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Department of Physiology (L.A.A.), Southern Illinois University School of Medicine, Carbondale, Illinois 62901-6512; Department of Pharmaceutical Sciences (G.V.S.), Texas Tech University Health Sciences Center, Amarillo, Texas 79106; and Department of Molecular and Integrative Physiology (J.L.V.), University of Kansas Medical Center, Kansas City, Kansas 66160-7401
Address all correspondence and requests for reprints to: Dr. Lydia A. Arbogast, Department of Physiology, School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-6512. E-mail: larbogast{at}som.siu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A likely role for the hypothalamic CT receptors is in the control of anterior pituitary function. CT or CT-like peptides can alter circulating PRL levels in vivo (15, 16, 17). The physiological significance of an inhibitory role is supported by the marked increase in circulating PRL levels of ovariectomized rats after administration of an anti-sCT serum (18). An sCT-like immunoreactive peptide, synthesized and secreted by the anterior pituitary, likely contributes to the inhibition of PRL secretion by a paracrine action within the anterior pituitary gland (3, 4, 12, 19). In addition, some evidence indicates that one or more sCT-like substances may also exert an indirect effect, possibly at the hypothalamic level, to control PRL release. First, the increase in PRL release induced by anti-sCT serum in ovariectomized rats is more profound in vivo than in vitro (18). Second, the sCT-induced decrease in PRL levels in male rats in prevented by median eminence lesions (15). Indeed, both the brain and pituitary are potential sources of sCT-like compounds (4, 5, 18). A possible site for sCT’s action is the tuberoinfundibular dopaminergic neurons. The dopamine released from these neurons is well established as a major PRL inhibiting hormone (20). Moreover, these dopaminergic neurons have their cell bodies in the arcuate nucleus of the hypothalamus (21), which has a high expression of CT receptor and high density of sCT binding sites (6, 22). The use of an in vitro model allows for isolation of brain area of interest and enhances the ability to explore the intracellular signaling mechanisms. Indeed, dopaminergic neurons in fetal hypothalamic cell cultures respond to many hormones and neuropeptides in a manner similar to the tuberoinfundibular dopaminergic neurons in an adult animal (23, 24, 25, 26, 27).
The primary goal of this study was to evaluate the effect of sCT on dopaminergic neurons in fetal hypothalamic cell cultures and to determine the intracellular signaling pathway(s) involved. Tyrosine hydroxylase (TH) is the rate limiting enzyme in catecholamine biosynthesis (28), and this study focused on this key control point in the dopaminergic neurons. The specific aims were: 1) to determine the effect of different concentrations of sCT on TH activity and cellular cAMP content; 2) to assess the expression of sCT receptors; 3) to investigate the intracellular signaling pathway by which sCT alters TH activity by using specific protein kinase inhibitors; and 4) to examine the effect of sCT on the phosphorylation state of TH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal hypothalamic cell cultures
Fetal hypothalamic cells were cultured as described previously
(23, 27, 29). Briefly, the medioventral hypothalami of day 19–20 rat
fetuses were excised, and the hypothalamic cells were dispersed with
trypsin. After dispersion, the cells were resuspended in a modified
phenol red-free, high glucose Dulbecco’s medium containing 2.5%
heat-inactivated FBS and 5% heat inactivated horse serum and were
plated at a density of 150,000 cells/well in 96-well plates for
determination of TH activity, or 1 x 106 cells/well
in 24-well plates for radiolabeled phosphate incorporation, cellular
cAMP content determinations or RNA isolation. After 18–24 h, the serum
containing medium was replaced with a serum-free, phenol red-free,
chemically defined, high glucose modified DMEM (27). The cells
were maintained under an atmosphere of 95% air-5% CO2.
The medium was changed every 1–2 days as needed.
Determination of TH activity
TH activity was determined on day 12–14 in vitro.
