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Department of Physiology and Biophysics (A.J.M., M.U.G.), the Neuroscience Program (A.E.H., M.U.G.), and the Department of Cell and Structural Biology (M.U.G.), University of Illinois-Urbana-Champaign, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. Martha U. Gillette, B107 Chemical and Life Sciences Laboratory, MC-123, University of Illinois, 601 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: mgillett{at}uiuc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEL is a neuroendocrine signal of darkness. The hormone is small (mol wt, 232.3) and lipophilic, and is rapidly carried by blood and cerebrospinal fluid to all tissues of the organism. Its production is tightly restricted to night, or the dark phase of the lighting cycle (3). The rise in plasma MEL levels signals the nocturnal state throughout the internal milieu, and the duration of elevated MEL levels is proportional to night length. As night length changes over the annual cycle, the alteration in MEL profile induces profound changes in the reproductive state of seasonally breeding animals.
Converging evidence suggests that MEL can modulate the mammalian circadian system as well. Although pinealectomy has little effect on circadian activity rhythms in mammals (5), pinealectomized rodents entrain to a reversed photoperiod regimen more quickly than intact animals (6). Also, pinealectomy dampens the amplitude of the firing rate rhythm in the SCN (7). Daily MEL administration can entrain activity rhythms in free running rats, independent of pinealectomy. This effect is temporally restricted: MEL at circadian time 10 (CT 10 = 10 h after lights on in the entraining 24-h lighting schedule), significantly advances behavioral rhythms (8). Armstrong also reported a single incident of phase advance at CT 22 during late subjective night (9). When the SCN of a rat is ablated, MEL has no entraining effect (8). These behavioral studies suggest that MEL can entrain circadian rhythms, and that it acts at the level of the SCN.
The hypothesis that MEL affects the SCN directly can be developed from anatomical and physiological evidence. First, the SCN is a major site of MEL binding in the rodent brain (10, 11). The SCN also exhibits circadian variation in the density of MEL-binding sites. Maximum [125I]MEL binding peaks late in the day (12). Second, MEL alters the metabolic activity of rat SCN in a bimodal fashion (i.e. the response is a function of the CT of administration) (13). MEL at late subjective day inhibits 2-deoxyglucose uptake, whereas treatment before lights on increases the metabolic rate in the SCN. Fos-like immunoreactivity is induced in the SCN in response to MEL treatment during late subjective night only (14). Third, single unit activity in rat and hamster SCN brain slices is acutely depressed when MEL is applied near the transition to subjective night (15, 16).
In the present report, a brain slice preparation from the rat is used to examine the interaction between MEL and SCN function. When the SCN is maintained in vitro, it continues to generate a near 24-h rhythm of the population firing rate (17). This rhythm matches that recorded in vivo with multiunit electrodes (18). Furthermore, this preparation achieves virtual isolation of the SCN pacemaker from signals integrated at other brain sites, permitting direct evaluation of responses at the SCN (19). In preliminary studies, we reported that MEL acts directly on the SCN clock in vitro, resetting the phase of the neuronal activity rhythm at late subjective day (20). Here we fully characterize the parameters of this effect and delineate more thoroughly the phase-dependent modulation of the SCN pacemaker by MEL. These studies reveal a second period of sensitivity at late subjective night. We also report experiments probing the transduction pathway(s) that transmits the MEL signal within SCN cells and provide evidence for protein kinase C (PKC) activation in this hormone’s mechanism of resetting the clock.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Brain slice preparation
The procedure for preparation, maintenance, and recording from
SCN hypothalamic slices was detailed previously (21, 22). Briefly,
animals were removed from the colony and quickly decapitated, and
500-µm coronal slices containing the SCN were prepared during the
donor’s subjective lights on period only. Slices were maintained at
the interface of the brain slice chamber, where they were continuously
perifused with Earle’s Balanced Salt Solution (EBSS; Life
Technologies, Grand Island, NY) supplemented to 24.6 mM
glucose and 26.2 mM sodium bicarbonate. Antibiotic (0.005%
gentamicin) was included in incubations longer than 24 h. The
medium, adjusted to pH 7.2, was warmed to 37 C and saturated with 95%
O2-5% CO2. The slices were prepared within 5
min of decapitation and allowed to equilibrate for at least 1 h
before experimental paradigms were initiated.
