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Department of Physiology, University of Occupational and Environmental Health, School of Medicine, Kitakyusyu 807, Japan
Address all correspondence and requests for reprints to: Hiroshi Yamashita, M.D., Ph.D., Department of Physiology, University of Occupational and Environmental Health, Kitakyusyu 807, Japan.
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Abstract |
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These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide originally isolated from ovine hypothalamus (10), has been reported to stimulate melanotroph secretion and POMC gene expression in rat and mouse PI (11, 12). Therefore, PACAP is a potential candidate as a secretagogue in melanotrophs. However, the possibility that PACAP reaches melanotrophs via a neural or endocrine pathway remains unclear. One clue relevant to this question was provided by a report of PACAP-like immunoreactive cells in the PI of rats (13). They found that the immunoreactivity was observed diffusely in the cytoplasmic matrix of cells in the PI, but not in the secretory granules.
There are two subclasses for the PACAP receptor: type I and type II receptors (14). The former has a higher affinity to PACAP over vasoactive intestinal polypeptide, and the latter shows similar affinities to PACAP and vasoactive intestinal polypeptide (14). The type I receptor messenger RNA (mRNA) has been found within the PI (12, 15). Although the involvement of protein kinase A (PKA) and protein kinase C (PKC) was suggested for POMC gene expression in response to PACAP (12), little is known about the mechanisms involved in PACAP stimulation of melanotroph secretion.
In the present study, in situ hybridization histochemistry was used to identify the source of PACAP that may be involved in the stimulation of melanotroph secretion. In addition, fura-2 [Ca2+]i imaging and patch-clamp techniques were used to analyze the signal transduction mechanisms after PACAP receptor activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization histochemistry
Rat pituitary glands were removed, and sections at 12 µm were
cut in a cryostat at -20 C. The probes used were 35S
3'end-labeled deoxyoligonucleotides complementary to rat PACAP and rat
PACAP type I receptor genes (base 813–861 and 1251–1289,
respectively). In situ hybridization histochemistry was
performed as described previously (16, 17). Hybridization was carried
out overnight at 37 C, and the hybridized sections were apposed to
autoradiographic film (Hyperfilm, Amersham, Aylesbury, UK) for 7 days
or dipped in nuclear emulsion (K-5, Ilford, Cheshire, UK) and exposed
for 4 weeks. The specificity of the PACAP mRNA has been described
previously (18) and confirmed by competition with a 100-fold excess of
the probe in the present study. The specificity of the PACAP type I
receptor mRNA has also been described previously (17).
Dissociation of rat melanotrophs
Rat melanotrophs were prepared using a modification of the
method described by Tomiko et al. (19). In brief, rats were
stunned by a blow on the back of the neck and rapidly decapitated. The
pituitary neurointermediate lobe was carefully isolated from the
anterior lobe so that endocrine cells in the anterior lobe could be
excluded. The neurointermediate lobe was transferred in a
HEPES-buffered solution (HBS), the composition of which was 140
mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10
mM HEPES, 10 mM glucose, and 0.1% BSA (pH
adjusted to 7.4 with NaOH) and then incubated in HBS containing 0.12%
trypsin (type III, Sigma Chemical Co., St. Louis, MO) at 37 C for 20
min with continuous shaking (100 cycles/min). After trypsin treatment,
the lobes were incubated in Ca2+-free HBS containing 0.14%
collagenase (type I, Sigma Chemical Co.) for 15 min and triturated with
glass pipettes (0.3- to 0.6-mm tip diameter). Isolated cells thus
obtained were washed twice with HBS and used for
[Ca2+]i measurement and recording of ion
currents.
[Ca2+]imeasurement
Isolated cells were suspended in HBS with the addition of
acetoxymethyl esters of fura-2 (fura-2/AM; 3 µM) at room
temperature (
23 C) for 1 h. The cells were then washed with
dye-free HBS and kept at room temperature until used.