The hypothalamic cells were preincubated for 30–45 min in phenol
red-free Earles’ Balanced Salt Solution (Life Technologies, Inc., Grand Island, NY) with 20 µM tyrosine and 20
mM potassium chloride added. When indicated, Rp-adenosine
3'5'-cyclic monophosphothioate triethylamine (Rp-cAMPS; Research Biochemicals International, Natick, MA),
N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide
dihydrochloride (H-8; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), Calphostin C (Kamiya Biomedical
Company, Thousand Oaks, CA; BIOMOL Research Laboratories, Inc.), 1-(6-((17ß-3-methoxyestra-1,3,5
(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
(U-73122, BIOMOL) or 1-(6-((17ß-3-methoxyestra-1,3,5
(10)-trien-17-yl)amino)hexyl-2,5-pyrrolidine-dione (U-73343;
BIOMOL) at the indicated concentration was included in the
preincubation medium. For experiments to reduce available intracellular
calcium, the preincubation medium was phenol red-free and calcium-free
Earle’s Balanced Salt Solution (Life Technologies, Inc.)
containing 1 mM ethylene glycol-bis
(ß-aminoethylether)N,N,N'N'-tetra-acetic acid (EGTA), 20
µM tyrosine and 20 mM potassium chloride.
Where indicated,
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetraacetoxy-methylester (BAPTA/AM; Molecular Probes, Inc., Eugene, OR) or thapsigargin (Molecular Probes, Inc.) at the indicated concentration was included in the
preincubation medium. The preincubation medium was removed and 120 µl
medium containing 100 µM brocresine
(4-bromo-hydroxybenzyloxyamine; gift from American Cyanamid Co., Pearl River, NY), an aromatic L-amino acid
decarboxylase inhibitor, and the indicated concentration of rat CT,
human CT, sCT (Bachem California, Inc., Torrance, CA),
forskolin (Sigma Chemical Co., St. Louis, MO), phorbol
12-myristate 13-acetate (PMA; Research Biochemicals International), 4-
-phorbol 12-myristate 13-acetate
(4--PMA; Research Biochemicals International) with or
without the kinase inhibitors was added. For experiments to reduce
available intracellular calcium, the medium was calcium-free with 1
mM EGTA and thapsigargin, but not BAPTA/AM, included in the
incubation medium as indicated. The cells were incubated for 1 h
at 37 C under an atmosphere of 10% CO2-90% air. The
medium was the removed, acidified with 12 µl 1 N
perchloric acid and frozen until samples were analyzed for
L-dihydroxyphenylalanine (DOPA) accumulation in the media by HPLC with
electrochemical detection (30).
Determination of cAMP content
The hypothalamic cells were preincubated for 15 min with 1 ml
Earle’s Balanced Salt Solution containing 20 µM tyrosine
and an additional 20 mM potassium chloride. The
preincubation was removed and medium containing 1 mM
isobutylmethylxanthine (IBMX; Sigma Chemical Co.), a
phosphodiesterase inhibitor, and the appropriate concentrations of sCT
was added. After 15 min, the medium was removed and assayed for cAMP
levels. Immediately thereafter, 500 µl 3% perchloric acid was added
to the cells. The cells were subjected to a freeze-thaw cycle, scraped
from the plate, and homogenized by sonication. The pH was adjusted to
pH 6.0 with 30% potassium bicarbonate. The samples were centrifuged at
10,000 x g for 10 min and the supernatant transferred
to the assay. The cAMP contents of the medium and cells were assayed
separately according to the manufacturer’s directions using a kit from
Diagnostic Products, Inc., (Los Angeles, CA) with
3H cAMP as the tracer. The lower limit of detection for the
assay was 0.55 pmol/1 x 106 cells. Each sample was
analyzed in duplicate and all samples from the same experiment were
analyzed in a single assay to avoid intraassay variability.
