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Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland
1Correspondence: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, U.K. E-mail: pb{at}rri.sari.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Changes in the level of
Gs or Gi G-protein subunits are ruled out
as part of the sensitization mechanism. However, changes in tyrosine
phosphorylation may be involved as tyrosine kinase inhibitors sensitize
ovine PT cells and tyrosine phosphatase inhibitors significantly blunt
adenylate cyclase activity, including the sensitized response to
melatonin. The adenylate cyclase isoforms mediating the sensitized
response may be broad as 7 of the 9 isoforms of adenylate cyclase are
expressed in the PT.—Barrett, P., Choi, W.-S., Morris, M., Morgan, P.
A role for tyrosine phosphorylation in the regulation and sensitization
of adenylate cyclase by melatonin.
Key Words: pars tuberalis • tyrosine kinase • G-protein • receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. The process of sensitization
involves chronic exposure to the receptor ligand, followed by
withdrawal, after which the production of cyclic AMP in response to
either a receptor mediated stimulus or the pharmacological tool
forskolin is enhanced. It has been suggested that the process of
sensitization may be involved in the process of drug of addiction
(cravings and relapse) (5)
.
Among these receptors are the melatonin receptors, which are involved
in mediating the effects of photoperiod in seasonal mammals
(6)
. Melatonin is a hormone that acts via a pertussis
toxin (PTX) -sensitive G-protein to inhibit adenylate cyclase (7
, 8)
. At one of its target sites, the pars tuberalis (PT) of the
pituitary, melatonin has been shown to sensitize PT cells to
stimulation by forskolin and produce enhanced levels of cyclic AMP
(9)
. Although melatonin has acute effects on the cyclic
AMP (cAMP) signal transduction system, what distinguishes melatonin
from other hormones is the requirement for a prolonged systemic
circulation of the hormone to bring about the seasonal changes in
physiology (6)
. The duration of the circulating hormone
depends on the length of the night, and in temperate zones will change
daily (10)
. The ability of seasonal animals to read and
decode the changing melatonin signal forms the basis for melatonin to
act as a seasonal timekeeper.
The phenomenon of sensitization has been shown to occur in cultured
cell lines through an activation of either endogenous or transfected
Gi protein-coupled receptors (1
2
3
4
, 11)
. Several mechanisms or potential components involved in the
cAMP signaling pathway have been investigated in these transfected
cells. However, no generalization of the mechanism initiated by
different receptors or between different cell lines used is yet
apparent. The possibility exists that the mechanism of sensitization
induced by melatonin in the PT cells could share common features with
sensitization induced in transfected cells. However, one important
difference exists between sensitization induced in the PT cells and
transfected cells, which is the prolonged duration of agonist
stimulation of the melatonin receptor required to achieve
sensitization. Exposure of PT cells to melatonin for up to 4 h has
little or no sensitizing effect. By contrast, 8–16 h of exposure to
melatonin produces maximal sensitization of adenylate cyclase
(9)
. This compares to 4 h or less for most other
Gi protein-coupled receptors in transfected cell
systems where a time course of sensitization has been examined
(1
, 4
, 12
, 13)
. Significantly this time-dependent
sensitization reflects summer to winter melatonin exposure in animals,
and therefore may be a physiological relevant mechanism in seasonal
mammals.
The molecular mechanism of the conditional sensitization by melatonin
is not known, but it requires depletion of a cellular protein(s) as
protein synthesis is required for the recovery of the basal adenylate
cyclase response (14)
. Furthermore, the establishment of
the sensitized state is assumed to take place through activation of
melatonin receptors coupled to the Gi inhibitory
protein. However, in PT cells only 20% of
125I-melatonin binding is susceptible to
pertussis toxin (7)
, suggesting a large proportion of the
melatonin receptors may not be coupled to Gi
proteins and hence sensitization may be achieved by activation of an
alternative G-protein.
