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0013-7227/04/$15.

00/0 Endocrinology 145(11):5056 –5067


Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0584

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 ANDERSON O. L. WONG
Department of Zoology, University of Hong Kong, Hong Kong, People’s Republic of China

Calmodulin (CaM) is a Ca2ⴙ-binding protein essential for bi- itary cells, activation of cAMP- or PKC-dependent pathways
ological functions mediated through Ca2ⴙ-dependent mecha- increased CaM mRNA levels, whereas the opposite was true
nisms. In the goldfish, CaM is involved in the signaling events for induction of Ca2ⴙ entry. Basal levels of CaM mRNA was
mediating pituitary hormone secretion induced by hypotha- accentuated by GnRH and pituitary adenylate cyclase-acti-
lamic factors. However, the structural identity of goldfish vating polypeptide but suppressed by dopaminergic stimula-
CaM has not been established, and the neuroendocrine mech- tion. Pharmacological studies using D1 and D2 analogs re-
anisms regulating CaM gene expression at the pituitary level vealed that dopaminergic inhibition of CaM mRNA expression
are still unknown. Here we cloned the goldfish CaM and tested was mediated through pituitary D2 receptors. At the pituitary
the hypothesis that pituitary expression of CaM transcripts level, D2 activation was also effective in blocking GnRH- and
can be the target of modulation by hypothalamic factors. pituitary adenylate cyclase-activating polypeptide-stimu-
Three goldfish CaM cDNAs, namely CaM-a, CaM-bS, and CaM- lated CaM mRNA expression. As a whole, the present study has
bL, were isolated by library screening. These cDNAs carry a confirmed that the molecular structure of CaM is highly con-
450-bp open reading frame encoding the same 149-amino acid served, and its mRNA expression at the pituitary level can be
CaM protein, the amino acid sequence of which is identical regulated by interactions among hypothalamic factors. (En-
with that of mammals, birds, and amphibians and is highly docrinology 145: 5056 –5067, 2004)
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
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
regulating cell division and differentiation, gene expression, compared with CaM molecules reported in lower verte-
programmed cell death, DNA replication and repairing, and brates, like the fish [e.g. electric eel (17)], only one amino acid
exocytosis of hormone/neurotransmitter are well docu- (a.a.) substitution in the primary sequence can be noted.
mented (1–3). The molecular structure of CaM is character- These findings indicate that CaM is highly conserved at the
ized by the presence of four Ca2⫹-binding motifs known as protein level during vertebrate evolution.
the helix-loop-helix EF-hands. Three-dimensional structural In the goldfish, gonadotropin (GTH), and GH secretion are
analysis has revealed that CaM is dumbbell shaped with two regulated by a multitude of neuroendocrine factors (18, 19).
similar domains located at either end, each with two EF- Among these regulators, most of them are hypothalamic
hands for Ca2⫹-binding. In the absence of Ca2⫹, these EF- factors that can exert regulatory actions on GTH and GH
hands are in a closed conformation. This Ca2⫹-free form of release simultaneously. For examples, GnRH and pituitary
CaM (or ApoCaM) is not functional but can still bind to a adenylate cyclase-activating polypeptide (PACAP) are
subset of target proteins, e.g. Nuf1p and Spc110p (4, 5). Upon known to stimulate GTH-II (or fish LH) and GH release
Ca2⫹ binding, CaM can shift to an open conformation. As a directly from goldfish pituitary cells (20 –23). Dopamine, on
result, two hydrophobic surfaces are exposed and allowed the contrary, can exert opposite effects on the release of these
for CaM interactions with Ca2⫹-sensitive target proteins (6, two hormones, being stimulatory to GH secretion via pitu-
7). CaM is known to be encoded by members of a multigene itary D1 receptors (24, 25) and inhibitory to GTH-II release
family. At present, three CaM genes (CaM I, II, and III) in
through D2 receptors (26). Apparently, the availability of
mammals (8 –13) and two CaM genes (CaM I and II) in birds
extracellular Ca2⫹ and its entry through voltage-sensitive
Ca2⫹ channels are essential for both basal and stimulated
Abbreviations: a.a., Amino acid; [Ca2⫹]i, intracellular Ca2⫹; CaM,
calmodulin; DIG, digoxigenin; DMSO, dimethylsulfoxide; GTH, gonad- GTH-II and GH release in goldfish pituitary cells (27). Using
otropin; LSD, least significance difference; ORF, open reading frame; a pharmacological approach, it has been shown that CaM
PACAP, pituitary adenylate cyclase-activating polypeptide; PKC, pro- antagonists and CaM kinase II inhibitors can block the reg-
tein kinase C; SDS, sodium dodecyl sulfate; SSC, saline sodium ci- ulatory effects on GTH-II and GH release by GnRH, PACAP,
trate; TPA, 12-O-tetra-decanoyl-phorbol-13-acetate; UTR, untranslated
region. and dopamine (28, 29), suggesting that CaM may be a key
Endocrinology is published monthly by The Endocrine Society (http://
component of the postreceptor signaling mechanisms for
www.endo-society.org), the foremost professional society serving the these regulators. Although neuroendocrine regulation of
endocrine community. CaM expression at the pituitary level has not been previously