[Ca2+]i measurement and cell perifusion were
performed as described previously (3, 20). In brief, a small portion of
the cell suspension (containing 100–300 cells) was transferred to a
temperature-controlled chamber (PDMI-2, Medical Systems Corp.,
Greenvale, NY) of small volume (150 µl). The chamber was positioned
on the stage of an inverted microscope (TMD-300, Nikon, Tokyo, Japan),
equipped with a Ca2+-imaging system (Quanticell/700, JEOL,
Tokyo, Japan). Perifusion was begun 5–10 min later when the cells had
settled and attached to the coverglass glued to the bottom of the
chamber. The perifusion fluid was standard HBS unless otherwise
indicated. The Ca2+-free solution used in the present study
was a modified HBS containing 1 mM EGTA and no
CaCl2. The flow rate of the perifusion fluid was adjusted
to 1 ml/min. Once cells were selected in the optical field with the aid
of a microscope, fluorescence intensities at 510 nm with excitation at
340 and 380 nm were recorded at an interval of 5 sec. The exposure time
and the interval between 340- and 380-nm excitations were 40 and 120
msec, respectively. [Ca2+]i in individual
melanotrophs was calculated from the ratio (R) of the fluorescence
images measured with excitation at 340 nm to those at 380 nm using the
following equation (21): [Ca2+]i
= Kd x (R - Rmin)/(Rmax - R) x
ß, where Kd is the dissociation constant for
fura-2 (224 nM); Rmax and Rmin are the ratio for unbound
and bound forms of the fura-2/Ca2+ complex, respectively;
and ß is the ratio between maximum and minimum fluorescence
intensities of fura-2 at 380 nm excitation. Rmax and Rmin were
estimated with the fluorescence intensities of fura-2 solution (3
µM) containing 10 mM CaCl2 and 10
mM EGTA, respectively. Autofluorescence in melanotrophs was
negligible compared with fluorescence in the fura-2-loaded cells.
[Ca2+]i measurement was performed at about 35
C.
Electrophysiological recording of ion currents
Melanotrophs were plated on a glass coverslip (11-mm diameter),
and electrophysiological experiments were begun about 30 min later when
the cells had become attached to the glass. The arrangements for
perifusing cells and recording membrane currents were described in
detail previously (22). The perifusion medium was HBS without BSA. The
inner pipette solution used in the recording electrodes contained 140
mM CsCl, 10 mM EGTA, 2 mM
CaCl2, 1 mM MgCl2, 2 mM
Mg-ATP, 0.3 mM GTP, and 10 mM HEPES (pH
adjusted to 7.2 with Tris). Membrane currents were recorded with a
patch-clamp amplifier (AxoPatch 200A, Axon Instruments, Foster City,
CA) and were digitized using Pclamp software (version 6.0.2, Axon
Instruments) and MacLab (version 3.4, BRC, Tokyo, Japan). Analysis of
the data was performed using Axograph software (version 3.0.11, Axon
Instruments). To examine the properties of currents induced by PACAP,
voltage ramps from a holding potential of -80 to +20 mV for 5 sec were
used. All electrophysiological measurements were made at room
temperature.
To assess the effects of PACAP on voltage-dependent Ca2+ channels, Ba2+ was used for the charge carrier. The pipette solution contained 140 mM CsCl, 10 mM EGTA, 1 mM MgCl2, 2 mM Mg-ATP, 0.3 mM GTP, and 10 mM HEPES (pH adjusted to 7.2 with Tris). After making a high resistance seal, the perifusion solution was switched from standard HBS to a Ba2+-containing solution, the composition of which was 10 mM BaCl2, 140 mM tetraethylammonium-Cl, 10 mM HEPES, 10 mM glucose, and 5 mM KCl (pH adjusted to 7.4 with Tris). Voltage-dependent Ba2+ currents were elicited by voltage steps from a holding potential of -80 mV to a test potential of 0 mV unless otherwise noted. The sampling rates were 1 and 10 kHz for the ramp-pulse and voltage-step experiments, respectively.