Electrophysiological recording and data analysis
Extracellular activity from single units was sampled with a
glass microelectrode filled with 5 M NaCl and positioned by
a hydraulic microdrive. A signal to noise ratio of 2:1 was the minimum
requirement for discerning individual cells. Each cell was monitored
for two or three sequential, 120-sec intervals. Spikes were counted in
10-sec bins, and the mean firing rate of the cell was calculated. On
the average, four to six cells were sampled throughout the SCN each
hour during a recording period of 8–14 h. The mean firing rate values
were then grouped into 2-h bins, and the pattern was smoothed by
plotting the data as 15-min running averages. The time of peak in the
electrical rhythm (based upon 28–61 U/experiment) was visually
discerned. Phase shifts in peak activity were evaluated as the
difference between the experimental result and the mean time of peak
electrical activity recorded in vehicle-treated controls (0.001%
ethanol for 1 h at CT 10, peak = CT 7.0 ± 0, n =
3; 0.05% dimethylsulfoxide (DMSO) for 40 min at CT 10, peak = CT
7.0, n = 1), which overlap with data from untreated slices.
Experimental treatments
Pharmacological treatments were administered by static
bath application of reagents in warmed oxygenated EBSS, pH 7.2. At
specified times, perifusion of the SCN slice was stopped, and the
medium in the dish was quickly removed and manually replaced with the
experimental treatment. Treatments were terminated by manual exchange
for normal EBSS, after which perifusion was resumed. Phase-shifting
treatments were 1 h in duration, unless otherwise indicated. 1)
MEL was dissolved at 1 mM in 95% ethanol and serially
diluted in EBSS to 1 nM MEL (ethanol equivalent 0.001%).
2) Treatments with 2-iodo-MEL (2-I-MEL; RBI, Natick, MA), a specific
MEL agonist, followed the same protocol. 3) Treatments with pertussis
toxin (PTX; 1 µg/ml, 6 h), which selectively inhibits a subclass
of G proteins including Gi, Go, and
Gq, and control incubations (EBSS exchange, 6 h) were
achieved by two sequential 3-h static pulses. In some experiments, MEL
was added for the last 10 min of PTX or EBSS static treatment. 4)
12-O-Tetradecanoylphorbol 13-acetate (TPA), a phorbol ester
that directly activates PKC, was bath-applied in a 10-min pulse (10
nM, ethanol equivalent 0.06%). 5) Calphostin C (CAL C), a
specific PKC inhibitor, was dissolved at 1 mM in DMSO and
serially diluted in EBSS to 0.1 µM (DMSO equivalent
0.05%), except for the PKC assays, in which CAL C was used at 1
µM. CAL C incubations lasted 40 min, and in some
experiments, treatments were followed by a 15-min pulse of 1
nM MEL in CAL C (CAL C/MEL). All experiments with CAL C
were carried out after direct exposure to normal laboratory fluorescent
lighting conditions. 6) Chelerythrine chloride (CC; Calbiochem, La
Jolla, CA), another specific inhibitor of PKC, was dissolved to 50
mM in aqueous DMSO and serially diluted in EBSS to 0.25
mM (0.05% DMSO equivalent). CC incubations lasted 20 min,
and in some experiments, treatments were followed by a 15-min pulse of
1 nM MEL in CC (CC/MEL). 7) Staurosporine (STP; 10
µM STP and 0.9% ethanol equivalent), a general kinase
inhibitor, was used alone for 1 h. In some experiments, STP
treatments were followed by a 10-min pulse of MEL (STP/MEL) or TPA
(STP/TPA). All chemicals not specifically noted were purchased from
Sigma Chemical Co. (St. Louis, MO).
PKC activity assays
Procedures for slice preparation and bath application follow
those stated in the Experimental treatments section, with
the exception that the duration of MEL treatments lasted only 1 min
after the time course of activation had been established. Reduced SCN
slices cut in half through the optic chiasm were treated with 1
µM MEL or control medium (EBSS) in the brain slice
chamber, and the reaction was stopped by rapid freezing of the tissue
on dry ice. In some cases, the inhibitors were added before MEL in the
brain slice chamber to evaluate the specificity of the PKC activation.
