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Endocrinology Vol. 145, No. 11 5056-5067
Copyright © 2004 by The Endocrine Society

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

Address all correspondence and requests for reprints to: Dr. Anderson 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{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Calmodulin (CaM) is a Ca2+-binding protein essential for biological functions mediated through Ca2+-dependent mechanisms. In the goldfish, CaM is involved in the signaling events mediating pituitary hormone secretion induced by hypothalamic factors. However, the structural identity of goldfish CaM has not been established, and the neuroendocrine mechanisms 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 pituitary cells, activation of cAMP- or PKC-dependent pathways increased CaM mRNA levels, whereas the opposite was true for induction of Ca2+ entry. Basal levels of CaM mRNA was accentuated by GnRH and pituitary adenylate cyclase-activating polypeptide but suppressed by dopaminergic stimulation. Pharmacological studies using D1 and D2 analogs revealed 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-stimulated CaM mRNA expression. As a whole, the present study has confirmed that the molecular structure of CaM is highly conserved, and its mRNA expression at the pituitary level can be regulated by interactions among hypothalamic factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
CALMODULIN (CAM), a heat-stable acidic protein expressed 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 documented (1, 2, 3). The molecular structure of CaM is characterized 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, 9, 10, 11, 12, 13) and two CaM genes (CaM I and II) in birds have been reported (14, 15, 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 vertebrates, 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, 21, 22, 23). Dopamine, on the contrary, can exert opposite effects on the release of these two hormones, being stimulatory to GH secretion via pituitary 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 regulatory 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 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 standard 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 sequences of CaM cDNAs obtained, a slot blot system was set up to quantify total CaM mRNA expression in primary cultures 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 incubation approach. In parallel experiments, the effects of activating cAMP-, protein kinase C (PKC)-, and Ca2+-dependent pathways on CaM mRNA expression at the pituitary level were also examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
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 according 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 purchased from Gibco Life Technologies (Grand Island, NY). PCR-digoxigenin (DIG) labeling kit, CDP-Star, and Anti-DIG-AP (Fab fragments) were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Salmon GnRH and PACAP-38 were obtained from Peninsula Laboratories Inc. (Belmont, CA). LY171555, (±) SKF38393, apomorphine, 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 dissolved 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 according 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, namely CaM-a, CaM-bS, and CaM-bL, were isolated. The cDNA inserts in these clones were sequenced using a BigDye terminator cycle sequencing 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 0.5x 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 ß-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 membrane (Roche) by vacuum blotting. After UV cross-linking, the membrane was incubated in 6x SSC with 1x 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 goldfish and washed three times in washing medium [Medium M199 with Hanks’ salts, 25 mM HEPES, 26 mM NaHCO3, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml Fungizone, and 0.3% BSA (pH 7.2)] to remove blood clots. After that, pituitaries were diced into 0.5-mm fragments using a McILwain tissue chopper (Mickle Laboratory Engineering, 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 penicillin, 100 µg/ml streptomycin, and 0.3% BSA (pH 7.2)]. Pituitary fragments 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 Laboratories, Richmond, CA). Pituitary cells were harvested by filtration through a nylon mesh (~30 µm pore size) followed by centrifugation at 200 x 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 µg/ml streptomycin (pH 7.2)] to give a concentration of approximately 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 µl 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 dopamine D1/D2 analogs were prepared in testing medium [Medium M199 with 25 mM HEPES, 26 mM NaHCO3, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1% BSA (pH 7.2)] at appropriate concentrations 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.

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 membrane was UV cross-linked, incubated for 3 h at 42 C in 50% formamide-containing 5x SSC with 1x 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 subjected to two washes in 2x SSC with 0.1% SDS at room temperature followed by two washes in 0.5x 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 ß-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 ß-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 considered significant at P < 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
After three rounds of library screening, three positive clones, namely CaM-a, CaM-bS, and CaM-bL, were isolated from the goldfish {lambda}-phage pituitary cDNA library. The inserts 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. 1Go). 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 sequence 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.



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  		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.