Statistics
Results are expressed as the mean ± SEM.
Statistical differences (P < 0.05) were determined by
Student’s t test for comparison between two groups or by
Scheffe’s F test for comparison among multiple groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B, shows the
expression pattern of PACAP mRNA in the pituitary gland. A high level
of expression was seen within the PI. By contrast, little or no
expression was detected in the pars distalis or pars nervosa. Figure 1, C and 1, shows the expression pattern of PACAP type I receptor mRNA in
the pituitary gland. As previously reported (15), a high level of
expression was noted in the PI, and moderate expression was seen in the
pars distalis. In the pars nervosa, no expression of PACAP type I
receptor mRNA was detected.
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) or initiated Ca2+
oscillations in 23.3% (Fig. 2B) of the total melanotrophs that
responded to PACAP. The onset of PACAP-induced
[Ca2+]i increase varied from 5 sec to 5 min
between cells (112.7 ± 8.1 sec); however, it was similar when
PACAP was added twice to the same cells. The increase in
[Ca2+]i was maintained for 10 min or longer
after PACAP application in 64.7% of the responding cells, and it
returned to the control level within 10 min in the remaining cells. The
slow onset and recovery in the response to PACAP contrasted with the
rapid response to 50 mM K+ tested in the same
perifusion system, which resulted in 2.4 ± 0.3 and 11.4 ±
1.2 sec (n = 118) for onset and recovery, respectively.
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) or potentiated the baseline Ca2+
oscillations with an 89.6 ± 15.6% (P < 0.05)
increase in the amplitude without a clear increase in the interspike
[Ca2+]i level (Fig. 2D) in 9 and 6 cells,
respectively. In contrast to the heterogeneity of the responses to PACAP, all cells tested regardless of the presence of spontaneous Ca2+ oscillations responded to 100 nM dopamine (n = 281); 10 µM nicardipine (n = 165), a dihydropyridine antagonist; or extracellular Ca2+ removal (n = 75) with a rapid reduction in [Ca2+]i or arrest of Ca2+ oscillations. The maximum reductions in [Ca2+]i observed with dopamine and Ca2+ removal were 55.3% and 55.8% of the basal [Ca2+]i, respectively, and were reached in 10–20 sec. In addition, all cells tested responded to high K+ (n = 227) and 10 µM BAY K 8644 (n = 72), a dihydropyridine agonist, with an increase in [Ca2+]i.
Effects of ionic environments and chemicals on PACAP-induced
[Ca2+]i increase
The PACAP-induced increase in [Ca2+]i
was abolished when extracellular Ca2+ was omitted (Fig. 3A), when extracellular Na+
was replaced with equimolar N-methyl-D-glucamine
(NMDG; Fig. 3B), or in the presence of 10 µM nicardipine
(Fig. 3C). In each instance it was confirmed that PACAP increased
[Ca2+]i in the same cells. Moreover,
nicardipine reversibly suppressed [Ca2+]i
increase in response to PACAP when nicardipine was added after PACAP
increased [Ca2+]i (Fig. 3D).
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), which maximally lowered basal
[Ca2+]i (3). Moreover, dopamine reversibly
suppressed the PACAP-induced [Ca2+]i increase
when it was added after PACAP increased
[Ca2+]i (Fig. 4B). In all 8 cells tested,
[Ca2+]i began to decrease rapidly after dopamine
application and began to recover rapidly upon dopamine removal.
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shows the effects of
staurosporine and Rp- diastereomer of cAMPS (Rp-cAMPS), PKC and PKA
inhibitors, respectively, on the S2/S1 ratio (see Fig. 5). The basal
[Ca2+]i and the maximum
[Ca2+]i in response to the first PACAP
application (S1) were 164.3 ± 6.6 and 319.1 ± 17.3
nM, respectively (n = 112). The S2/S1 ratio, which was
90.4 ± 6.9% in the absence of inhibitors, was significantly
decreased by 1 µM staurosporine or 100 µM
Rp-cAMPS to 35.7 ± 10.5% and 34.9 ± 7.0%, respectively.