Slices were thawed in assay dilution buffer and briefly sonicated.
Total PKC phosphotransferase activity was assayed on this broken tissue
preparation using a PKC assay kit (Upstate Biotechnology, Lake Placid,
NY). Activity was based on incorporation of [
-32P]ATP
(DuPont-New England Nuclear, Boston, MA) into a synthetic substrate
peptide (QKRPSQRSKYL) corresponding to amino acid residues 4–14 of
myelin basic protein in the absence of lipids. This assay has been
fully validated for the selective assay of PKC in crude tissue extracts
(23). A cocktail of protease inhibitors and phosphatase inhibitors was
added to the reaction mixture after slices had been thawed and
sonicated (0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 1 µg/ml
aprotinin, 40 µg/ml bestatin, 0.5 mM
phenylmethylsulfonylfluoride, 1 mM EGTA, 5 µM
microcystin, and 1 mM NaF). The protein content of each
slice was determined using the Bradford assay (24), and activity is
expressed as counts per min/µg protein.
Statistical analysis
Data are presented as the mean ± SEM or
SD where indicated. Statistical significance between or
among groups was tested using Student’s t test (unpaired)
or one-way ANOVA followed by preplanned comparisons or the Tukey
post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, replotted from 25 . In
untreated or vehicle-treated controls, the peak occurred near CT 7 (CT
6.9 ± 0.1; n = 8). The experiments contributing to this mean
were performed for this study by the authors. The time of peak is
consistent with that of the control peaks reported in earlier studies
on these inbred rats obtained by our recording methods (26).
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) and 3
in vitro (Fig. 1B; separate experiment). This phase advance
is significant on both day 2 (
A = 3.8 ± 0.1
h; n = 6; P < 0.0001, by Student’s t
test) and day 3 (A = 3.8 ± 0.4 h; n = 4;
P < 0.0001). The new phase of the rhythm is stable;
that is, the two subsequent peaks following treatment occur at the same
circadian time. Thus, the clock is fully reset during the interval
between the treatment and the time the next peak appears. A similar
effect was observed when MEL was applied at CT 23 (subjective dawn;
Fig. 1C). The mean phase advance was 3.7 ± 0.1 h (n =
3) on day 2 and = 3.6 h (n = 2) on day 3. When the period of exposure to MEL either at CT 10 or CT 23 was reduced from 1 h to 15 min, the phase advances were 4.25 h (n = 2) at CT 10 and 4.00 ± 0.00 h (n = 3) at CT 23 (data not shown), which overlap those seen with 1-h incubations. This indicates that the processes underlying MEL induction of phase shifts are fully stimulated within 15 min of SCN exposure to MEL.
SCN sensitivity to the MEL agonist, 2-I-MEL, was temporally similar to
that of MEL. 2-I-MEL treatment (1 nM) at CT 10 induced a
robust phase advance of 6.8 ± 0.1 h (n = 3; Fig. 1D).
2-I-MEL treatment early in the subjective day at CT 2, a
MEL-insensitive time, did not affect the electrical activity rhythm
(A = 0.3 ± 0.2 h; n = 3). The means of
the results of these experiments at CT 2 and CT 10 with 2-I-MEL have
previously been reported (20).
Ten different time points across the circadian cycle were evaluated for
MEL sensitivity (CT 0, 2, 6, 7, 10, 11, 14, 18, 22, and 23). A total of
38 experiments were performed to construct the MEL phase response curve
(Fig. 2); five of the points in this curve originally
appeared in Ref. 20. A phase response curve is obtained by plotting the
magnitude of the phase shift in hours vs. the circadian time
of treatment. These experiments reveal two separate periods of SCN
sensitivity to MEL that correlate with points of transition in the
entrained lighting cycle. The first, a relatively broad window, appears
to surround the subjective dusk period (CT 10–14). A second period of
sensitivity appears near subjective dawn (CT 23–0). Although this
morning window of sensitivity is narrower than the one at dusk, the
magnitudes of the phase advances are equivalent (
4 h).