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Huo et al. • CaM mRNA Expression in Pituitary Cells Endocrinology, November 2004, 145(11):5056 –5067 5057

examined, the findings in the goldfish have prompted us to namely CaM-a, CaM-bS, and CaM-bL, were isolated. The cDNA inserts
speculate that CaM gene expression at the pituitary level may in these clones were sequenced using a BigDye terminator cycle se-
quencing kit (Applied Biosystems, Foster City, CA) in a 310 Genetic
be the target of modulation by hypophysiotropic factors, analyzer (PerkinElmer, Boston, MA). Based on the sequences obtained,
which may have direct consequences on pituitary hormone a pair of primers (PU1, 5⬘-CAGATATGGCTGACCAACTCAC-3⬘ and
synthesis and secretion. PD1, 5⬘-ACAGAAGAGCTTCACTTTGCCG-3⬘) were designed to cover
In the present study, the structural identity of goldfish a common sequence of CaM-a, CaM-bS, and CaM-bL. After that, a
CaM was established by molecular cloning using the stan- DIG-labeled cDNA probe that can recognize the mRNA transcripts
corresponding to these CaM clones was prepared for subsequent studies
dard techniques of cDNA library screening. The transcript using a PCR-DIG labeling kit (Roche Diagnostics, Mannheim, Germany).
expression of CaM in various tissues and brain areas was
examined by Northern blot. Based on the nucleotide se- Northern blot of CaM mRNA
quences of CaM cDNAs obtained, a slot blot system was set
up to quantify total CaM mRNA expression in primary cul- Total RNA was extracted with TRIzol from selected tissues and brain
areas of the goldfish. After that, mRNA was purified from total RNA
tures of goldfish pituitary cells. Using this assay system, the using a PolyATract mRNA isolation system III (Promega, Madison, WI).
effects of hypophysiotropic factors in fish models, including These mRNA samples were denatured, size fractionated in 1% agarose
GnRH, PACAP, and dopamine, on CaM gene expression in gel with 0.22 m formaldehyde, and blotted onto a positively charged
goldfish pituitary cells were investigated using a static in- nylon membrane (Roche) using a VacuGene vacuum blotting system
(Pharmacia Biotech, Piscataway, NJ). The membrane was UV cross-
cubation approach. In parallel experiments, the effects of linked using a Stratalinker 2400 (Stratagene), prehybridized for 3 h in
activating cAMP-, protein kinase C (PKC)-, and Ca2⫹-depen- 50% formamide-containing hybridization buffer, and incubated with the
dent pathways on CaM mRNA expression at the pituitary DIG-labeled CaM cDNA probe overnight at 42 C. On the following day,
level were also examined. 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)
Materials and Methods with CPD-Star as the substrate. Unless stated otherwise, Northern blot
was conducted using the DIG-labeled probe common for CaM-a, CaM-
Animals bS, and CaM-bL. In this study, Northern blot of ␤-actin was used as an
Goldfish (Carassius auratus) with body weight ranging from 35 to 50 g internal control.
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 Southern blot of genomic DNA
holding period, the fish were fed to satiation daily with commercial fish
feed. After the acclimation, pituitaries were collected for cell dispersion Genomic DNA was isolated from whole blood freshly collected from
from goldfish anesthetized in MS222 followed by spinosectomy accord- the goldfish. Briefly, blood cells were collected from 0.5 ml whole blood
ing to the regulations of animal use at the University of Hong Kong. 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
Reagents and test substances 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
TRIZOL, medium M199, and horse serum for cell culture were pur- (pH 8.0) and one time with chloroform. After that, the genomic DNA in