RT-PCR and Southern blot for CT receptor
The hypothalamic cells (2 x 106 cells) were
lysed in 800 µl RNAzol B (Tel-Test, Inc., Friendswood,
TX) and total RNA isolated according to the protocol provided by the
manufacturer. RNA (5 µg) was used for first strand complementary DNA
(cDNA) synthesis using the Superscript Preamplification System
(Life Technologies, Inc.) with oligo dT primers. The cDNA
was amplified through 30 cycles of PCR with Taq polymerase
using 62.5 C as the annealing temperature. The 21 mer primers were
described by Albrandt et al. (7) and were 5'-GTT GAG GTT GTG
CCC AAT GGA-3' and 5' CCC TGG AAA TGA ATC AGA GAG-3'. These primers
generate a 545-bp product for the C1a receptor and a 656-bp product for
the C1b receptor. The PCR samples were run along with a 100-bp ladder
(Life Technologies, Inc.) in an agarose gel containing
ethidium bromide. After depurination of DNA fragments with 0.25
M HCl, denaturation with buffer containing 0.5
M sodium hydroxide and 1.5 M sodium chloride
and neutralization with buffer containing 0.5 M Tris HCl pH
7.0 and 1.5 M sodium chloride, the DNA from the PCR
reaction was transferred to a NYTRAN membrane using the
Schleicher & Schuell Turboblotter downward transfer
apparatus (Midwest Scientific, St. Louis, MO). The PCR product was
verified by Southern blot hybridization. The partial pituitary cDNA
probe for sCT receptor was generated by RT-PCR of a total RNA
preparation from rat anterior pituitary glands using gene-specific
sense and antisense amplimers as previously described (7). The
amplified cDNA was subcloned in pGem-T vector (Promega Corp., Madison, WI) and the DNA sequence was determined. The
partial pituitary CT receptor cDNA was 546 bp long and displayed 99%
homology with rat C1a receptor (544/546) and C1b receptor (503/504)
cDNA sequences (7).
Evaluation of radiolabeled phosphate incorporation into TH
The procedure to determine the phosphorylation state of TH is
adapted from the procedures described by Cahill and Perlman (31) and
Porter (32). The basic incubation medium contained 116 mM
sodium chloride, 5.3 mM potassium chloride, 2.5
mM calcium chloride, 0.8 mM magnesium sulfate,
5 mM glucose, and 25 mM HEPES buffer pH 7.4.
Fetal hypothalamic cells (1 x 106 cells/well) were
washed 2 x 5 min with medium and then incubated for 1 h in
0.25 ml medium containing 32P orthophosphoric acid (500
µC/ml). The radiolabeled phosphate was the sole source of phosphate
in the medium. Hypothalamic cells were then incubated an additional 30
min with 1 mM Rp-cAMPS as indicated and subsequently for 15
min with 10 nM sCT and/or 1 mM Rp-cAMPS as
indicated. At the end of the incubation time, cells were quickly rinsed
four times with medium. Ice-cold phosphatase-inhibiting buffer (80
µl), consisting of 30 mM potassium phosphate, pH 7.5, 25
mM sodium fluoride, 5 mM sodium pyrophosphate,
0.5 µM okadaic acid, 1 mM EDTA, 0. 5
mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin and
0.5% IGEPAL CA-630, was added to each well. The cells were subjected
to a freeze-thaw cycle and scraped from the plate. Individual wells
were rinsed with an additional 40 µl of ice-cold phosphatase
inhibiting buffer. Homogenates were centrifuged for 1 min and 100 µl
of the homogenate was used for immunoprecipitation with 2 µl TH
antibody (East Acre Biologicals, Southbridge, MA) at 4 C for 18 h.
The antigen-antibody complex was then precipitated at 4 C for 30 min
with 100 µl of a 10% solution of Staphylococcus aureus
cells (Pansorbin; Calbiochem). The S.
aureus-antibody-TH pellet was vigorously washed 4 times, sonicated
in 20 µl of sample buffer, boiled for 2 min, and centrifuged at
10,000 x g for 5 min. The sample (20 µl) was subject
to electrophoresis on a 7.5% polyacrylamide gel slab using a buffer
containing 0.375 M Tris buffer pH 8.8 and 0.1% SDS. The
gels was calibrated with molecular weight standards between 14,000 and
100,000. After electrophoresis, the slab gel was dried and exposed to a
phosphor screen (Molecular Dynamics, Inc., Sunnyvale, CA).