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) demonstrates that the
1-nM concentration used in these experiments elicits the
maximal response and is well within the plateau range. Remarkably, a
robust phase advance in electrical activity was induced at 0.2
pM MEL (A = 3.15 h; n = 2),
whereas no effect was found with 0.1 pM MEL
(A = 0.15 h; n = 2). This sharp transition is
indicative of an "all or none" physiological response to ligand
binding.
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) did not alter the time of
peak (A = 0.1 ± 0.1 h; n = 3),
indicating that this treatment is neither toxic to the SCN tissue nor
are the G proteins that it affects involved in time-keeping during
subjective day (CT 4–10). The SCN tissue was still responsive to
exogenous treatment of MEL after a 6-h static bath of normal EBSS (Fig. 4B). PTX incubation before a 10-min PTX/MEL pulse at CT 10 blocked the
phase-shifting effects of MEL (Fig. 4C). This indicates that MEL acts
through a PTX-sensitive G protein pathway in the SCN. The time of peak
in these experiments occurred at CT 6.6 ± 0.1 h (n =
5), which is comparable to the time of peak in vehicle controls and
untreated slices.
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). Near midsubjective day, a
MEL-insensitive time, TPA was relatively ineffective (CT 6; mean
A = 1.0 h; n = 2). Treatment at CT 10 or CT 23
resulted in significant phase shifts. When applied at CT 10, TPA
produced a A = 4.8 ± 0.4 h (n = 3);
treatment at CT 23, subjective dawn, induced a A =
4.0 ± 0.4 h (n = 3). These phase advances are not
significantly different from those seen with MEL.
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),
which indicates that these treatments do not affect ongoing pacemaker
function at those times (CT 10: Cal C A = 0.1 ±
0.4 h, n = 3; CC A = 0.0 ±
0.3 h, n = 3; CT 23: Cal C A = -0.1
± 0.1 h, n = 4; CC A = 0.4 ± 0.1
h, n = 3). However, when a 40-min Cal C treatment was followed by
a 15-min pulse of CAL C/MEL, at dusk or dawn, the phase-advancing
effect of MEL was completely blocked (CT 10 A = 0.2
± 0.2 h, n = 3; CT 23 A = 0.1 ±
0.1 h, n = 3). CC, when applied 20 min before a 15-min
CC/MEL pulse, also completely blocked the effect of MEL (CT 10
A = 0.1 ± 0.1 h, n = 3; CT 23
A = 0.1 ± 0.1 h, n = 3). The nonspecific
kinase inhibitor STP produced the same results as those seen with CAL C
and CC as well as blocking the phase advances seen with TPA at dusk and
dawn (data not shown). These results support a role for PKC in
transduction of the MEL signal for phase resetting in the SCN.
MEL increases PKC phosphotransferase activity
To directly test the hypothesis that MEL activates PKC in the SCN,
PKC enzyme activity was measured at CT 6, 10, and 23. An assay was used
that is based on the transfer of radiophosphate [32P]ATP
from [-32P]ATP into a substrate peptide that
corresponds to a fragment of myelin basic protein that is specifically
phosphorylated by PKC. During midday (CT 6), PKC activity was low and
not altered by 1-min MEL treatment (Fig. 6; basal activity, 341.8
± 98.13 cpm/µg protein; MEL, 300.49 ± 23.35 cpm/µg).
However, at dusk (CT 10), MEL nearly doubled PKC phosphotransferase
activity (basal activity, 598.93 ± 160.29 cpm/µg; MEL,
1204.65 ± 7.23 cpm/µg). The same effect was seen at CT 23
(basal, 948.34 ± 226.33 cpm/µg; MEL, 1769.43 ± 40.82
cpm/µg). Results are plotted as the mean ± SD and
n = 3 in all cases.
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). When
SCN slices were incubated with the specific PKC inhibitors, CAL C and
CC, alone or before a 1-min pulse of MEL, activity was either not
significantly different from basal levels (CT 10; CAL C =
371.67 ± 132.50 cpm/µg; CAL C/MEL, 647.00 ± 206.00
cpm/µg; CC, 414.33 ± 319.66 cpm/µg; CC/MEL, 865.67 ±
111.73 cpm/µg) or lower than them (CT 23; CAL C, 563.78 ±
167.14 cpm/µg; CAL C/MEL, 465.23 ± 97.33 cpm/µg; CC,
314.47 ± 130.59 cpm/µg; CC/MEL, 390.24 ± 143.90 cpm/µg)
These results demonstrate that MEL activates PKC in the SCN at both
dusk and dawn, but not at midday. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The response of the SCN in vitro to MEL correlates in several respects with responses of animals in vivo to MEL treatment. Firstly, rats free running in constant darkness entrain to daily MEL injections during late subjective day (CT 8–12) by phase advancing their wheel-running activity (27). To date, little evidence has been shown for a late night entraining effect of MEL in vivo, although Armstrong reports one incident of a phase advance with MEL application at dawn (9).