chased from Gibco Life Technologies (Grand Island, NY). PCR-digoxi- the aqueous phase was precipitated with equal volume of isopropanol
genin (DIG) labeling kit, CDP-Star, and Anti-DIG-AP (Fab fragments) and 1:3 volume of 3 m sodium acetate (pH 5.2). The DNA pellet was
were obtained from Roche Molecular Biochemicals (Mannheim, Ger- harvested, soaked in 70% ethanol for 5 h, and dried in a fume hood. After
many). Salmon GnRH and PACAP-38 were obtained from Peninsula that, the genomic DNA obtained was dissolved in 1 ml TE buffer and
Laboratories Inc. (Belmont, CA). LY171555, (⫾) SKF38393, apomor- digested with restriction enzymes including PvuII, HindIII, PstI, and
phine, domperidone, SCH23390, forskolin, and 12-O-tetra-decanoyl- HincII, respectively. The digested products were size fractionized in a
phorbol-13-acetate (TPA) were purchased from RBI Sigma (St. Louis, 0.7% agarose gel and transferred onto a positively charged nylon mem-
MO). A23187 was obtained from Calbiochem (La Jolla, CA). GnRH and brane (Roche) by vacuum blotting. After UV cross-linking, the mem-
PACAP were dissolved in double-distilled deionized water to prepare brane was incubated in 6⫻ SSC with 1⫻ blocking reagent (Roche) for 3 h
1-mm stock solutions, aliquoted in small volume, and stored frozen at followed by hybridization overnight with the DIG-labeled CaM cDNA
⫺80 C. Stock solutions of forskolin, TPA, and A23187 were prepared in probe at 42 C. On the following day, the membrane was washed, and
a similar manner except that they were dissolved in dimethylsulfoxide signal development was carried out as described for Northern blot.
(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- Static incubation of goldfish pituitary cells
solved in DMSO to give a 10-mm stock solution, which was then diluted Primary cultures of pituitary cells were prepared from the goldfish
with culture medium to appropriated concentrations 15 min before drug as described previously (30). Briefly, pituitaries were excised from gold-
treatment. The final concentration of DMSO in the medium was less than fish and washed three times in washing medium [Medium M199 with
0.1% and had no effect on CaM mRNA expression. 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
Screening of goldfish pituitary cDNA library remove blood clots. After that, pituitaries were diced into 0.5-mm frag-
ments using a McILwain tissue chopper (Mickle Laboratory Engineer-
A 32P-labeled probe prepared from bovine CaM cDNA was used for ing, Gomshall, UK) and exposed to trypsin (40 mg/10 ml, Sigma) for 30
the screening of a goldfish pituitary Zap Express cDNA library accord- min at 28 C. After the incubation, trypsin digestion was terminated by
ing to the instructions by the manufacturer (Stratagene, La Jolla, CA). adding soybean trypsin inhibitor (25 mg/10 ml, Sigma) and pituitary
Briefly, the 32P-labeled probe was allowed to hybridize with nylon fragments were rinsed with DNase II (0.1 mg/10 ml, Sigma) followed
membranes lifted from agar plates with phage colonies of the goldfish by a two-step washing with EDTA (2 mm and 1 mm, respectively) in
pituitary cDNA library. The areas corresponding to positive signals on Ca2⫹-free medium [Medium M199 with Hanks’ salts without CaCl2,
the membranes were identified on the original plates. The plaques in supplemented with 25 mm HEPES, 26 mm NaHCO3, 100 U/ml peni-
these areas were cored out, extracted, and spread on agar plates for cillin, 100 ␮g/ml streptomycin, and 0.3% BSA (pH 7.2)]. Pituitary frag-
secondary and tertiary screening until single colonies were isolated. As ments were then dispersed in Ca2⫹-free medium with DNase II (0.1
a result of library screening, three positive clones of goldfish CaM, mg/50 ml) by gentle trituration using a DPTP transfer pipette (Bio-Rad