Here we investigated whether sensitization of the adenylate cyclase
pathway occurs through activation of a melatonin receptor coupled to an
inhibitory Gi protein and if changes in levels of
G-protein subunits may be involved. We also provide evidence to
suggest that a tyrosine kinase may be involved in this mechanism as the
tyrosine kinase inhibitor herbimycin also induces sensitization. This
is supported by the opposing effect of tyrosine phosphatase inhibitors,
which reverse sensitization induced by herbimycin and melatonin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Cell pretreatments
After cell dispersal from whole tissue, cells were incubated
overnight and harvested the next day. After counting the cells,
0.5 x 106 cells were seeded into
poly-D-lysine-coated 24-well plates. At an appropriate time, the media
on the cells was changed for media containing the required drugs (or
appropriate vehicle) for the overnight treatment. Sensitization of
cells with herbimycin and/or melatonin was achieved by the addition of
these compounds to 1 µM and 1 nM, respectively, for 16 h. Where
appropriate, addition of the tyrosine phosphatase inhibitors was made
at the same time as the melatonin or herbimycin treatments. The
tyrosine phosphatase inhibitors used in the experiments described here
were sodium vanadate or potassium
bisperoxo(1,10-phenanthroline)oxovanadate(V) (bpVphen). The experiment
to demonstrate PTX sensitivity was carried out with cells in suspension
as described previously (7)
. Pertussis toxin-treated cells
were incubated with 100 µg/ml PTX for 16 h.
Measurement of intracellular levels of cAMP.
For the cells stimulated in plates, after sensitization with
herbimycin and/or melatonin, the cells were washed three times with 500
µl of cold media. Five hundred microliters of cold media containing
0.1 mM IBMX with forskolin (1 µM) or melatonin (10 nM) as appropriate
was added to the cells. The reactions were started by transferring the
plate to a 37°C water bath. The cells were incubated for 30 min and
then washed three times with ice-cold phosphate-buffered saline (PBS:
137 mM NaCl, 2.7 mM KCl, 4.3
Na2HPO4, 1.47 mM
KH2PO4, pH 7.1). The cells
were lysed by the addition of 200 µl of 5% trichloroacetic acid at
room temperature for 30 min. cAMP was determined as described
previously using 10 µl of the lysate or appropriate dilutions thereof
(16)
.
For the PTX treatment experiments, the media was removed after pertussis toxin treatment of the PT cells; the cells were washed once and replaced with media with or without 1 nM melatonin to sensitize the cells. The next day, cells were harvested and counted before use for stimulation and assessment of the cAMP responses.
Western blot analysis for G-protein content
Aliquots of 20 x 106 cells were
placed in 90 mm petri dishes and treated for 16 h with either 100
ng/ml PTX; 5 µg/ml cholera toxin (CTX); herbimycin; 1 nM melatonin;
herbimycin plus melatonin or received no treatment. Cells were
harvested and then pelleted at 700 g for 10 min. The cell
pellet was resuspended in 1 ml of cold PBS, transferred to a Microfuge
tube, and repelleted. The cells were washed twice more with PBS, then
frozen at -70°C. Crude membrane fractions were prepared as follows:
cells were thawed and resuspended in 0.5 ml of ice-cold homogenization
buffer (1 mM EGTA, 10 mM Tris-HCl, pH 7.5) and homogenized with 20
strokes in a hand-held homogenizer. The homogenized cells were
transferred to a 14 ml polypropylene tube made up to 1 ml and spun at
500 g for 15 min at 4°C. The supernatant was transferred
to a Microfuge tube and spun at 40,000 g for 5 min at 4°C.
The pellet was washed three times by resuspension in homogenization
buffer and pelleting at 40,000 g. On the third wash, a
fraction of the resuspended membrane preparation was taken for protein
determination. The crude membrane was resuspended at 2 µg/µl in 1x
sodium dodecyl sulfate (SDS) loading buffer, boiled for 5 min, and
sonicated for 5 s at 5 microns. Twenty micrograms of protein was
separated on a 10% polyacrylamide gel according to Laemmli
(17)
. Proteins were transferred to Immobilon P
polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass.)
using the Bio-Rad transfer apparatus and the buffer of Towbin et al.