 
Sequence alignment at the a.a. level (Fig. 2Go) 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 modern bony fish (e.g. perch and medaka) and highly homologous to that reported in early evolved bony fish (e.g. eel), cyclostome (e.g. hagfish), mollusca (e.g. aplysia), cephalochordate (e.g. brachiostoma), urochordate (e.g. ciona), nematode (e.g. Caenorhabditis elegans), porifera (e.g. sponge), and protozoa (e.g. trypanosome). Except for a single a.a. substitution 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, 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 evolutionary constraints/selection pressure imposed by the essential functions of CaM in eukaryotic cells.



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  		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.

 
Although the protein structure of CaM is highly conserved, 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 protein have been reported in the human (8, 9, 31), rat (13, 32), chicken (15, 16), and frog (33), respectively. Based on sequence comparison, these nonallelic CaM genes can be divided into CaM I, CaM II, and CaM III subfamilies (8, 9, 10, 11, 12, 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 occurred 1400 million years ago (35, 36). Using phylogenetic analysis based on nucleotide sequence comparison (Fig. 3Go), 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+-bindings domains are highly conserved at the protein level (Fig. 4AGo). Parallel comparison of nucleotide sequences of these EF-hands (Fig. 4BGo) 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 (≥ 95%) was noted between 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 lineage (39).



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  		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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  		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 algorithm with MacDNAsis 3.1 program.

 
A DIG-labeled probe covering the common sequence of CaM-a and CaM-b was used to examine the tissue distribution of CaM gene expression in the goldfish (Fig. 5AGo). Except for the testes, a 1.6-kb transcript was predominantly expressed 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 the brain and liver; to a lesser extent in the pituitary, gills, kidney, muscle, and ovary; and to a lower level in the intestine, spleen, and testes. An additional transcript of 0.8 kb in size was also detected in the gonads. In the case of the testes, the dominant transcript for CaM was the one with 0.8 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 transcripts (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. 5BGo), 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), indicating that, except in the testes, CaM-bL mRNA is predominantly expressed in the goldfish.



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  		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 goldfish 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, including the telencephalon, olfactory bulbs, hypothalamus, cerebellum, optic tectum, medulla oblongata, 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 experiments, parallel blotting of ß-actin mRNA was used as an internal control.

 
In mammals, CaM is highly expressed in the central nervous 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 examine the brain distribution of CaM mRNA, Northern blot was performed in different brain areas of the goldfish, including the olfactory bulbs, optic tectum, telencephalon, hypothalamus, cerebellum, medulla oblongata, and spinal cord (Fig. 5CGo). 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 oblongata and to a lesser extent in the telencephalon, cerebellum, 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 of neurotransmitters (46, 47, 48, 49). Besides, CaM is also known to increase nitric oxide production in various neuronal pathways by activating neuronal NO synthase coupled to Ca2+ influx through N-methyl-D-aspartate receptors (50). This functional coupling can be modulated by CaM kinase II-induced phosphorylation of neuronal NO synthase associated with N-methyl-D-aspartate receptors via the postsynaptic 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, respectively (Fig. 6Go). 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 preceding section, the scattering pattern of nucleotide substitutions 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.



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  		FIG. 6. Southern blot of goldfish genomic DNA for CaM genes. Genomic DNA prepared from the goldfish was digested with restriction enzymes including PvuII, HindIII, PstI, and HincII. After size fractionation in 0.7% agarose gel and transblotting onto a nylon membrane, positive signals for goldfish CaM genes were detected by hybridization with a DIG-labeled CaM cDNA probe.

 
In this study, using goldfish pituitary cells as a cell model, signal transduction mechanisms regulating CaM gene expression at the pituitary level were examined using a pharmacological approach. In this case, pituitary cells were exposed to increasing doses of the adenylate cyclase activator forskolin (0.1–30 µM, Fig. 7AGo), PKC-activator TPA (10–300 nM, Fig. 7BGo) and Ca2+ ionophore A23187 (1 nM, 10 nM, and 10 µM, Fig. 8Go). Treatment with forskolin and TPA dose-dependently increased CaM mRNA levels in goldfish pituitary cells, suggesting that cAMP- and PKC-dependent pathways 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. Unlike forskolin and TPA, A23187 was effective in reducing CaM mRNA expression in the nanomolar dose range (Fig. 8AGo). When a high dose of A23187 (10 µM) was used, basal levels of CaM mRNA were almost undetectable (Fig. 8BGo). 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 prolonged 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+ ionophores). In previous studies by other research groups, micromolar 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 effects. Because CaM is known to bind with L-type Ca2+ channel 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 become less sensitive to Ca2+ stimulation by reducing CaM expression.