The phosphor screen was scanned with a Molecular Dynamics, Inc. Phosphorimager 445SI. Individual TH bands on the gels were
identified and the volume quantified with the associated FragmeNT
analysis software. Total protein concentration was determined with 10
µl of the original homogenate by the method of Bradford (33), and the
volume values obtained from the phosphorimager were normalized to the
total protein content of the individual samples. Subsequently, the gels
were exposed at -70 C for 3–4 h to Kodak XAR-2 film
(Eastman Kodak Co., Rochester, NY) using an intensifying
screen, the film was developed, and the 32P-labeled TH
bands on the gel localized.
Statistical analysis
To normalize data, experimental values were adjusted to a
percentage of control values for individual experiments. For TH
activity, the control values were a mean of 9–12 wells and each
experimental value was the mean of triplicate wells for each
hypothalamic culture. For radiolabeled phosphate incorporation
experiments, control and experimental values were determined from
duplicate wells. The results are expressed as the mean ±
SE of determinations from five to eight different
experiments. Data were evaluated by ANOVA, and multiple comparisons
were made with Fisher’s least significant procedures (34, 35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; top panel). There was no
significant increase in enzyme activity with 1 and 10 pM
sCT, but 100 pM sCT increased TH activity to 130% of
control values. The sCT-induced increase in TH activity reached near
plateau levels between 1–100 nM sCT with values 145–157%
of control levels. In a separate experiment, the hypothalamic cells
were incubated with either rat CT or sCT to determine if the homologous
hormone would have the same effect. Rat CT and sCT (10 nM)
increased TH activity similarly to 142 ± 6% and 149 ± 4%
of control values, respectively, indicating CT from both species was
effective in increasing TH activity.
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; bottom panel). A
2.2-fold increase in cellular cAMP accumulation was observed with 100
pM sCT, whereas 1 and 10 nM sCT resulted in a
4.7-fold and 9.5-fold increase, respectively. Although the cell extract
contained approximate 90% of the accumulated cAMP, cAMP accumulation
in the medium showed an identical trend. Control cAMP accumulation
(pmol/1 x 106 cells/15 min) was 4.06 ± 0.34 and
cAMP accumulation was increased to 7.02 ± 0.22, 14.78 ±
0.84 and 28.18 ± 1.37 with 100 pM, 1 nM
and 10 nM sCT, respectively. The sCT-induced increase in
cAMP occurred within the same concentration range as the changes in TH
activity.
Expression of CT receptor in hypothalamic cells
RT-PCR was used to determine whether the CT receptor was present
in the hypothalamic cell cultures. Expression of the CT receptor was
similar in adult mediobasal hypothalamic tissue and in fetal
hypothalamic cell cultures (Fig. 2, bottom panel). Both the C1a and C1b forms of the CT receptor
were expressed, as evidenced by the 545-bp and 656-bp PCR products,
respectively. No PCR product was detected with the omission of reverse
transcriptase. The identity of the PCR products were confirmed by
Southern blot analysis using a specific 32P-labeled probe
for the CT receptor (Fig. 2, top panel).
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).
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, top panel). TH activity was
not significantly different from control values in the presence of 100
µM Rp-cAMPS with or without sCT. The 1 mM
concentration of Rp-cAMPS completely abolished the response and also
reduced basal TH activity to 69% of control values. In a similar
manner, H-8 also suppressed the sCT-induced elevation in TH activity in
a concentration dependent manner (Fig. 4, bottom panel). The
response was significantly suppressed with 0.5 µM H-8 and
completely blocked with 5 µM H-8. At the highest
concentration (50 µM), H-8 alone suppressed TH activity
to 82% of control values, in addition to inhibiting the sCT-induced
rise. These data suggest that cAMP and protein kinase A are involved in
the sCT-induced increase in TH activity in hypothalamic cells.