Secondly, behavioral entrainment to MEL is dose dependent (ED50, 5 µg/kg) and quantal (28), with rats either fully responding or not at all. A similar all or none response describes the relationship between MEL concentration and its effects on SCN phase resetting in vitro. This supports the idea that MEL’s action on behavioral entrainment is at the level of the SCN.
Thirdly, the threshold level for maximal response to MEL’s action in vitro (4.64 pg/ml) is near daytime levels of plasma MEL in rats (4.00 pg/ml) (29), which do not shift circadian rhythms. It is possible that the absence of MEL in the brain slice perfusion medium may make the SCN in vitro hypersensitive to MEL. In vivo, the sensitivity of rats to MEL entrainment is increased by lack of exposure to MEL. Rats that have been pinealectomized and are free running in constant darkness can be entrained by daily MEL injections of 100 ng/kg, which is one third the amount needed to entrain sham-operated controls (30). Both groups responded to single MEL treatments.
Our results also demonstrate that recent exposure to endogenous MEL is not a prerequisite for SCN sensitivity to exogenous MEL treatment. Brain slices are prepared during the subjective day, when MEL production is inhibited in the animal, and there is no available source of endogenous MEL to the brain slice. In our hands, the SCN can survive for up to 3 days (without exposure to MEL), and maintain stable rhythms. Experiments with MEL treatment at CT 23 require slices to be without MEL for 23 h or more, and yet the dawn window of sensitivity appears. Dusk sensitivity, followed by a long period of MEL insensitivity through the night, then the reappearance of sensitivity at dawn, indicate that the response to MEL is a clock-regulated phenomenon and is not induced by the hormone itself. Margraf and Lynch (16) have reported that the circadian profile of MEL sensitivity in hamster SCN single unit activity is also independent of prior exposure to endogenous MEL. MEL-induced suppression of the firing rate in SCN neurons disappeared during night 1 in vitro, but reappeared in their preparation at subjective dusk on the second day in vitro.
It is curious that the MEL agonist induces a greater change than MEL itself. A similar effect, however, is seen with serotonin and its agonists, 5-carboxamidotryptamine and 8-hydroxy-dipropylaminotetralin. When serotonin is applied to an SCN slice at CT 9, it produces a phase advance of 4.6 ± 0.5 h, whereas 5-carboxamidotryptamine advances the rhythm by 6.0 ± 0.1 h, and 8-hydroxy-dipropylaminotetralin advances it by 7.0 ± 0.0 h (22). However, no difference in the maximal amplitude of the phase-shifting response is seen with a wide range of cholinergic agonists (31). This suggests an unusual feature of 2-I-MEL action. The difference may be due to 2-I-MEL’s greater binding affinity at the receptor (32, 33), or the agonist could be coactivating an additional pathway, due to its differential structure, and producing an additive response.
Mechanisms of MEL signal transduction show both species and tissue specificity. Many studies have shown that MEL alters cellular function by modifying cyclic nucleotide levels. The cloned mammalian MEL receptor, in fact, has been shown to be coupled to inhibition of adenylate cyclase in COS-7 cells (33). However, our investigations into MEL signal transduction mechanisms in the rat SCN in vitro did not demonstrate cyclic nucleotide involvement. The phase-response curves for reagents stimulating cAMP and cGMP are out of phase with each other, and their periods of sensitivity are temporally distinct from the MEL phase-response curve (19, 26, 34). Further, we found no convincing evidence that MEL treatment at late subjective day altered basal levels of cAMP or cGMP, or that forskolin-stimulated cAMP levels in the SCN were affected by MEL treatment (35). As the SCN has a low level of MEL receptors (36), it is possible that local changes in cAMP levels may occur in some SCN cells, but these changes may be undetectable when the whole SCN is assayed. Thus, we cannot exclude the possibility that MEL acts to inhibit adenylate cyclase locally within the SCN.