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  		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 forskolin (0.1–30 µM, A) and TPA (10–300 nM, 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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  		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 µM, 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).

 
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 µM, Fig. 9AGo) and PACAP (0.001–10 µM, Fig. 9BGo) 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 µM, Fig. 10AGo). In contrast to GnRH and PACAP, apomorphine suppressed CaM mRNA levels in a dose-related fashion. In the presence of apomorphine (1 µM), the stimulatory effects of GnRH (1 µM) and PACAP (1 µM) on CaM mRNA expression were totally abolished (Fig. 10BGo).



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  		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 µM, A) and PACAP (0.001–10 µM, B) for 48 h under static incubation. After drug treatment, total RNA samples were extracted and assayed for CaM mRNA levels as described in Materials and Methods. In these experiments, parallel blotting of ß-actin mRNA 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). Representative results of slot blots are also included in these figures for reference.

 


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  		FIG. 10. Dopaminergic inhibition of CaM mRNA expression in goldfish pituitary cells. A, Dopaminergic inhibition of basal CaM mRNA expression. Pituitary cells were incubated for 48 h with increasing doses (0.001–10 µM) of the nonselective dopamine agonist apomorphine (Apo). B, Dopaminergic inhibition of GnRH- and PACAP-stimulated CaM mRNA expression. Pituitary cells were incubated for 48 h with GnRH (1 µM) or PACAP (1 µM) with or without simultaneous treatment of apomorphine (1 µM). 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).

 
To elucidate the receptor specificity mediating apomorphine’s action on CaM mRNA expression, goldfish pituitary cells were treated with increasing doses (0.01–10 µM) of the dopamine D1 agonist SKF38393 or D2 agonist Ly171555 (Fig. 11AGo). The dose-dependence of apomorphine inhibition on CaM mRNA expression was mimicked by the D2 agonist Ly171555 but not by the D1 agonist SKF38393. Besides, the inhibitory action of apomorphine (1 µM) on CaM mRNA expression was blocked by the dopamine D2 antagonist domperidone (1 µM, Fig. 11BGo) and the D1 antagonist SCH23390 was not effective in this regard (data not shown). Similar to apomorphine, the D2 agonist Ly171555 (1 µM) also blocked the stimulatory effects of GnRH (1 µM) and PACAP (1 µM) on CaM mRNA expression (Fig. 12Go).



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  		FIG. 11. Receptor specificity of dopaminergic inhibition of CaM mRNA expression in goldfish pituitary cells. A, Effects of the dopamine 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 µM) or Ly171555 (0.01–10 µM). B, Blockade of dopaminergic inhibition of CaM mRNA expression by the D2 antagonist domperidone (Domp). Pituitary cells were incubated for 48 h with the nonselective dopamine agonist apomorphine (Apo, 1 µM) in the presence or absence of domperidone (1 µM). 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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  		FIG. 12. Blockade of GnRH- and PACAP-stimulated CaM mRNA expression in goldfish pituitary cells by the dopamine D2 agonist Ly171555. Pituitary cells were incubated for 48 h with GnRH (1 µM) or PACAP (1 µM) in the presence or absence of Ly171555 (1 µM). 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).

 
These results, taken together, confirm that dopamine’s action on CaM mRNA expression is mediated through pituitary D2 receptors. The present study has also demonstrated for the first time that CaM gene expression in the pituitary is under the control of hypothalamic factors. Apparently, the stimulatory effects of GnRH and PACAP on CaM gene expression are negatively regulated by dopaminergic 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 stimulatory 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 mobilization (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 modulation 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 regulate 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 and CaM-b are putative members of the CaM I gene subfamily. 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. Besides, 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 dopaminergic 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.


   Footnotes
 
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.

Abbreviations: a.a., Amino acid; [Ca2+]i, intracellular Ca2+; CaM, calmodulin; DIG, digoxigenin; DMSO, dimethylsulfoxide; GTH, gonadotropin; LSD, least significance difference; ORF, open reading frame; PACAP, pituitary adenylate cyclase-activating polypeptide; PKC, protein kinase C; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TPA, 12-O-tetra-decanoyl-phorbol-13-acetate; UTR, untranslated region.