(18)
. PVDF filters were incubated in blocking buffer
(Tris-buffered saline [TBS]: 20 mM Tris, 136 mM NaCl, pH 7.6] with
0.1% Tween-20 and 5% nonfat dry milk) for 1 h at room
temperature. After blocking, the filters were incubated with primary
antibodies at 4°C overnight in blocking buffer. The primary
antibodies used in these studies were to Gi1/2
(Upstate Biotechnology, Lake Placid, N.Y.; 1:1000 dilution);
Gi3/0 (Upstate Biotechnology; 1:2000 dilution);
Gs (Santa Cruz Biotechnology, Santa Cruz,
Calif., 1:1000 dilution); and Gz (Santa Cruz,
1:1000 dilution). After the primary antibody incubation, the filters
were washed three times, 5 min per wash, with TBS containing 0.1%
Tween-20 and incubated with an horseradish peroxidase-conjugated
anti-rabbit second antibody (Scottish antibody production unit) for
1 h at room temperature in blocking buffer. The filters were again
washed three times for 5 min per wash in TBS plus 0.1% Tween-20. For
chemiluminescent detection, the immunostained membrane was incubated
with the Pierce Super-Signal chemiluminescent substrate for 5 min and
exposed to X-Omat S film (Kodak).
PCR and Northern analysis for adenylate cyclase isoforms
Amplification of adenylate cyclase isoforms was carried out
using three sets of polymerase chain reaction (PCR) primers: Set 1: 5'
primer ATGAGCTCTTCGGSAAGTTTGAC; (NELFGKFD) 3' primer
GGWCACRTCGTKGGACCACAC (VWSNDVT); primer set 2: 5' primer
AAGATCCTNGGNGAYTGYTACTAC (KILGDCYY); 3' primer GCCANDGTSACATCRKKRGACCA
(WSNDVTLA); primer set 3: 5' primer
GAAGCTTAARATIAARACIATIGGIWSIACITAYATGGC (KIKTIGSTYMA); 3' primer
GGGATCCACRTTIACIGTRTTICCCCAIATRTCRTA (YDIWGDTVN). The amplification
reaction contained 100 ng of randomly primed PT cDNA, 100 pM of each
primer, 2.5 U of Taq DNA polymerase (Promega, Madison, Wis.)
10 mM Tris-HCl (pH 9 at 25°C), 1.5 mM MgCl2, 50
mM KCl, 0.1% Triton, and 250 µM each dNTP. DNA fragments of the
appropriate size were cloned into pGEM-T and sequenced. Plasmids
containing identified adenylate cyclase sequences were used for
generation of DNA fragments for probes in Northern analysis.
For Northern analysis, 15 µg of poly(A)+ RNA isolated from the sheep pars tuberalis, pars distalis (PD), cortex, and cerebellum were separated on a 1% denaturing formaldehyde gel. The RNA was transferred to nylon and probed with 32P-labeled DNA fragments for each adenylate cyclase isoform obtained by PCR. Probes were hybridized at 65°C for 2 h in Quickhyb solution (Stratagene, San Diego, Calif.) and washed per the manufacturer’s instructions with a final wash in 0.1x SSC, 0.5% SDS at 65°C. Filters were exposed to Kodak Biomax film with the appropriate intensifying screen for 1–4 days.
Protein estimations
Samples for protein estimation were dissolved in 0.5M NaOH.
Protein estimation was made by the method of Bradford
(19)
.