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

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.
FIG. 4. Interdomain comparison of EF-hands in goldfish CaM. A,
Homology analysis based on Clustal W alignment of a.a. sequences of
Data transformation and statistical analysis
EF-I, EF-II, EF-III, and EF-IV domains. Identical a.a. residues were
For quantitation of CaM gene expression in goldfish pituitary cells, marked by dark gray color and conserved substitutions were shaded
CaM mRNA levels were measured in terms of arbitrary density units by light gray. B, Comparison of the nucleotide sequences of EF-hands.
and normalized against ␤-actin mRNA or 18S rRNA data of the same Percentage homology was calculated using the Haggin & Sharp al-
sample. These ratio data were then transformed into a percentage of the gorithm with MacDNAsis 3.1 program.
mean value in the control group without drug treatment for statistical
analysis (as percent control). This data transformation was performed to Results and Discussion
allow for pooling of data from separate experiments together without
increasing the overall variability of the data in individual groups. In this After three rounds of library screening, three positive
study, data were analyzed using Student’s t test or ANOVA followed by clones, namely CaM-a, CaM-bS, and CaM-bL, were isolated
Fisher’s least significance difference (LSD) test. Differences were con- from the goldfish ␭-phage pituitary cDNA library. The in-
sidered significant at P ⬍ 0.05. 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
FIG. 3. Phylogenetic analysis of CaM cDNAs from the goldfish and protozoa (e.g. trypanosome). Except for a single a.a. substi-
other animals. The cDNA sequences of representative species were tution in the hagfish and eel CaMs, the primary structure of
downloaded from the GenBank and unrooted analysis was conducted CaM is conserved in vertebrates. The a.a. sequences of CaMs
using the neighbor-joining method after 100 bootstraps. The guide
tree was constructed with PHYLIP and TreeView V.32 programs. The
in invertebrates, however, are more diverse. When compared
stippled oval indicates the branch of CAM I gene subfamily, whereas with the mammalian counterpart, multiple substitutions
the scale bar represents the genetic distance for evolution. (3–15 a.a.) could be noted in the CaMs of brachiostoma,

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Huo et al. • CaM mRNA Expression in Pituitary Cells Endocrinology, November 2004, 145(11):5056 –5067 5061

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


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

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Huo et al. • CaM mRNA Expression in Pituitary Cells Endocrinology, November 2004, 145(11):5056 –5067 5063