Received May 10, 2004.

Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Van Eldik LJ, Roberts DM 1988 Calcium modulated proteins in pathophysiology. In: Thompson MP, ed. Calcium-binding proteins. Vol II. Biological functions. Washington, DC: CRC Press; 401–403
  2. Van Eldik LJ, Watterson, DM 1998 Calmodulin and signal transduction. In: Van Eldik LJ, Watterson DM, eds. Calmodulin and signal transduction. New York: Academic Press; 1–19
  3. Bachs O, Agell N 1995 Calmodulin and calmoudlin-binding proteins in the cell nucleus. In: Bachs O, Agell N, eds. Calcium and calmodulin function in the cell nucleus. New York: Springer-Verlag; 217–240
  4. Cyert MS 2001 Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae. Annu Rev Genet 35:647–672[CrossRef][Medline]
  5. Jurado LA, Chockalingam PS, Jarrett HW 1999 Apocalmodulin. Physiol Rev 79:661–682[Abstract/Free Full Text]
  6. Chin D, Means AR 2000 Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322–328[CrossRef][Medline]
  7. Nelson MR, Chazin WJ 1998 Calmodulin as a calcium sensor. In: Van Eldik LJ, Watterson DM, eds. Calmodulin and signal transduction. New York: Academic Press; 17–65
  8. SenGupta B, Friedberg F, Detera-Wadleigh SD 1987 Molecular analysis of human and rat calmodulin complementary DNA clones. Evidence for additional active genes in these species. J Biol Chem 262:16663–16670[Abstract/Free Full Text]
  9. Fischer R, Koller M, Flura M, Mathews S, Strehler-Page MA, Krebs J, Penniston JT, Carafoli E, Strehler EE 1988 Multiple divergent mRNAs code for a single human calmodulin. J Biol Chem 263:17055–17062[Abstract/Free Full Text]
  10. Koller M, Schnyder B, Strehler EE 1990 Structural organization of the human CaM III calmodulin gene. Biochim Biophys Acta 1087:180–189[Medline]
  11. Berchtold MW, Egli R, Rhyner JA, Hameister H, Strehler EE 1993 Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24–q31, 2p21.1-p21.3, and 19q13.2-q13.3. Genomics 16:461–465[CrossRef][Medline]
  12. Toutenhoofd SL, Foletti D, Wicki R, Rhyner JA, Garcia F, Tolon R, Strehler EE 1998 Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3. Cell Calcium 23:323–338[CrossRef][Medline]
  13. Nojima H 1989 Structural organization of multiple rat calmodulin genes. J Mol Biol 208:269–282[CrossRef][Medline]
  14. Putkey JA, Ts’ui KF, Tanaka T, Lagace L, Stein JP, Lai EC, Means AR 1983 Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone. J Biol Chem 258:11864–11870[Abstract/Free Full Text]
  15. Simmen RC, Tanaka T, Ts’ui KF, Putkey JA, Scott MJ, Lai EC, Means AR 1985 The structural organization of the chicken calmodulin gene. J Biol Chem 260:907–912[Abstract/Free Full Text]
  16. Ye Q, Berchtold MW 1997 Structure and expression of the chicken calmodulin I gene. Gene 194:63–68[CrossRef][Medline]
  17. Lagace L, Chandra T, Woo SL, Means AR 1983 Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA. J Biol Chem 258:1684–1688[Abstract/Free Full Text]
  18. Trudeau VL 1997 Neuroendocrine regulation of gonadotrophin II release and gonadal growth in the goldfish, Carassius auratus. Rev Reprod 2:55–68[Abstract]
  19. Peng C, Peter RE 1997 Neuropeptide regulation of growth hormone secretion and growth in fish. Zool Stud 36:79–89
  20. Marchant TA, Chang JP, Nahorniak CS, Peter RE 1989 Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish. Endocrinology 124:2509–2518[Abstract]
  21. Wong AOL, Li WS, Lee EK, Leung MY, Tse LY, Chow BK, Lin HR, Chang JP 2000 Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish. Biochem Cell Biol 78:329–343[CrossRef][Medline]