Statistics
The effect of treatments on cAMP responses was analyzed by a
one-way ANOVA using the Sigma-Stat software package (Jandel, Errath,
Germany). Differences were assessed using the Tukey test for multiple
comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the control cells receiving neither pretreatment with PTX nor
melatonin, only a modest increase in cAMP levels was obtained in
response to forskolin stimulation (Fig. 1)
. This increase is retarded
by simultaneous treatment with 10 nM melatonin. In contrast, melatonin
pretreatment without PTX pretreatment sensitized the cells to forskolin
stimulation and resulted in an enhanced increase in cAMP. Melatonin
again blocked the response to forskolin, indicating that the maximal
response to melatonin is unaffected by chronic exposure to melatonin
for 16 h. Where cells were pretreated with PTX but not exposed to
melatonin, a sensitization response to forskolin occurred that was of
greater magnitude than cells receiving no PTX treatment. In these
PTX-treated cells, melatonin still reduced the forskolin-induced
response. In PT cells treated with both melatonin and pertussis toxin,
a sensitized response to forskolin was obtained, but this was no larger
than the increase obtained with PTX alone. These data suggest that
chronic melatonin treatment in the absence of PTX sensitizes the
adenylate cyclase system, which remains fully responsive to further
melatonin action. However, pertussis toxin, which induces sensitization
alone, abrogates the ability of melatonin to sensitize the cells but
does not inhibit the ability of melatonin to inhibit forskolin-induced
increase in cAMP. Such an interpretation is consistent with the
demonstration that only a small percentage of melatonin receptors in
the PT are PTX sensitive. These data also suggest that a proportion of
the melatonin receptors may be coupled to an alternative inhibitory
G-protein.
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Sensitization of PT cells by herbimycin
Next we asked whether tyrosine phosphorylation may be involved in
sensitization of the adenylate cyclase system in PT cells. Initial
comparisons were made after pretreatment with the src kinase selective
inhibitor herbimycin (20
21
22
23)
or melatonin relative to
control cells receiving no treatment. The response of PT cells to
forskolin stimulation in the presence or absence of melatonin was
examined. Figure 2A
shows a typical cAMP analysis; when compared to cells
receiving no pretreatment, an enhanced cAMP response to forskolin is
observed in cells pretreated with melatonin; this response can be
inhibited by melatonin. A similar effect was observed for cells treated
with herbimycin. Thus, herbimycin also sensitized PT cells to
stimulation with forskolin. Like the melatonin sensitized cells,
melatonin still inhibited the forskolin response. In both the
melatonin- and herbimycin-sensitized cells, small increases in basal
cAMP values could often be observed (see also Fig. 1
).
|
In addition to the individual sensitization effects of herbimycin and
melatonin, pretreatment with the two compounds in combination for
16 h incubation further enhanced the response to forskolin (Fig. 2A
). We have called the enhanced effect super sensitization.
The sensitization of adenylate cyclase is achieved not only with the
pharmacological tool forskolin, but is also obtained with cholera
toxin-activated Gs stimulation of adenylate
cyclase (Fig. 2B
). Another consistent observation in the
super-sensitized cells are the higher basal levels of cAMP relative to
either the control or cells sensitized with herbimycin or melatonin
alone.
The ability of melatonin to inhibit the forskolin response is not impeded in the herbimycin- or super-sensitized cells, demonstrating that herbimycin does not affect the acute signal transduction mechanisms associated with the melatonin receptor.
Protein tyrosine phosphatase inhibitors reverse the sensitizing
effect of herbimycin and melatonin
If tyrosine kinase inhibition sensitizes adenylate cyclase in PT
cells, then tyrosine phosphatase inhibition may have an opposing
action. To assess this, the effect of the protein tyrosine phosphatase
inhibitors vanadate (24)
and bpV(phen) (25)
on the sensitization of adenylate cyclase by melatonin and herbimycin
was examined. When preincubated with the PT cells for 16 h, both
phosphatase inhibitors reduced the ability of forskolin to stimulate
cAMP synthesis in PT cells independently of adenylate cyclase
sensitization treatments or after sensitization with melatonin,
herbimycin, or a combination of the two compounds (Fig. 3
). The reduction in forskolin-stimulated cAMP levels was not due to
reduced cell viability as neither vanadate nor bp(V)phen had any effect
on cell viability under the conditions used (data not shown).
|
The effect of vanadate we observed with whole cells is in contrast to
the effect of vanadate in PT membrane preparations. In PT membrane
preparations, vanadate potentiated the effect of forskolin (data not
shown). This is consistent with the previously reported effect of
vanadate on membrane preparation of rat adipose tissue
(26)
.