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Effect of sCT on TH phosphorylation
Given the rapid action of sCT on TH activity, the next experiment
was to examine whether there was a change in radiolabeled phosphate
incorporation into the TH molecule after sCT treatment and if cAMP was
involved in the changes in phosphate incorporation. The
autoradiographic film showed a band at approximately a molecular weight
of 60,000, corresponding to TH. As shown in the inset in
Fig. 5, the intensity of this band
increased after sCT treatment and was reduced by prior and concomitant
treatment with Rp-cAMPS. The 60,000 molecular weight band was
completely absent when TH antiserum was omitted during the
immunoprecipitation procedure (data not shown). sCT treatment increased
32P incorporation into TH to 169% of control levels and 1
mM Rp-cAMPS completely prevented this increase (Fig. 5).
Rp-cAMPS alone had no effect on phosphate incorporation.
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, top panel), indicating that
sCT is not coupled to the protein kinase C pathway to exert its effect
on TH activity in the hypothalamic cells.
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, bottom panel). In contrast, 4--PMA, a negative control
for phorbol ester activation of protein kinase C, did not alter TH
activity. Calphostin C (1 µM) completely prevented
the decrease in TH activity induced by 1 and 10 nM PMA and
partially blocked the inhibitory effect of 0.1 and 1 µM
PMA.
The involvement of intracellular calcium mobilization was evaluated in
a calcium-free EGTA containing medium by depletion of the intracellular
calcium stores and by chelating intracellular calcium. The hypothalamic
cells were preloadedwith BAPTA/AM, a cell permeant calcium selective
chelator (39), for 45 min and then incubated with sCT for 1 h to
determine TH activity. Treatment with 10 nM sCT increased
TH activity in wells without BAPTA/AM treatment to 152% of control
values (Fig. 7, top right
panel). Pretreatment of the cells with 1 µM BAPTA/AM
did not significantly alter TH activity or the ability of sCT to
increase TH activity. However, even though basal TH activity was
decreased to 68% of control values after 10 µM BAPTA/AM
pretreatment, sCT increased TH activity to 163% of the
BAPTA/AM-treated wells. Thapsigargin, an inhibitor of the endoplasmic
reticulum calcium ATPase, an enzyme responsible for pumping the calcium
into intracellular stores (40) was used to evaluate the depletion of
intracellular calcium stores. Thapsigargin caused a modest decrease in
TH activity, but did not alter the sCT-induced increase in TH activity
(Fig. 7, top left panel). It should also be noted that sCT
caused a similar increase in TH activity with calcium-containing medium
(Fig. 7, bottom panel) and calcium-free, EGTA-containing
medium (Fig. 7, top left panel), suggesting that
extracellular calcium does not have a role in the response.
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, bottom panel).
The structurally related compound, U73343, which does not inhibit
phopholipase C, modestly reduced basal and sCT-induced levels of TH
activity. |
Discussion |
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This study identifies dopaminergic neurons as a site of action for CT in fetal hypothalamic cells. The marked stimulatory effect was observed on the enzyme, TH, which serves as a key control point in dopamine biosynthesis. sCT caused a significant increase in TH activity at concentrations as low as 100 pM and a near maximal effect at 1–10 nM. The concentration dependence for the effect of sCT on TH was very similar to concentrations of sCT that displaced 125I sCT binding to brain or hypothalamic membranes (13, 14) and the cloned rat CT receptor (7, 8). Thus, the sensitivity for the biological response reported in this study corresponds to CT receptor binding characteristics reported by other investigators (13, 14). Both the homologous rat CT and the heterologous sCT were effective in increasing TH activity, even though the amino acid sequences of these peptides are not identical (5). It is not clear what peptide serves as the endogenous ligand for the sCT-sensitive receptor(s) in the hypothalamus. The principal product of the CT gene itself in the brain is calcitonin gene-related peptide (CGRP), which has binding sites with a specificity and distribution distinct from the conventional CT binding sites (1). However, peptides that are biologically and immunologically similar to sCT have been identified in the brain (5, 42). Moreover, the identification of a sCT-like immunoreactive peptide, which is synthesized and release by the anterior pituitary (4, 18), raises the possibility of pituitary control of hypothalamic function.