  22. Chang JP, Wirachowsky NR, Kwong P, Johnson JD 2001 PACAP stimulation of gonadotropin-II secretion in goldfish pituitary cells: mechanisms of action and interaction with gonadotropin releasing hormone signalling. J Neuroendocrinol 13:540–550[CrossRef][Medline]
  23. Wong AOL, Leung MY, Shea WL, Tse LY, Chang JP, Chow BK 1998 Hypophysiotropic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: immunohistochemical demonstration of PACAP in the pituitary, PACAP stimulation of growth hormone release from pituitary cells, and molecular cloning of pituitary type I PACAP receptor. Endocrinology 139:3465–3479[Abstract/Free Full Text]
  24. Wong AOL, Van Goor F, Chang JP 1994 Entry of extracellular calcium mediates dopamine D1-stimulated growth hormone release from goldfish pituitary cells. Gen Comp Endocrinol 94:316–328[CrossRef][Medline]
  25. Wong AOL, van der Kraak G, Chang JP 1994 Cyclic 3',5'-adenosine monophosphate mediates dopamine D1-stimulated growth hormone release from goldfish pituitary cells. Neuroendocrinology 60:410–417[Medline]
  26. Chang JP, Van Goor F, Lo A, Johnson JC, Jobin RM, Goldberg JI 1997 Signal transduction in gonadotropin-II secretion in goldfish pituitary cells. In: Kikuyama Ska S, ed. Advances in comparative endocrinology. Bologna: Monduzzi Editore; 29–33
  27. Chang JP, Johnson JD, Van Goor F, Wong CJ, Yunker WK, Uretsky AD, Taylor D, Jobin RM, Wong AOL, Goldberg JI 2000 Signal transduction mechanisms mediating secretion in goldfish gonadotropes and somatotropes. Biochem Cell Biol 78:139–153[CrossRef][Medline]
  28. Chang JP, Abele JT, Van Goor F, Wong AO, Neumann CM 1996 Role of arachidonic acid and calmodulin in mediating dopamine D1- and GnRH-stimulated growth hormone release in goldfish pituitary cells. Gen Comp Endocrinol 102:88–101[CrossRef][Medline]
  29. Jobin RM, Neumann CM, Chang JP 1996 Roles of calcium and calmodulin in the mediation of acute and sustained GnRH-stimulated gonadotropin secretion from dispersed goldfish pituitary cells. Gen Comp Endocrinol 101:91–106[CrossRef][Medline]
  30. Lee EK, Chan VC, Chang JP, Yunker WK, Wong AOL 2000 Norepinephrine regulation of growth hormone release from goldfish pituitary cells. I. Involvement of {alpha}2 adrenoreceptor and interactions with dopamine and salmon gonadotropin-releasing hormone. J Neuroendocrinol 12:311–322[CrossRef][Medline]
  31. Senterre-Lesenfants S, Alag AS, Sobel ME 1995 Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin-I gene. J Cell Biochem 58:445–454[CrossRef][Medline]
  32. Nojima H, Sokabe H 1987 Structure of a gene for rat calmodulin. J Mol Biol 193:439–445[CrossRef][Medline]
  33. Chien YH, Dawid IB 1984 Isolation and characterization of calmodulin genes from Xenopus laevis. Mol Cell Biol 4:507–513[Abstract/Free Full Text]
  34. Nojima H 1987 Molecular evolution of the calmodulin gene. FEBS Lett 217:187–190[CrossRef][Medline]
  35. Baba ML, Goodman M, Berger-Cohn J, Demaille JG, Matsuda G 1984 The early adaptive evolution of calmodulin. Mol Biol Evol 1:442–455[Abstract]
  36. Hardy DO, Bender PK, Kretsinger RH 1988 Two calmodulin genes are expressed in Arbacia punctulata. An ancient gene duplication is indicated. J Mol Biol 199:223–227[CrossRef][Medline]
  37. Erickson BW, Watterson DM, Marshak DR 1980 Sequence alignment of calmodulin domains by metric analysis. Ann NY Acad Sci 356:378–379[CrossRef][Medline]
  38. Watterson DM, Sharief F, Vanaman TC 1980 The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem 255:962–975[Abstract/Free Full Text]