The opposing actions of the protein tyrosine phosphatase inhibitors with the action of melatonin and herbimycin suggest that the effect of forskolin to stimulate larger increases in cAMP in sensitized cells may be due to a post-translational tyrosine dephosphorylation event. We therefore assessed whether melatonin, herbimycin, or a combination of the two could sensitize the forskolin-stimulated cAMP in membranes prepared from pretreated cells.
Although this experiment was repeated six times and in some experiments a small enhanced response to forskolin was observed in melatonin-pretreated cells (~twofold), we were unable to obtain a consistent response and conclude that the sensitizing effect of melatonin or herbimycin requires the intact cell.
Sensitization does not involve a change in
Gi/o or Gs
Both the stimulatory (Gs) and inhibitory
(Gi1/2, Gi3/0, and
Gz) components of the cAMP signal transduction
pathway are present in the PT cells (Fig. 4
). One potential mechanism of sensitization is a quantitative change in
these G-protein subunits to alter the balance between the
stimulatory and inhibitory components regulating cAMP synthesis. Thus,
we investigated the possibility of this mechanism by examining the
relative levels of these G-protein subunits by Western blot analysis.
PT cells were treated for 16 h with either no stimulant,
herbimycin, melatonin, combination of both, pertussis toxin, or cholera
toxin overnight. Four antibodies were used that recognize
Gs, Gi1/2,
Gi3/0, and Gz. As
demonstrated previously (27)
, 16 h treatment with 5
µg/ml cholera toxin down-regulates Gs
G-protein subunit (Fig. 4)
. However, no effect of herbimycin,
melatonin, or a combination of herbimycin and melatonin or PTX was
obtained on the Gs G-protein subunit.
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Similarly, inhibitory G-protein subunits were also unaffected by the
sensitizing treatments. As expected, CTX had no effect on
Gi subunits; however, PTX did cause a small
(but statistically insignificant) increase in
Gi1/2.
Adenylate isoforms in PT cells
One of several potential mechanisms used by other members of the
Gi-coupled, protein-coupled receptors may depend
on the isoform of adenylate cyclase present within the cell. Recent
evidence (28)
demonstrates that four of the nine isoforms
(I, V, VI, and VIII) can be sensitized by chronic quinpirole treatment
of Cos7 cells transiently coexpressing the dopamine
D2 receptor with each of the individual isoforms
of adenylate cyclase. Therefore, the isoform of adenylate cyclase
expressed in the PT may have a role in the mechanism of sensitization.
PCR amplification was carried out on PT cDNA, with PCR primers based on
several conserved regions of the adenylate cyclase isoforms. Several
PCR fragments were obtained, which were cloned and sequenced. This
revealed the presence of adenylate isoforms I, III–VI (a region common
to both isoforms), VII, and IX. These PCR fragments were used for
probing a Northern blot containing 15 µg of
poly(A)+ RNA isolated from the ovine PT, PD,
cerebellum, and cortex. This analysis showed adenylate isoform I was
barely detectable in the PT and PD but was detected in the cerebellum
and cortex. Isoform IV was barely detectable in all tissues, whereas
isoforms III, V/VI, VII, and IX could be detected in all four tissues
(Fig. 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 16
, 29
30
31
32
33)
. In particular,
elevation of cAMP has been shown to contribute to the regulation of the
Mel 1a melatonin receptor mRNA and the clock gene oPer1 mRNA levels in
PT cells (15
, 34
, 35)
. These observations support a role
of central importance for cAMP signal transduction in regulating the
state of activation of PT cells. However, whereas melatonin can inhibit
cAMP production acutely, paradoxically melatonin can enhance cAMP
synthesis in PT cells after prolonged exposure and subsequent
withdrawal of melatonin. This process is called sensitization
(9)
.