Studies in two systems have now shown that the MEL-receptor complex
uses a PTX-sensitive G protein pathway; PTX disrupts signaling pathways
in COS-7 cells transfected with the cloned mammalian MEL receptor (33),
and PTX completely blocks the MEL-induced phase advance at CT 10 in our
system. PTX uncouples receptor-ligand complexes from their usual G
protein interactions by inducing ADP-ribosylation of the regulatory
subunit of multiple G proteins. PTX-sensitive G proteins include
Gi, which inhibits adenylate cyclase and modulates
potassium and calcium channels; Go, which activates PLC and
regulates ion channels; and Gq, which activates PLC. Recent
reports suggest that activation of PLC by PTX-sensitive G proteins
occurs via the ß-subunit, and not the 
-subunit, as is usually
the case (37, 38).
Diacylglycerol (DAG) and PKC are key elements in the PLC pathway. PLC has been measured in moderate amounts in rat SCN (39). Activation of PLC by PTX-sensitive G proteins leads to the production of DAG. DAG, in combination with calcium, can activate PKC. The SCN contains significant amounts of PKC (Hunt, A., unpublished results). Thus, should MEL act at a PTX-sensitive G protein-coupled receptor to activate PLC in the SCN, it could stimulate PKC. Alternatively, should the MEL-activated receptor act primarily via a G protein-linked ion channel, the resulting ionic fluxes could lead to phospholipase A2 activation. Phospholipase A2 can subsequently activate PKC via ecosanoids such as arachidonic acid. The elements of these linkages mediating MEL activation of PKC in the SCN are yet to be determined.
The phorbol ester, TPA, directly activates PKC by mimicking unsaturated DAG (40). Our electrophysiological experiments examining the ability of TPA to phase shift the ensemble firing rate rhythm revealed that the SCN has a profile of sensitivity to TPA comparable to that of MEL with regard to time of day and magnitude of response. These experiments demonstrate that the clock regulates a permissive element in the PKC pathway at an intracellular level (41) downstream from membrane phospholipids. This gate to PKC activation and phase resetting is closed at CT 6, but opens at both dusk and dawn. MEL or TPA exposure at these times can access the clock mechanism via a PKC-mediated cascade and adjust clock timing.
PKC inhibitors have been developed that can interact with the substrate-binding site (ATP or protein) or with the regulatory domain binding sites (DAG or phorbol ester). CAL C, a compound isolated from the bacterium Cladosporium cladosporides, binds to the DAG-binding site of PKC, thereby inactivating the enzyme. CAL C is highly specific for PKC, with an IC50 value of 0.05 µM, and shows virtually no inactivation of other protein kinases (42). A benzophenanthridine alkaloid, CC, binds and blocks the ATP-binding site of the PKC molecule and is again a highly specific inhibitor of PKC, with an IC50 of 0.66 µM (43). STP, a Streptomyces-derived indole alkaloid, binds directly to the regulatory subunit of PKC, thereby preventing activation of this enzyme (44), but is nonspecific for PKC, interacting with other protein kinases. When treatment with any of these inhibitors was coupled with a subsequent pulse of MEL at either window of SCN sensitivity, blockade of both PKC activation and the phase advance was observed in all cases. Thus, activation of PKC must be a primary path by which MEL resets the SCN pacemaker.
Finally, PKC activity assays showed that MEL increases kinase activity in the SCN at dusk and dawn, but not at midday. In both cases, PKC phosphotransferase activity was doubled upon the addition of MEL. The response to MEL was very rapid and transient; it could be maximally measured within 1 min of treatment, and at that time activation was significant and robust. PKC activity after the addition of inhibitors was not significantly different from basal levels of PKC activity at CT 10. This may be related to the fact that inhibition of PKC at CT 10 has no effect on circadian rhythmicity, suggesting that the clock itself is not using PKC at this time. It is through MEL exposure that PKC has an effect on clock timing. At CT 23, inhibitors suppressed PKC activity below basal levels, suggesting a subtle difference in the PKC interaction with the inhibitors at this time. It is possible that different isoforms or regulators of PKC may be present at dusk and dawn.
