ionophores is known to be toxic in cell cultures (56), the
drop in CaM mRNA levels as a result of cytotoxicity is rather
unlikely as the inhibitory effect of A23187 could be noted at
nanomolar doses (i.e. the nontoxic dose range of Ca2⫹ iono-
phores). In previous studies by other research groups, mi-
cromolar doses of A23187 and ionomycin have been reported
to induce GTH (57) and GH release (58) in goldfish pituitary
cells without causing any noticeable levels of cytotoxic ef-
fects. Because CaM is known to bind with L-type Ca2⫹ chan-
nel to inhibit its channel activity on Ca2⫹ activation (59), we
speculate that a feedback control on CaM expression by
Ca2⫹-dependent mechanisms may be present in the fish
model. This feedback mechanism may help to nullify the
cytotoxic effects of high [Ca2⫹]i because the cells will be-
come less sensitive to Ca2⫹ stimulation by reducing CaM
expression.
To examine whether CaM gene expression at the pituitary
level could be the target of modulation by hypophysiotropic
factors, goldfish pituitary cells were exposed to increasing
concentrations of GnRH (0.001–10 ␮m, Fig. 9A) and PACAP
(0.001–10 ␮m, Fig. 9B) under static incubation conditions. In
this case, basal levels of CaM mRNA were elevated in a
dose-dependent manner by treatment with GnRH and
PACAP. In parallel experiments, pituitary cells were also
exposed to increasing levels of the nonselective dopamine
agonist apomorphine (0.001–10 ␮m, Fig. 10A). In contrast to
GnRH and PACAP, apomorphine suppressed CaM mRNA
levels in a dose-related fashion. In the presence of apomor-
phine (1 ␮m), the stimulatory effects of GnRH (1 ␮m) and
5064 Endocrinology, November 2004, 145(11):5056 –5067
FIG. 9. Stimulation of CaM mRNA expression in goldfish pituitary
cells by GnRH and PACAP. Pituitary cells were exposed to increasing
concentrations of GnRH (0.001–10 ␮M, A) and PACAP (0.001–10 ␮M,
B) for 48 h under static incubation. After drug treatment, total RNA
samples were extracted and assayed for CaM mRNA levels as de-
scribed in Materials and Methods. In these experiments, parallel
blotting of ␤-actin mRNA was used as an internal control. Data pre-
sented are expressed as mean ⫾ SEM (n ⫽ 9) and are the pooled results
of three separate experiments. Individual groups denoted by the same
letter represent a similar magnitude of CaM mRNA expression (P ⬎
0.05, ANOVA followed by Fisher’s LSD test). Representative results
of slot blots are also included in these figures for reference.
PACAP (1 ␮m) on CaM mRNA expression were totally abol-
ished (Fig. 10B).
To elucidate the receptor specificity mediating apomor-
phine’s action on CaM mRNA expression, goldfish pituitary
cells were treated with increasing doses (0.01–10 ␮m) of the
dopamine D1 agonist SKF38393 or D2 agonist Ly171555 (Fig.
11A). The dose-dependence of apomorphine inhibition on
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Huo et al. • CaM mRNA Expression in Pituitary Cells
FIG. 10. Dopaminergic inhibition of CaM mRNA expression in gold-
fish pituitary cells. A, Dopaminergic inhibition of basal CaM mRNA
expression. Pituitary cells were incubated for 48 h with increasing
doses (0.001–10 ␮M) of the nonselective dopamine agonist apomor-
phine (Apo). B, Dopaminergic inhibition of GnRH- and PACAP-stim-
ulated CaM mRNA expression. Pituitary cells were incubated for 48 h
with GnRH (1 ␮M) or PACAP (1 ␮M) with or without simultaneous
treatment of apomorphine (1 ␮M). In these experiments, parallel blot-
ting of 18S rRNA was used as an internal control. Data presented are
expressed as mean ⫾ SEM (n ⫽ 9) and are the pooled results of three
separate experiments. Individual groups denoted by the same letter
represent a similar magnitude of CaM mRNA expression (P ⬎ 0.05,
ANOVA followed by Fisher’s LSD test).
CaM mRNA expression was mimicked by the D2 agonist
Ly171555 but not by the D1 agonist SKF38393. Besides, the
inhibitory action of apomorphine (1 ␮m) on CaM mRNA
expression was blocked by the dopamine D2 antagonist
domperidone (1 ␮m, Fig. 11B) and the D1 antagonist
SCH23390 was not effective in this regard (data not shown).
Huo et al. • CaM mRNA Expression in Pituitary Cells
FIG. 11. Receptor specificity of dopaminergic inhibition of CaM
mRNA expression in goldfish pituitary cells. A, Effects of the dopa-
mine D1 agonist SKF38393 and D2 agonist Ly171555 on CaM mRNA
expression. Pituitary cells were incubated for 48 h with increasing
concentrations of SKF38393 (0.01–10 ␮M) or Ly171555 (0.01–10 ␮M).