Since melatonin receptors appear to couple to both PTX-sensitive and
-insensitive mechanisms in the PT (7)
, we sought to
determine whether the sensitization of the adenylate cyclase response
was due to activation of the PTX-sensitive
(Gi-coupled) receptors. The abolition of the
sensitization effect by PTX strongly supports the involvement of
Gi in this mechanism in ovine PT cells. This is
in accordance with other Gi-coupled receptors
expressed in transfected cells, including the melatonin receptor
expressed in CHO cells. The sensitization of PT cells to forskolin
stimulation by PTX treatment alone probably results from the removal of
the tonic inhibition of adenylate cyclase by the PTX-inactivated
Gi proteins.
In addition to these findings, this study also reveals that the
remaining PTX-insensitive melatonin receptors in the PT are able to
inhibit cAMP synthesis after PTX treatment. These data are consistent
with 125I-melatonin binding data after PTX
treatment of PT cells, where only a 20% reduction in
125I-melatonin binding is achieved. One
explanation for these data may be that the existence of the melatonin
receptor in a large protein complex (36)
restricts the
access of PTX to the G-protein. Alternatively, the melatonin receptor
is coupled to an alternative G-protein. One such candidate is
Gz: this G-protein inhibits adenylate cyclase
through a G-mediated event; it is PTX
insensitive and is present in the PT cells. Furthermore, melatonin
receptors have been demonstrated to couple to Gz
in transfected cells (37)
.
With the growth of information on the molecular mechanisms involved in
signaling pathways, it is becoming increasingly apparent that tyrosine
kinases play an important role in signal transduction events
(38
39
40
41)
. This study revealed that the tyrosine kinase
inhibitor herbimycin was able to induce sensitization of adenylate
cyclase to stimulation by forskolin or cholera toxin and that this is
as effective as melatonin sensitization.
The ability of cholera toxin to produce enhanced levels of cAMP in sensitized relative to nonsensitized PT cells has potentially important physiological implications. These data suggest that activation of a Gs-coupled receptor would lead to an enhanced cAMP response. Hypothetically, this would result in enhanced levels of cAMP during short photoperiods, which may be important to the seasonal physiological responses in mammals.
Tyrosine kinases are known to be involved in the regulation of the cAMP
signaling and enhanced cAMP production has been reported in the
presence of the tyrosine kinase inhibitors genestein and erbstatin.
These tyrosine kinase inhibitors have been shown to inhibit
phosphodiesterases, resulting in elevated levels of cAMP
(38)
. Sensitization of adenylate cyclase by herbimycin in
Jukat T lymphoblasts has been reported (39)
where
herbimycin enhanced cAMP synthesis in response to several G-protein
receptor agonists. However, the effect of forskolin was not enhanced in
that study, and therefore it may suggest a different mechanism of
sensitization in the Jukat T lymphoblasts.
As forskolin activates adenylate cyclase through a direct interaction with the enzyme, the sensitization of adenylate cyclase by herbimycin in PT cells would suggest that the effect is at the level of the adenylate cyclase itself. However, the mechanism of sensitization may not involve direct change in the phosphorylation state of the adenylate cyclase enzyme since the enhanced response of the adenylate cyclase enzyme is not obtained in membrane preparations of sensitized PT cells. Possible explanations for this are phosphorylation of adenylate cyclase by other kinases reduces enzyme activity or the loss of an additional component during the membrane preparation.
The involvement of tyrosine dephosphorylation in the mechanism of sensitization is supported by the contrasting effect of two tyrosine phosphatase inhibitors vanadate and bp(V)phen on sensitization by herbimycin or melatonin. These compounds reduced the effectiveness of forskolin to stimulate cAMP in either herbimycin- or melatonin-treated cells. Taken together, the data on the tyrosine kinase and tyrosine phosphatase inhibitor implicate a tyrosine dephosphorylation in the mechanism of sensitization.