It is noteworthy that the recently cloned mammalian MEL 1a receptor is a G protein-linked receptor bearing two consensus sequences for PKC phosphorylation on its cytoplasmic domain (33). This observation coupled with our results demonstrating a role for PKC in MEL signal transduction suggest an intracellular feedback role of this pathway in the receptor function, possibly through regulation of receptor desensitization.
In conclusion, the results of the present study support a new signal transduction mechanism for MEL that involves PTX-sensitive activation of PKC. The sensitivity of the SCN to MEL is clock-driven, rather than a response to previous exposure to the hormone. Circadian clock-controlled sensitivity results in precise temporal ordering of the MEL-sensitive periods so that they fall at the crepuscular transitions (19). MEL activation of the PKC pathway alters the clock function at subjective dawn and dusk, periods when seasonal changes in night length alter the endogenous MEL profile. MEL acts rapidly on the SCN at these times, advancing the clock and thus facilitating physiological changes in anticipation of environmental demands and maintaining adaptive synchrony with nature.
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Acknowledgments |
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Footnotes |
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2 These two authors contributed equally to this work.
3 Present address: Department of Psychiatry, L-469, Oregon Health
Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon
97201.
Received May 24, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dimers as well as subunits.
Cell 71:1069–1072[CrossRef][Medline]
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 X. Jin, C. von Gall, R. L. Pieschl, V. K. Gribkoff, J. H. Stehle, S. M. Reppert, and D. R. Weaver Targeted Disruption of the Mouse Mel1b Melatonin Receptor Mol. Cell. Biol., February 1, 2003; 23(3): 1054 - 1060. [Abstract] [Full Text]
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H. Arima, S. B. House, H. Gainer, and G. Aguilera Neuronal Activity Is Required for the Circadian Rhythm of Vasopressin Gene Transcription in the Suprachiasmatic Nucleus in Vitro Endocrinology, November 1, 2002; 143(11): 4165 - 4171. [Abstract] [Full Text] [PDF]
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 D. Roy and D. D. Belsham Melatonin Receptor Activation Regulates GnRH Gene Expression and Secretion in GT1-7 GnRH Neurons. SIGNAL TRANSDUCTION MECHANISMS J. Biol. Chem., January 4, 2002; 277(1): 251 - 258. [Abstract] [Full Text]
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C. S. Nelson, M. Ikeda, H. S. Gompf, M. L. Robinson, N. K. Fuchs, T. Yoshioka, K. A. Neve, and C. N. Allen Regulation of Melatonin 1a Receptor Signaling and Trafficking by Asparagine-124 Mol. Endocrinol., August 1, 2001; 15(8): 1306 - 1317. [Abstract] [Full Text] [PDF]
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 A. E. Hunt, W. M. Al-Ghoul, M. U. Gillette, and M. L. Dubocovich Activation of MT2 melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock Am J Physiol Cell Physiol, January 1, 2001; 280(1): C110 - C118. [Abstract] [Full Text] [PDF]
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 A. J. McArthur, A. N. Coogan, S. Ajpru, D. Sugden, S. M. Biello, and H. D. Piggins Gastrin-Releasing Peptide Phase-Shifts Suprachiasmatic Nuclei Neuronal Rhythms In Vitro J. Neurosci., July 15, 2000; 20(14): 5496 - 5502. [Abstract] [Full Text] [PDF]
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 M. Akashi and E. Nishida Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock Genes & Dev., March 15, 2000; 14(6): 645 - 649. [Abstract] [Full Text]
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L. Brydon, F. Roka, L. Petit, P. de Coppet, M. Tissot, P. Barrett, P. J. Morgan, C. Nanoff, A. D. Strosberg, and R. Jockers Dual Signaling of Human Mel1a Melatonin Receptors via Gi2, Gi3, and Gq/11 Proteins Mol. Endocrinol., December 1, 1999; 13(12): 2025 - 2038. [Abstract] [Full Text]