Moreover, the S2/S1 ratio in the presence of both staurosporine and
Rp-cAMPS (15.3 ± 6.1%) was smaller than the ratios in the
presence of staurosporine and Rp-cAMPS alone, although the former ratio
was not significantly different from either of the latter ratios. The
selectivity of staurosporine and Rp-cAMPS was examined by observing
effects of the inhibitors on the [Ca2+]i
response to 50 mM K+. The responses to
K+ in the presence of staurosporine (1 µM;
n = 23) or Rp-cAMPS (100 µM; n = 30) were
92.7 ± 8.1% and 92.6 ± 4.0%, respectively, of the control
responses without the inhibitors.
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, B and C).
PACAP-induced changes in ion currents
To examine the possible ionic mechanisms underlying the
PACAP-induced [Ca2+]i increase, membrane
currents were recorded with the whole cell configuration of the
patch-clamp technique. The perifusion of melanotrophs with PACAP (300
nM) induced gradual and long lasting inward currents in 6
of 13 melanotrophs tested at a holding potential of -80 mV (Fig. 6A). The current-voltage relationship of
PACAP-induced currents is shown in Fig. 6B, and the reversal potential
was -0.79 ± 3.57 mV (n = 3). The PACAP-induced inward
currents were inhibited when 1 µM staurosporine was added
2.5–9 min before PACAP application (Fig. 6B). Staurosporine at the
same concentration had little or no effect on currents elicited by
voltage ramps from -80 to 20 mV in the absence of PACAP (n = 6),
suggesting that nonspecific effects of staurosporine on the currents
are negligible, and the contribution of basal PKC activity to the
currents is minor. Moreover, 1 µM staurosporine did not
affect voltage-dependent Ba2+ currents elicited by voltage
steps from -80 to 0 mV (102.2 ± 1.9%; n = 8). On the other
hand, 100 µM Rp-cAMPS reduced Ba2+ currents
elicited by voltage steps from -80 to 0 mV in 4 of 6 cells, and the
mean Ba2+ currents obtained in the presence of Rp-cAMP from
the 6 cells was 96.7 ± 1.3% of the control current.
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). The mean ± SEM facilitation was estimated
to be 31.2 ± 11.7% (n = 5). The effect of PACAP on
Ba2+ currents appeared within 2 min and was reversible
within 5 min. When the voltage dependency of the PACAP-induced
facilitation was examined with voltage steps from a holding potential
of -80 mV to various voltages (-60 to 30 mV at 5- to 10-mV intervals;
n = 5), facilitation was observed at voltages between -40 and 0
mV.
Effects of OAG and Sp-cAMPS on ion currents
Application of OAG (100 µM) caused inward currents
in 5 of 11 melanotrophs tested at a holding potential of -80 mV (Fig. 7A). The peak amplitude of the
OAG-induced inward currents observed at the holding potential of -80
mV was 14.3 ± 8.7 pA (n = 5), which was larger than but not
significantly different from that of the currents induced by 300
nM PACAP (10.2 ± 2.5 pA; n = 6). The
current-voltage relationship of OAG-induced currents is shown in Fig. 7B, and the reversal potential was -5.56 ± 7.26 mV (n = 3).