Both the C1a and C1b forms of the CT receptor were expressed similarly in the hypothalamic cell cultures and the mediobasal hypothalamus of adult rats, suggesting that the findings in this study may have implications in the control of hypothalamic function in vivo. This high level of CT receptor expression in this study agrees with previous reports of significant CT receptor messenger RNA levels (8, 22) and a high density of CT binding sites (1, 6) in the hypothalamus. The primers used in this study were designed by Albrandt et al. (7) and flank the 111 bp nucleotide insert in the C1b receptor isoform found predominantly in the brain (7, 8). Thus, the primers were able to recognize both forms of the receptor and differentiate between the isoforms by the size of the PCR product. The specific receptor subtype associated with activation of the dopaminergic neurons was not identified in this study, but the receptor recognizes both rat CT and sCT. These data argue against the involvement of the C1b isoform, which has a poor interaction with human and rat CT (8). Furthermore, the concentration response characteristics for human CT and sCT are similar to sCT binding and cAMP production profiles of the cloned C1a CT receptor (8). In general, these data support the notion that the C1a CT receptor mediates the effects of sCT on the hypothalamic dopaminergic neurons. This study did not address the issue of whether sCT receptors are located on dopaminergic neurons, but the response occurs rapidly, supporting the notion of a direct action.
Our results indicate that a CT receptor coupled to adenylate cyclase activation is present in the hypothalamic cell culture, as evidenced by the sCT-induced concentration dependent increase in cellular cAMP content. These data contrast with previous studies showing that high concentrations of sCT cause a decrease in adenylate cyclase activity in hypothalamic membrane preparations (13, 14). However, when both of the identified isoforms of the CT receptor were transfected into COS cells, sCT caused a concentration-dependent increase in cAMP production (7, 8). Thus, the use of intact cells may be required for functional coupling to adenylate cyclase.
The increased TH activity in hypothalamic dopaminergic neurons appears to be exclusively due to activation of protein kinase A. The involvement of the adenylate cyclase/cAMP/protein kinase A pathway based is supported by a couple of lines of evidence. First, sCT caused a concentration-dependent increase in cellular cAMP content, indicating activation of adenylate cyclase by sCT. Moreover, the range of sCT concentrations necessary to elicit increased cAMP content was similar to that required to increase TH activity in the hypothalamic neurons. Second, two specific inhibitors to this signal transduction pathway blocked the sCT-induced increase in TH activity. H-8 is a potent inhibitor of cyclic nucleotide kinases, acting by competing with the ATP binding site (43). H-8 caused a significant reduction in the sCT-induced increase in TH activity at 0.5 µM and completely blocked the response at 5 µM. Although the present study used intact cells, the effect H-8 concentrations are consistent with the reported Ki of 1.2 µM for cAMP-dependent protein kinase and well below the reported Ki of 15 µM for protein kinase C (43). However, H-8 has an even greater potency (Ki=0.5 µM) inhibiting cGMP-dependent protein kinase (43). Strong support for the involvement of cAMP-dependent protein kinase was provided with Rp-cAMPS, a competitive antagonist for cAMP (36, 37), which completely blocked the sCT-induced increase in TH activity. The high concentrations required for complete inhibition of the response likely stem from the relative inability of this compound to penetrate cell membranes.