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 B. Pitrosky, R. Kirsch, A. Malan, E. Mocaer, and P. Pevet Organization of rat circadian rhythms during daily infusion of melatonin or S20098, a melatonin agonist Am J Physiol Regulatory Integrative Comp Physiol, September 1, 1999; 277(3): R812 - R828. [Abstract] [Full Text] [PDF]
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 S. Benloucif, M. I. Masana, K. Yun, and M. L. Dubocovich Interactions between Light and Melatonin on the Circadian Clock of Mice J Biol Rhythms, August 1, 1999; 14(4): 281 - 289. [Abstract] [PDF]
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 L. L. Haak Metabotropic Glutamate Receptor Modulation of Glutamate Responses in the Suprachiasmatic Nucleus J Neurophysiol, March 1, 1999; 81(3): 1308 - 1317. [Abstract] [Full Text] [PDF]
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M. D. A. Kopp, C. Schomerus, F. Dehghani, H.-W. Korf, and H. Meissl Pituitary Adenylate Cyclase-Activating Polypeptide and Melatonin in the Suprachiasmatic Nucleus: Effects on the Calcium Signal Transduction Cascade J. Neurosci., January 1, 1999; 19(1): 206 - 219. [Abstract] [Full Text] [PDF]
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M. L. Dubocovich, K. Yun, W. M. Al-ghoul, S. Benloucif, and M. I. Masana Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms FASEB J, September 1, 1998; 12(12): 1211 - 1220. [Abstract] [Full Text]
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P. A. Witt-Enderby, M. I. Masana, and M. L. Dubocovich Physiological Exposure to Melatonin Supersensitizes the Cyclic Adenosine 3',5'-Monophosphate-Dependent Signal Transduction Cascade in Chinese Hamster Ovary Cells Expressing the Human mt1 Melatonin Receptor Endocrinology, July 1, 1998; 139(7): 3064 - 3071. [Abstract] [Full Text] [PDF]
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 J. VANECEK Cellular Mechanisms of Melatonin Action Physiol Rev, July 1, 1998; 78(3): 687 - 721. [Abstract] [Full Text] [PDF]
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A. W. Ross, C. A. Webster, M. Thompson, P. Barrett, and P. J. Morgan A Novel Interaction Between Inhibitory Melatonin Receptors and Protein Kinase C-Dependent Signal Transduction in Ovine Pars Tuberalis Cells Endocrinology, April 1, 1998; 139(4): 1723 - 1730. [Abstract] [Full Text] [PDF]
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J. R. Redman and A. J. P. Francis Entrainment of Rat Circadian Rhythms by the Melatonin Agonist S-20098 Requires Intact Suprachiasmatic Nuclei But Not the Pineal J Biol Rhythms, February 1, 1998; 13(1): 39 - 51. [Abstract] [PDF]
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P. Barrett, G. Davidson, D. G. Hazlerigg, M. A. Morris, A. W. Ross, and P. J. Morgan Mel 1a Melatonin Receptor Expression Is Regulated by Protein Kinase C and an Additional Pathway Addressed by the Protein Kinase C Inhibitor Ro 31-8220 in Ovine Pars Tuberalis Cells Endocrinology, January 1, 1998; 139(1): 163 - 171. [Abstract] [Full Text] [PDF]
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J. R. Redman Circadian Entrainment and Phase Shifting in Mammals with Melatonin J Biol Rhythms, December 1, 1997; 12(6): 581 - 587. [Abstract] [PDF]
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S. M. Biello, D. A. Golombek, K. M. Schak, and M. E. Harrington Circadian Phase Shifts to Neuropeptide Y In Vitro: Cellular Communication and Signal Transduction J. Neurosci., November 1, 1997; 17(21): 8468 - 8475. [Abstract] [Full Text] [PDF]
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S.M.W. Rajaratnam and J.R. Redman Effects of Daily Melatonin Administration on Circadian Activity Rhythms in the Diurnal Indian Palm Squirrel (Funambulus pennanti J Biol Rhythms, August 1, 1997; 12(4): 339 - 347. [Abstract] [PDF]
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C. Liu, J. M. Ding, L. E. Faiman, and M. U. Gillette Coupling of Muscarinic Cholinergic Receptors and cGMP in Nocturnal Regulation of the Suprachiasmatic Circadian Clock J. Neurosci., January 15, 1997; 17(2): 659 - 666. [Abstract] [Full Text] [PDF]
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