induced phosphorylation of neuronal NO synthase associ- Unlike forskolin and TPA, A23187 was effective in reducing
ated with N-methyl-D-aspartate receptors via the postsyn- CaM mRNA expression in the nanomolar dose range (Fig.
aptic density protein PSD-95 (51). To shed light on the copy 8A). When a high dose of A23187 (10 ␮m) was used, basal
number of CaM gene(s) in the goldfish, Southern blot was levels of CaM mRNA were almost undetectable (Fig. 8B).
conducted using genomic DNA digested with restriction These results, however, are at variance with the stimulatory
enzymes including PvuII, HindIII, PstI, and HincII, respec- effects of Ca2⫹ ionophores on CaM expression reported in
tively (Fig. 6). In the samples examined, multiple bands (five mammalian cells, e.g. in NRK-44F cells (55). Although a pro-
to seven bands) were consistently detected, implying that longed elevation of intracellular Ca2⫹ ([Ca2⫹]i) caused by
there are multiple copies of CaM genes in the goldfish. In Ca2⫹ ionophores is known to be toxic in cell cultures (56), the
mammals, CaM genes, including CaM I, CaM II, and CaM III, drop in CaM mRNA levels as a result of cytotoxicity is rather
are consisted of six exons interrupted by introns of varying unlikely as the inhibitory effect of A23187 could be noted at
sizes (52), and multiple bands are commonly observed in the nanomolar doses (i.e. the nontoxic dose range of Ca2⫹ iono-
results of Southern blot (13, 53). As mentioned in the pre- phores). In previous studies by other research groups, mi-
ceding section, the scattering pattern of nucleotide substitu- cromolar doses of A23187 and ionomycin have been reported
tions observed in the cDNAs of CaM-a and CaM-b argues for to induce GTH (57) and GH release (58) in goldfish pituitary
the presence of at least two CaM genes in the goldfish. Given cells without causing any noticeable levels of cytotoxic ef-
that the CaM-a and CaM-b genes have not been cloned in this
fects. Because CaM is known to bind with L-type Ca2⫹ chan-
study, a detailed analysis of exon-intron structures related to
nel to inhibit its channel activity on Ca2⫹ activation (59), we
the interpretation of the results in Southern blot was not
speculate that a feedback control on CaM expression by
performed.
Ca2⫹-dependent mechanisms may be present in the fish
In this study, using goldfish pituitary cells as a cell model,
model. This feedback mechanism may help to nullify the
signal transduction mechanisms regulating CaM gene ex-
pression at the pituitary level were examined using a phar- cytotoxic effects of high [Ca2⫹]i because the cells will be-
macological approach. In this case, pituitary cells were ex- come less sensitive to Ca2⫹ stimulation by reducing CaM
posed to increasing doses of the adenylate cyclase activator expression.
forskolin (0.1–30 ␮m, Fig. 7A), PKC-activator TPA (10 –300 To examine whether CaM gene expression at the pituitary
nm, Fig. 7B) and Ca2⫹ ionophore A23187 (1 nm, 10 nm, and level could be the target of modulation by hypophysiotropic
10 ␮m, Fig. 8). Treatment with forskolin and TPA dose- factors, goldfish pituitary cells were exposed to increasing
dependently increased CaM mRNA levels in goldfish pitu- concentrations of GnRH (0.001–10 ␮m, Fig. 9A) and PACAP
itary cells, suggesting that cAMP- and PKC-dependent path- (0.001–10 ␮m, Fig. 9B) under static incubation conditions. In
ways can activate pituitary expression of CaM gene in fish this case, basal levels of CaM mRNA were elevated in a
species. In mammalian cell models, e.g. PC12 cells, cAMP dose-dependent manner by treatment with GnRH and
analogs can up-regulate CaM I and CaM II mRNA levels PACAP. In parallel experiments, pituitary cells were also
without affecting CaM III transcripts (54). In NRK-44F cells, exposed to increasing levels of the nonselective dopamine
CaM mRNA levels can be elevated by TPA-induced PKC agonist apomorphine (0.001–10 ␮m, Fig. 10A). In contrast to
activation (55). Our findings in the goldfish may indicate that GnRH and PACAP, apomorphine suppressed CaM mRNA
the involvement of cAMP- and PKC-dependent mechanisms levels in a dose-related fashion. In the presence of apomor-
in CaM gene expression is highly conserved in vertebrates. phine (1 ␮m), the stimulatory effects of GnRH (1 ␮m) and

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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5064 Endocrinology, November 2004, 145(11):5056 –5067 Huo et al. • CaM mRNA Expression in Pituitary Cells