The enhanced effect of herbimycin with melatonin could be due to both
compounds having an action on a common component or to the fact that
neither compound is 100% effective. The sensitization induced by
melatonin may involve the down-regulation of an inhibitory protein,
since we have previously shown that the recovery from sensitization is
cycloheximide dependent (14)
. Since the extent of
enhancement of cAMP synthesis with melatonin combined with herbimycin
varied between experiments (Figs. 2A
and 3
), this may
suggest that the sensitization induced by melatonin or herbimycin
involves a single protein, possibly a tyrosine kinase, that is not
completely down-regulated by melatonin but then is effectively
neutralized by the addition of herbimycin. Alternatively, each compound
has an effect on different components involved in the regulation of
adenylate cyclase activity, but with each component sensitizing
adenylate cyclase to a fraction of the total maximum of the sensitized
response.
The identity of the tyrosine kinase(s) and tyrosine phosphatase(s) that
may be involved in regulating the sensitization process is not known.
It is possible that these kinases and phosphatases are similar or are
the same kinases and phosphatases that are associated with single
transmembrane receptors such as the IGF-1 receptor. Although IGF-1
receptors are present on the PT cells, it unlikely that a kinase or
phosphatase associated with this receptor signal transduction pathway
is involved, since IGF-1 does not affect either basal or forskolin
stimulated cAMP levels (32)
. A diagrammatic representation
of the possible interactions to bring about sensitization is shown in
Fig. 6
.
|
Another possible mechanism of sensitization may involve a change in the
quantity of the appropriate G-proteins regulating the adenylate cyclase
enzyme as observed in SH-SY5Y cells during chronic opioid receptor
activation (42)
. However, this appears unlikely for either
melatonin or herbimycin, as neither Gs nor
Gi change in expression with these treatments.
A critical component of the sensitization process must be the adenylate
cyclase enzyme, but not all isoforms of the adenylate cyclase enzymes
are susceptible to sensitization (28)
. Isoforms I V, VI,
and VIII can be sensitized by chronic activation of the dopamine
D2 receptor when coexpressed in Cos7 cells,
whereas isoforms II, III IV VII cannot. No previous data regarding the
expression of isoforms of adenylate cyclase in the PT exist. Therefore,
we sought to determine which isoforms of adenylate cyclase are
expressed in PT cells. Using a reverse transcription (RT) -PCR
technique with several pairs of primers based on conserved regions of
the known adenylate cyclases, we were able to amplify and clone
fragments of six (V and VI were represented by a common fragment) of
the nine adenylate cyclase isoforms (I, III–VII, IX). Of the isoforms
that can be sensitized by dopamine D2 receptor
activation in Cos7 cells, isoforms I and VIII were not found by
Northern blotting (I) or PCR amplification (VIII) in PT cells. Type
V/VI was present by Northern analysis, but is probably not the most
abundant of the isoforms found. Moreover none of the adenylate cyclase
species may be in abundance, since the Northern blots required 15 µg
of poly(A)+ RNA to observe a signal for any of
the isoforms found by RT-PCR. This leaves open the possibility of
another adenylate cyclase present in PT cells that mediates the
sensitizing effects of melatonin.
In conclusion, the discovery that the treatment of PT cells with
herbimycin sensitizes the adenylate cyclase system suggests a mechanism
by which the sensitizing effect of both herbimycin and melatonin may
occur, as the characterized action of herbimycin is as an inhibitor of
the src family of tyrosine kinases. An action via a tyrosine kinase
inhibition is supported by the reversal of the effect of herbimycin by
tyrosine phosphatase inhibitors vanadate and bpV(phen). The possibility
that sensitization by either melatonin or herbimycin may arise due to
changes in expression of key G-protein subunits has been excluded.
However, the expression of a specific isoform of adenylate cyclase that
is specifically sensitized by the action of melatonin (and herbimycin)
in PT cells is a possibility, but it may not be one of the known
isoforms of adenylate cyclase.
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ACKNOWLEDGMENTS |
|---|
Received for publication September 16, 1999.
Revision received January 5, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mol. Pharmacol. 52,993-999
subunits in transducing enhanced activity of the type VI isoform. Mol. Pharmacol. 49,907-914[Abstract]
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