B, Blockade of dopaminergic inhibition of CaM mRNA expression by
the D2 antagonist domperidone (Domp). Pituitary cells were incu-
bated for 48 h with the nonselective dopamine agonist apomorphine
(Apo, 1 ␮M) in the presence or absence of domperidone (1 ␮M). In these
experiments, parallel blotting of 18S rRNA was used as an internal
control. Data presented are expressed as mean ⫾ SEM (n ⫽ 9) and are
the pooled results of three separate experiments. Individual groups
denoted by the same letter represent a similar magnitude of CaM
mRNA expression (P ⬎ 0.05, ANOVA followed by Fisher’s LSD test).
Similar to apomorphine, the D2 agonist Ly171555 (1 ␮m) also
blocked the stimulatory effects of GnRH (1 ␮m) and PACAP
(1 ␮m) on CaM mRNA expression (Fig. 12).
These results, taken together, confirm that dopamine’s
action on CaM mRNA expression is mediated through pi-
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Endocrinology, November 2004, 145(11):5056 –5067 5065
FIG. 12. Blockade of GnRH- and PACAP-stimulated CaM mRNA ex-
pression in goldfish pituitary cells by the dopamine D2 agonist
Ly171555. Pituitary cells were incubated for 48 h with GnRH (1 ␮M)
or PACAP (1 ␮M) in the presence or absence of Ly171555 (1 ␮M). In
these experiments, parallel blotting of 18S rRNA was used as an
internal control. Data presented are expressed as mean ⫾ SEM (n ⫽
9) and are the pooled results of three separate experiments. Individ-
ual groups denoted by the same letter represent a similar magnitude
of CaM mRNA expression (P ⬎ 0.05, ANOVA followed by Fisher’s LSD
test).
tuitary D2 receptors. The present study has also demon-
strated for the first time that CaM gene expression in the
pituitary is under the control of hypothalamic factors. Ap-
parently, the stimulatory effects of GnRH and PACAP on
CaM gene expression are negatively regulated by dopami-
nergic input through activation of pituitary D2 receptors. In
the goldfish, GnRH and PACAP are known to stimulate
GTH-II and GH secretion at the pituitary level mainly
through the PKC- (27) and cAMP-dependent mechanisms
(21), respectively. In the same animal model, dopamine can
exert differential effects on these two hormones, being stim-
ulatory to GH release through D1 receptors coupled to the
adenylyl cyclase/cAMP/protein kinase A pathway (25) and
inhibitory to GTH-II release via D2 receptors coupled to
suppression of Ca2⫹ channel activity and [Ca2⫹]i mobiliza-
tion (26, 60, 61). Because CaM and CaM kinase II are known
to be involved in GTH-II (29) and GH release in goldfish
pituitary cells (28), it is tempting to speculate that modula-
tion of CaM expression may represent a novel mechanism for
GTH-II and GH regulation by hypothalamic factors. This
possibility clearly warrants future investigations, especially
to clarify whether GnRH, PACAP, and dopamine can reg-
ulate CaM gene expression in goldfish gonadotrophs and
somatotrophs.
In summary, we have isolated two forms of goldfish CaM
cDNA, CaM-a and CaM-b, by library screening. These two
cDNAs encode the same CaM protein with identical a.a.
sequence, compared with that reported in other vertebrates
including mammals, birds, and amphibians. The nucleotide
sequences of these cDNAs also reveal that goldfish CaM-a
5066 Endocrinology, November 2004, 145(11):5056 –5067
and CaM-b are putative members of the CaM I gene sub-
family. Furthermore, multiple copies of CaM genes have
been implicated in the goldfish genome and CaM transcripts
are ubiquitously expressed in various tissues and brain areas
of the goldfish. Using goldfish pituitary cells as a cell model,
we have shown that CaM mRNA expression at the pituitary
level can be induced by the cAMP- and PKC-dependent
pathways but inhibited by Ca2⫹-dependent mechanisms. Be-
sides, pituitary expression of CaM mRNA is also under the
control of hypothalamic factors. GnRH and PACAP can up-
regulate CaM mRNA expression in goldfish pituitary cells,
and these stimulatory effects can be blocked by dopaminer-
gic activation through D2 receptors. These results, as a
whole, provide evidence that CaM gene expression at the
pituitary level can be a target of modulation by hypothalamic
factors, which may have direct consequence on pituitary
functions in fish models.
Acknowledgments
Received May 10, 2004. Accepted July 28, 2004.
Address all correspondence and requests for reprints to: Dr. Ander-
son O. L. Wong, Associate Professor, Department of Zoology, University
of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of
China. E-mail: olwong@hkucc.hku.hk.
This work was supported by Research Grant Council (Hong Kong)
and Committee on Research and Conference Grants from University of
Hong Kong (to A.O.L.W.). Financial support from the Department of
Zoology, University of Hong Kong (to L.H.) in the form of a Ph.D.
studentship is also acknowledged.
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