FIG. 9. Stimulation of CaM mRNA expression in goldfish pituitary


cells by GnRH and PACAP. Pituitary cells were exposed to increasing FIG. 10. Dopaminergic inhibition of CaM mRNA expression in gold-
concentrations of GnRH (0.001–10 ␮M, A) and PACAP (0.001–10 ␮M, fish pituitary cells. A, Dopaminergic inhibition of basal CaM mRNA
B) for 48 h under static incubation. After drug treatment, total RNA expression. Pituitary cells were incubated for 48 h with increasing
samples were extracted and assayed for CaM mRNA levels as de- doses (0.001–10 ␮M) of the nonselective dopamine agonist apomor-
scribed in Materials and Methods. In these experiments, parallel phine (Apo). B, Dopaminergic inhibition of GnRH- and PACAP-stim-
blotting of ␤-actin mRNA was used as an internal control. Data pre- ulated CaM mRNA expression. Pituitary cells were incubated for 48 h
sented are expressed as mean ⫾ SEM (n ⫽ 9) and are the pooled results with GnRH (1 ␮M) or PACAP (1 ␮M) with or without simultaneous
of three separate experiments. Individual groups denoted by the same treatment of apomorphine (1 ␮M). In these experiments, parallel blot-
letter represent a similar magnitude of CaM mRNA expression (P ⬎ ting of 18S rRNA was used as an internal control. Data presented are
0.05, ANOVA followed by Fisher’s LSD test). Representative results expressed as mean ⫾ SEM (n ⫽ 9) and are the pooled results of three
of slot blots are also included in these figures for reference. 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).
PACAP (1 ␮m) on CaM mRNA expression were totally abol-
ished (Fig. 10B). CaM mRNA expression was mimicked by the D2 agonist
To elucidate the receptor specificity mediating apomor- Ly171555 but not by the D1 agonist SKF38393. Besides, the
phine’s action on CaM mRNA expression, goldfish pituitary inhibitory action of apomorphine (1 ␮m) on CaM mRNA
cells were treated with increasing doses (0.01–10 ␮m) of the expression was blocked by the dopamine D2 antagonist
dopamine D1 agonist SKF38393 or D2 agonist Ly171555 (Fig. domperidone (1 ␮m, Fig. 11B) and the D1 antagonist
11A). The dose-dependence of apomorphine inhibition on SCH23390 was not effective in this regard (data not shown).

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Huo et al. • CaM mRNA Expression in Pituitary Cells 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
FIG. 11. Receptor specificity of dopaminergic inhibition of CaM adenylyl cyclase/cAMP/protein kinase A pathway (25) and
mRNA expression in goldfish pituitary cells. A, Effects of the dopa- inhibitory to GTH-II release via D2 receptors coupled to
mine D1 agonist SKF38393 and D2 agonist Ly171555 on CaM mRNA
expression. Pituitary cells were incubated for 48 h with increasing
suppression of Ca2⫹ channel activity and [Ca2⫹]i mobiliza-
concentrations of SKF38393 (0.01–10 ␮M) or Ly171555 (0.01–10 ␮M). tion (26, 60, 61). Because CaM and CaM kinase II are known
B, Blockade of dopaminergic inhibition of CaM mRNA expression by to be involved in GTH-II (29) and GH release in goldfish
the D2 antagonist domperidone (Domp). Pituitary cells were incu- pituitary cells (28), it is tempting to speculate that modula-
bated for 48 h with the nonselective dopamine agonist apomorphine tion of CaM expression may represent a novel mechanism for
(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 GTH-II and GH regulation by hypothalamic factors. This
control. Data presented are expressed as mean ⫾ SEM (n ⫽ 9) and are possibility clearly warrants future investigations, especially
the pooled results of three separate experiments. Individual groups to clarify whether GnRH, PACAP, and dopamine can reg-
denoted by the same letter represent a similar magnitude of CaM ulate CaM gene expression in goldfish gonadotrophs and
mRNA expression (P ⬎ 0.05, ANOVA followed by Fisher’s LSD test).
somatotrophs.
In summary, we have isolated two forms of goldfish CaM
Similar to apomorphine, the D2 agonist Ly171555 (1 ␮m) also cDNA, CaM-a and CaM-b, by library screening. These two
blocked the stimulatory effects of GnRH (1 ␮m) and PACAP cDNAs encode the same CaM protein with identical a.a.
(1 ␮m) on CaM mRNA expression (Fig. 12). sequence, compared with that reported in other vertebrates
These results, taken together, confirm that dopamine’s including mammals, birds, and amphibians. The nucleotide
action on CaM mRNA expression is mediated through pi- sequences of these cDNAs also reveal that goldfish CaM-a