  39. Larhammar D, Risinger C 1994 Molecular genetic aspects of tetraploidy in the common carp Cyprinus carpio. Mol Phylogenet Evol 3:59–68[CrossRef][Medline]
  40. Brown IR, Gurd JW, Ni B 1992 Molecular cloning of neuronal calmodulin mRNA species. Biochem Soc Trans 20:382–385[Medline]
  41. Palfi A, Kortvely E, Fekete E, Kovacs B, Varszegi S, Gulya K 2002 Differential calmodulin gene expression in the rodent brain. Life Sci 70:2829–2855[CrossRef][Medline]
  42. Toutenhoofd SL, Strehler EE 2000 The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools? Cell Calcium 28:83–96[CrossRef][Medline]
  43. Saimi Y, Kung C 2002 Calmodulin as an ion channel subunit. Annu Rev Physiol 64:289–311[CrossRef][Medline]
  44. Sola C, Barron S, Tusell JM, Serratosa J 1999 The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells. Prog Neurobiol 58:207–232[CrossRef][Medline]
  45. Sola C, Barron S, Tusell JM, Serratosa J 2001 The Ca2+/calmodulin system in neuronal hyperexcitability. Int J Biochem Cell Biol 33:439–455[CrossRef][Medline]
  46. DeLorenzo RJ 1980 Role of calmodulin in neurotransmitter release and synaptic function. Ann NY Acad Sci 356:92–109[Medline]
  47. DeLorenzo RJ 1982 Calmodulin in neurotransmitter release and synaptic function. Fed Proc 41:2265–2272[Medline]
  48. Brailoiu E, Miyamoto MD, Dun NJ 2002 Calmodulin increases transmitter release by mobilizing quanta at the frog motor nerve terminal. Br J Pharmacol 137:719–727[CrossRef][Medline]
  49. Fujisawa H, Yamauchi T, Nakata H, Okuno S 1984 Role of calmodulin in neurotransmitter synthesis. Fed Proc 43:3011–3014[Medline]
  50. Dawson VL, Dawson TM 1996 Nitric oxide neurotoxicity. J Chem Neuroanat 10:179–190[CrossRef][Medline]
  51. Watanabe Y, Song T, Sugimoto K, Horii M, Araki N, Tokumitsu H, Tezuka T, Yamamoto T, Tokuda M 2003 Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase. Biochem J 372:465–471[CrossRef][Medline]
  52. Friedberg F, Rhoads AR 2001 Evolutionary aspects of calmodulin. IUBMB Life 51:215–221[Medline]
  53. Rhyner JA, Ottiger M, Wicki R, Greenwood TM, Strehler EE 1994 Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2. Eur J Biochem 225:71–82[Medline]
  54. Bai G, Nichols RA, Weiss B 1992 Cyclic AMP selectively up-regulates calmodulin genes I and II in PC12 cells. Biochim Biophys Acta 1130:189–196[Medline]
  55. Bosch M, Lopez-Girona A, Bachs O, Agell N 1994 Protein kinase C regulates calmodulin expression in NRK cells activated to proliferate from quiescence. Cell Calcium 16:446–454[CrossRef][Medline]
  56. Clapham DE 1995 Calcium signaling. Cell 80:259–268[CrossRef][Medline]
  57. Chang JP, de Leeuw R 1990 In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible roles of extracellular calcium and arachidonic acid metabolism? Gen Comp Endocrinol 80:155–164[CrossRef][Medline]
  58. Chang JP, Freedman GL, de Leeuw R 1990 Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. II. Extracellular calcium dependence and dopaminergic inhibition of gonadotropin responses. Gen Comp Endocrinol 77:274–282[CrossRef][Medline]
  59. Mori MX, Erickson MG, Yue DT 2004 Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels. Science 304:432–435[Abstract/Free Full Text]
  60. Chang JP, Van Goor F, Jobin RM, Lo A 1996 GnRH signaling in goldfish pituitary cells. Biol Signals 5:70–80[Medline]
  61. Van Goor F, Goldberg JI, Chang JP 1998 Dopamine-D2 actions on voltage-dependent calcium current and gonadotropin-II secretion in cultured goldfish gonadotrophs. J Neuroendocrinol 10:175–186[Medline]



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