On the other hand, Sp-cAMPS (100 µM) increased
voltage-dependent Ba2+ currents in 3 of 7 melanotrophs
tested (Fig. 7C), and the mean ± SEM increase was
estimated to be 23.4 ± 4.1% (n = 3). A representative
example of the time course of Sp-cAMPS-induced facilitation of
Ba2+ currents is shown in Fig. 7D. Facilitation appeared
within 1 min, reached a peak level in 1.5 min, and returned to the
control level 4 min after the application of Sp-cAMPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The source of the [Ca2+]i increase in response to PACAP appears to be exclusively extracellular, as the PACAP-induced [Ca2+]i increase was completely abolished by the removal of extracellular Ca2+. The lack of Ca2+ release in response to PACAP in melanotrophs shows clear contrast with the effects of PACAP observed in bovine adrenal chromaffin cells or rat gonadotrophs, where rapid Ca2+ release from ryanodine-sensitive Ca2+ stores preceded the long lasting Ca2+ entry (20), and Ca2+ release was blocked by heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) receptors (23), respectively. The difference in the PACAP responses between melanotrophs and these two cell types could be due to the difference in the PACAP receptor subtype. There are five different isoforms for the PACAP type I receptors, and some difference in coupling with Gs or Gq proteins has been reported for these receptor subtypes (15). Moreover, it has recently been reported that the novel PACAP receptor TM4 selectively activates Ca2+ entry through dihydropyridine-sensitive Ca2+ channels without affecting the production of cAMP or inositol phosphates (IPs) (24). However, the possibility that such receptors mediate Ca2+ entry in rat melanotrophs does not seem likely, as it has been reported that PACAP increases the formation of cAMP and IPs in rat and mouse melanotrophs (11, 12), and the present study revealed that both PKA and PKC are involved in the PACAP-induced Ca2+ entry mechanism. An alternative explanation for the lack of Ca2+ release in the PACAP response of rat melanotrophs is that these cells may possess Ca2+ stores of small capacity. The suggestion is supported by previous observations that both [Ca2+]i under basal conditions and increases in [Ca2+]i in response to various stimuli, such as high K+, glutamate, or cAMP, were critically dependent on Ca2+ entry through Ca2+ channels in melanotrophs (3, 4, 5, 8, 25) and also by a report that rat melanotrophs apparently have no caffeine-sensitive Ca2+ stores, and the depolarization-induced [Ca2+]i increase is due to Ca2+ influx across the cell membrane (26). On the other hand, both thapsigargin and cyclopiazonic acid, inhibitors of Ca2+-adenosine triphosphatase in the endoplasmic reticulum, have been reported to increase [Ca2+]i in rat melanotrophs (27), indicating the existence of Ca2+ stores in these cells. This result together with the finding that PACAP increased IPs in the PI (12) suggest that Ca2+ release from IP3-sensitive stores may be more relevant to the PACAP-induced [Ca2+]i response in rat melanotrophs. It is possible that Ca2+ stores in melanotrophs were already depleted during the short period (2–3 min) of extracellular Ca2+ removal or during the addition of dopamine or nicardipine before PACAP application.
The present results indicate that the mechanism of Ca2+ entry during PACAP receptor activation in melanotrophs is due to two independent effects of PACAP, namely induction of long lasting inward currents and facilitation of voltage-dependent Ca2+ channel currents. These effects show close similarity to the effects of PACAP observed in adrenal chromaffin cells (20) and thus seem to be common mechanisms by which PACAP enhances Ca2+ entry in endocrine cells. The former effects appear to be due to activation of nonselective cation channels because the reversal potential of the currents was near 0 mV, which is almost identical to the reversal potential of such channels reported in rat melanotrophs (28). This is also supported by the results obtained from [Ca2+]i measurements showing that the PACAP-induced [Ca2+]i response was abolished when extracellular Na+ was replaced with NMDG. The slow time course of the inward currents is in good agreement with that of the Ca2+ increase and suggests that some intracellular messengers and activation of protein kinases are involved in the effects. The latter effects, namely receptor-operated facilitation of voltage-dependent Ca2+ channels, have not been reported previously for melanotrophs. However, these are well known phenomena for L-type channels in other types of cells, such as adrenal chromaffin cells or cardiac myocytes (29, 30). One possible explanation for the present finding that the L-type Ca2+ channel blocker, nicardipine, potently suppressed the PACAP-induced Ca2+ increase despite other types of Ca2+ channels being present in these cells (31, 32) is that PACAP selectively facilitated L-type Ca2+ channels. Taken together, the PACAP-induced [Ca2+]i increase may be accounted for by Ca2+ entry promoted by a combination of depolarization produced by positive charge entry through cation channels and the facilitation of voltage-dependent Ca2+ channels.