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5066 Endocrinology, November 2004, 145(11):5056 –5067 Huo et al. • CaM mRNA Expression in Pituitary Cells

and CaM-b are putative members of the CaM I gene sub- The structural organization of the chicken calmodulin gene. J Biol Chem
260:907–912
family. Furthermore, multiple copies of CaM genes have 16. Ye Q, Berchtold MW 1997 Structure and expression of the chicken calmodulin
been implicated in the goldfish genome and CaM transcripts I gene. Gene 194:63– 68
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19. Peng C, Peter RE 1997 Neuropeptide regulation of growth hormone secretion
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dotropin-releasing hormone also functions as a growth hormone-releasing
control of hypothalamic factors. GnRH and PACAP can up- factor in the goldfish. Endocrinology 124:2509 –2518
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gic activation through D2 receptors. These results, as a 22. Chang JP, Wirachowsky NR, Kwong P, Johnson JD 2001 PACAP stimulation
whole, provide evidence that CaM gene expression at the of gonadotropin-II secretion in goldfish pituitary cells: mechanisms of action
pituitary level can be a target of modulation by hypothalamic and interaction with gonadotropin releasing hormone signalling. J Neuroen-
docrinol 13:540 –550
factors, which may have direct consequence on pituitary 23. Wong AOL, Leung MY, Shea WL, Tse LY, Chang JP, Chow BK 1998 Hy-
functions in fish models. pophysiotropic action of pituitary adenylate cyclase-activating polypeptide
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24. Wong AOL, Van Goor F, Chang JP 1994 Entry of extracellular calcium me-
Received May 10, 2004. Accepted July 28, 2004. diates dopamine D1-stimulated growth hormone release from goldfish pitu-
Address all correspondence and requests for reprints to: Dr. Ander- itary cells. Gen Comp Endocrinol 94:316 –328
son O. L. Wong, Associate Professor, Department of Zoology, University 25. Wong AOL, van der Kraak G, Chang JP 1994 Cyclic 3⬘,5⬘-adenosine mono-
of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of phosphate mediates dopamine D1-stimulated growth hormone release from
China. E-mail: olwong@hkucc.hku.hk. goldfish pituitary cells. Neuroendocrinology 60:410 – 417
This work was supported by Research Grant Council (Hong Kong) 26. Chang JP, Van Goor F, Lo A, Johnson JC, Jobin RM, Goldberg JI 1997 Signal
and Committee on Research and Conference Grants from University of transduction in gonadotropin-II secretion in goldfish pituitary cells. In:
Kikuyama Ska S, ed. Advances in comparative endocrinology. Bologna: Mon-
Hong Kong (to A.O.L.W.). Financial support from the Department of duzzi Editore; 29 –33
Zoology, University of Hong Kong (to L.H.) in the form of a Ph.D. 27. Chang JP, Johnson JD, Van Goor F, Wong CJ, Yunker WK, Uretsky AD,
studentship is also acknowledged. Taylor D, Jobin RM, Wong AOL, Goldberg JI 2000 Signal transduction
mechanisms mediating secretion in goldfish gonadotropes and somatotropes.
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