The involvement of the phospholipase C/protein kinase C signal transduction pathway in the sCT-induced increase in TH activity is not supported by the data in the present study. Specific inhibitors for each step in the signal transduction pathway were not effective in blocking the ability of sCT to increase TH activity. U-73122, a phospholipase C inhibitor, did not alter the stimulatory effect of sCT at a dose equal to or above that required to inhibit other phospholipase C-mediated events (41, 44). Our data also argue against the involvement of calcium mobilization to increase TH activity. The ability of sCT to increase TH activity was retained when available calcium was lowered by depleting the calcium stores with thapsigargin or chelating intracellular calcium with BAPTA/AM. The 1 µM concentration of thapsigargin has been reported to deplete intracellular stores in less than 10 min (45). It would be anticipated that the intracellular calcium stores would remain depleted during the present study because thapsigargin was included during both the 45-min preincubation and the 1-h incubation period. Additionally, calcium was nominally absent from the extracellular medium to prevent restoration of the calcium stores. The 10 µM concentration of BAPTA/AM suppressed basal TH levels, but did not alter the stimulatory effect of sCT. Although the absolute levels of TH activity after sCT treatment were lower and not significantly different from control levels in the 10 µM BAPTA/AM-treated group, sCT’s ability to stimulate TH activity was apparent. This concentration of BAPTA/AM has been reported to completely suppress the increase in dopamine release in striatal slices induced by ionomycin, but not the nitric oxide (46). Finally, protein kinase C involvement is not supported. In contrast to the sCT-induced increase in TH activity, phorbol ester activation of protein kinase C resulted in a marked decrease in TH activity that was specific to the active isomer. Thus, the effects of PMA and sCT on hypothalamic TH activity are diametrically opposed. Furthermore, calphostin C, a potent and specific inhibitor of protein kinase C (38), did not alter the ability of sCT to increase TH activity in the hypothalamic cells. The Ki for Calphostin C with respect to protein kinase C is 0.05 µM and 1 µM Calphostin C completely inhibits protein kinase C, whereas 50 µM has no effect on protein kinase A (38). In the present study, concentrations as high as 1 µM did not alter sCT’s action on TH activity. In contrast, calphostin C reversed the inhibitory effect of PMA on TH activity. While we cannot rule out that sCT affects multiple hypothalamic cell types and activates different signaling pathways in other cells, it appears that sCT regulation of TH is governed entirely by a cAMP-dependent mechanism.
A role for augmented phosphorylation of the TH protein in response to sCT stimulation is implicated in the present study. Indeed, sCT caused a marked increase in radiolabeled phosphate incorporation into a 60,000 molecular weight protein immunoprecipitated with a specific TH anti-TH sera. Moreover, the enhanced phosphorylation state is driven by a cAMP-dependent mechanism, as evidence by the ability of Rp-cAMPS, a cAMP antagonist, to prevent the change. The involvement of a phosphorylation/dephosphorylation mechanism is not surprising, given the very rapid action of sCT on TH activity. Indeed, TH is a well established target for cAMP-dependent protein kinase, which phosphorylates TH on serine 40 and results in enzyme activation (47, 48, 49, 50, 51, 52).
The data in this study may have broader implications in the neuroendocrine control of PRL release. The tuberoinfundibular dopaminergic neurons in the hypothalamus have a primary role in the tonic inhibition PRL secretion (20), and an increase in dopaminergic neuronal activity would result in suppressed PRL levels. Indeed, sCT administration has been reported to reduce circulating PRL levels in some cases (15, 16). Although the endogenous ligand is not known, an anti-sCT serum causes a marked increase in circulating PRL levels in vivo but caused a more modest increase in PRL release of anterior pituitary cells in vitro (18). This suggests an extra-pituitary action of a sCT-like peptide, possibly on the hypothalamic dopaminergic neurons. This prospect requires further investigation.
In conclusion, our data support the notion that the adenylate cyclase/cAMP/protein kinase A signaling pathway mediates the marked sCT-induced increase in TH activity in hypothalamic dopaminergic neurons. Furthermore, phosphorylation of the TH protein is a major intracellular mechanism for the enhanced enzyme activity and is driven by cAMP-dependent protein kinase. The identification of the dopaminergic neurons as a specific target for sCT supports an indirect hypothalamic action for sCT to influence PRL secretion.
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Acknowledgments |
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Footnotes |
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Received November 9, 1998.
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