Both OAG and Sp-cAMPS increased [Ca2+]i, and staurosporine and Rp-cAMPS inhibited the PACAP-induced [Ca2+]i increase. Moreover, staurosporine and Rp-cAMPS did not significantly inhibit the high K+-induced [Ca2+]i increase. These results suggest that the effects of the inhibitors were selective for the PACAP-induced [Ca2+]i increase and that both PKC and PKA are involved in Ca2+ entry mechanisms that are activated by PACAP. These findings show a close similarity to PACAP-induced stimulation of POMC gene transcription, which was mimicked by forskolin, an activator of adenylate cyclase, and by phorbol 12-myristate 13-acetate, an activator of PKC (12). The close similarity between these two phenomena may suggest that one is the cause of the other. For example, the increase in [Ca2+]i may trigger POMC gene transcription. Such a link between the [Ca2+]i increase and gene transcription has been reported (33). The present results further indicate that nonselective cation channels may be one of the sites of PKC action, as OAG activated inward currents of the reversal potential of -5.6 mV, which was almost identical to that of PACAP-induced inward currents, and the latter currents were suppressed by staurosporine. Moreover, voltage-dependent Ca2+ channels seem to be one of the sites of PKA action, as Sp-cAMPS facilitated Ba2+ currents.
The single cell [Ca2+]i imaging technique
used in the present study revealed that PACAP increased
[Ca2+]i by more than 30 nM for
less than half of the melanotrophs examined. Moreover, the pattern of
the responses to PACAP was variable. One possible explanation for the
heterogeneity in the [Ca2+]i responses to
PACAP is that the dissociated cells used in the present study contained
cells other than melanotrophs, such as folliculo-stellate cells or
pituicytes, nonsecretory glial-like cells in the PI and pars nervosa,
respectively. However, the finding that the same population of cells
consistently responded to dopamine, nicardipine, BAY K 8644, or high
K+, just as melanotrophs would respond to these
manipulations (1), suggests that the cells examined in the present
study were indeed melanotrophs. Although the PI is known to be composed
of a vast majority of melanotrophs (34), some heterogeneity among
melanotrophs has been suggested from the appearance at the
ultrastructural level (35), POMC mRNA staining intensity (36), a long
isoform of dopamine D2 receptors (37), or mRNA for the
1A Ca2+ channel subunit (38). Such
heterogeneity might explain the heterogeneity in the
[Ca2+]i responses to PACAP. It should be
noted that heterogeneity in functional responses in melanotrophs is not
without a precedent; a glutamate-induced
[Ca2+]i increase was observed in only 20% of
acutely dissociated rat melanotrophs under basal conditions (8). The
heterogeneity in the [Ca2+]i responses could
be due to the fact that the cells used in the above-mentioned study as
well as those in the present study were acutely dissociated. It is
reported that L-type Ca2+ channel activity increased during
culture when the physiological restraint for melanotrophs mediated by
the secreto-inhibitory neurotransmitter, dopamine, was removed (39, 40). Other studies showed that the L-type Ca2+ channel
carries the major Ca2+ currents in mammalian melanotrophs
(31, 41) and is importantly involved in peptide secretion in these
cells (42). Moreover, the present results show that the channel is
responsible for the PACAP-induced [Ca2+]i
responses.
Besides the heterogeneity in the [Ca2+]i responses, there was heterogeneity in [Ca2+]i behavior under unstimulated conditions. About 13% of the cells examined showed spontaneous Ca2+ oscillations, whereas the majority of cells showed relatively stable basal [Ca2+]i. These results clearly differ from previous results obtained from melanotrophs of Xenopus laevis, where 73% of melanotrophs obtained from toads adapted to a black background exhibited slow, large, spontaneous Ca2+ oscillations (Ca2+ pulsing) (43, 44). The difference in the percentage of spontaneously oscillating cells seems to match the difference in the secretory activity between melanotrophs of the two species; the percentages of the maximum inhibition of secretion by extracellular Ca2+ omission in melanotrophs of black-adapted toads and rats were over 90% and about 60%, respectively (19, 45). It should be noted, however, that rat cells showing no spontaneous Ca2+ oscillations also responded to dopamine or extracellular Ca2+ removal with a rapid and large reduction in [Ca2+]i. These results suggest that rat melanotrophs, regardless of their basal [Ca2+]i behavior, possess spontaneous Ca2+ entry mechanisms and that such spontaneous Ca2+ entry may drive the spontaneous secretion characteristic of these cells.
The dense PACAP mRNA distribution in the PI observed in the present study is in good agreement with immunohistochemical staining of this peptide reported in the PI (13). These data suggest that PACAP is synthesized in the PI and is presumably secreted to the PI tissue. The similarly dense expression of PACAP type I receptor mRNA observed in the PI confirmed a previous report that the highest expression of PACAP type I receptor was present in the PI (12, 15). These two pieces of evidence suggest that PACAP may mediate autocrine and/or paracrine roles for melanotrophs. The findings that dopamine promptly suppressed the PACAP-induced [Ca2+]i increase and that PACAP did not increase [Ca2+]i in the presence of dopamine indicate that the inhibitory action of dopamine can overcome the secretagogue action of PACAP. These results are consistent with the previous observation that exposure to the dopamine D2 agonist, bromocryptine, blunted the secretory response to PACAP by more than 90% in rat melanotrophs (11). It has been reported that dopamine withdrawal consistently induced a rebound increase in [Ca2+]i and POMC peptide release from melanotrophs (1, 3, 8, 44, 46), a phenomenon common to other adenohypophyseal cells (47, 48). Therefore, if PACAP is coreleased with POMC peptides, it would be expected to be released during the rebound effect of dopamine removal and stimulate melanotroph secretion via an autocrine and/or paracrine mechanism. As PACAP has also been found in other endocrine cells, such as adrenal chromaffin and pancreatic ß-cells, as well as in neurons innervating endocrine cells (49, 50), the autocrine and/or paracrine pathways may be a common mechanism by which this peptide regulates endocrine cells. The mechanism seems to be particularly important in melanotrophs, as the PI is known to be virtually avascular (35), and therefore, peptides released to the tissue would tend to stay around melanotrophs for some period of time. Moreover, the long lasting effects of PACAP on membrane currents and [Ca2+]i observed in the present study would be effective to maintain secretion at a high level for a long period of time. Although the release of PACAP from melanotrophs needs to be confirmed to prove this hypothesis, PACAP appears to be a good candidate for the long sought endogenous secretagogue for mammalian melanotrophs. To date, three ligands have been reported to possess secretagogue actions in rat melanotrophs: CRF, ß-adrenergic agonists, and glutamate (6, 7, 8). However, none of these has been shown to naturally occur in the PI.
In conclusion, these data suggest that PACAP potentiates the Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA. In addition, the results suggest that PACAP may serve as an endogenous secretagogue via autocrine and/or paracrine mechanisms in the PI.
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Acknowledgments |
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Footnotes |
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Received February 17, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1A-type Ca2+ channel mRNA and protein are
differentially regulated by melanotrope dopamine receptor stimulation.
26th Annual Meeting of the Society for Neuroscience, Washington DC,
1996, p 1244 (Abstract)
kanson R, Sundler F 1994 Pituitary adenylate cyclase-activating peptide in the adrenal gland of
mammals: distribution, characterization and responses to drugs.
Neuroendocrinology 59:113–119[Medline]
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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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K. Tanaka, I. Shibuya, N. Kabashima, Y. Ueta, and H. Yamashita Inhibition of Voltage-Dependent Calcium Channels by Prostaglandin E2 in Rat Melanotrophs Endocrinology, December 1, 1998; 139(12): 4801 - 4810. [Abstract] [Full Text] [PDF]
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