1 Obesity Research Center, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118; and 2 Immunology Division, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Culturing clonal -cells (HIT-T15)
overnight in the presence of phorbol ester [phorbol myristate acetate
(PMA)] enhanced insulin secretion while causing downregulation of some
protein kinase C (PKC) isoforms and most PKC activity. We show here
that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-
, -
II, and -
, but could be because of the continued
activation of the two remaining diacylglycerol-sensitive isoforms
and µ. The enhanced secretion did not involve changes in glucose
metabolism, cell membrane potential, or intracellular Ca2+
handling, suggesting a distal effect. PMA washout caused the loss of
the enhanced response, but secretion was then stimulated by acute
readdition of PMA or bombesin. The magnitude of this restimulation
appeared dependent on the mass of PKC-
, which was rapidly
resynthesized during PMA washout. Therefore, stimulation of insulin
secretion by PMA, and presumably by endogenous diacylglycerol, involves
the activation of PKC isoforms
and/or µ, and also PKC-
.
insulin secretion; protein kinase C isoforms; phorbol 12-myristate 13-acetate; bombesin
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INTRODUCTION |
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THE CONSENSUS MODEL
of glucose stimulus-secretion coupling involves closure of
ATP-sensitive K+ channels, resulting in a voltage-dependent
gating of Ca2+ channels and a rise in intracellular
Ca2+ concentration ([Ca2+]i; see
Ref. 22). In addition to this Ca2+ rise,
glucose metabolism elicits a rise in other second messengers, such as
cAMP and long-chain fatty acyl-CoA (LC-CoA) and its derivatives (5). Nutrient stimulation is known to switch the -cell
metabolism of fatty acids from
-oxidation to the accumulation of
LC-CoA and the synthesis of complex lipids, such as phosphatidate or diacylglycerol (DAG). These lipids, which can target and activate protein kinase C (PKC), could play a role in either the action of
glucose to stimulate insulin secretion or in potentiating such secretion (21, 27, 28). Two potentiating agents,
acetylcholine or cholecystokinin, are widely held to augment the effect
of nutrient secretagogues by triggering the generation of DAG and the
activation of PKC (10, 32), indicating that the activation
of PKC provides a positive signal for insulin secretion.
The complexity of signal transduction via PKC is increased considerably
by the existence of 11 isozymes. These isozymes differ in their
requirements for the activators, Ca2+ and lipids, and in
their intracellular location (19). The known PKC isozymes
can be divided into the following three major classes: the conventional
(cPKC), which are activated by both Ca2+ and DAG; the novel
(nPKC), which require only DAG; and the atypical (aPKC), which bind
neither Ca2+ nor DAG. Clonal -cells (HIT-T15 and MIN6)
have been shown to express seven PKC isoforms, including cPKC (
and
II), nPKC (
,
, and µ), and aPKC (
and
; see Refs.
26 and 29). Phorbol esters, such as phorbol myristate
acetate (PMA), can substitute for DAG as high-affinity ligands for cPKC
and nPKC isoforms, and the acute addition of PMA to
-cells
stimulates secretion (7). Although most isoforms in other
kinase families share a similar activation mechanism, this is clearly
not the case for PKC. This observation has led to the assumption that
the different PKC isoforms underpin different functions within a given
cell (15). However, very little is known about the role of
PKC isoforms that are expressed in
-cells and specifically how and
which PKC isoforms couple intracellular lipid signals to the
stimulation of exocytosis (26, 29).
Downregulation of PKC activity, because of chronic stimulation
(12-24 h) by phorbol esters, has been used widely as a tool to
examine the involvement of PKC in insulin secretion (1, 2, 13,
17, 18, 25, 26, 29, 31). Some studies have treated PKC as a
single enzyme activity and have correlated the reduction of total PKC
activity to a given change in the secretory response of the -cell
(1, 2, 13, 17, 18, 25). More recent studies have
demonstrated that chronic stimulation by phorbol ester resulted in the
differential downregulation of PKC isoform mass (26, 29).
In HIT cells, overnight exposure to PMA selectively downregulated
PKC-
, -
II, and -
while preserving PKC-
, -µ, -
, and
-
. This differential loss of PKC isoform mass was correlated with
the loss of Ca2+-sensitive PKC activity (29).
Interestingly, not only did chronic PMA treatment and PKC
downregulation not diminish glucose-stimulated secretion but they
resulted in an enhanced secretory response to stimulation by glucose,
cell depolarization, or cholinergic stimulation, suggesting an
increased sensitivity in a signaling step common to various
secretagogues (17, 29, 31). The mechanism of this
increased secretion is unknown but has been previously attributed to
either the removal of an inhibitory input by PKC or the long-lived
phosphorylation of cellular substrates by the activated and
subsequently downregulated isoforms, in particular PKC-
(17,
31).
The studies presented here support a different explanation. After
chronic PMA treatment, -cells (HIT-T15) no longer responded to the
acute addition of PMA but exhibited a heightened release of insulin,
which required the presence of residual PMA in the cell. These data
indicated that increased secretion was the result of the continued
PMA-induced activity of one or more of the DAG-sensitive PKC isoforms
that are not downregulated. Removal of the residual cellular PMA after
downregulation caused a slight resynthesis of PKC-
and -
. The
functional consequence of PKC resynthesis was that acute PMA again
stimulated secretion, and the magnitude of this response correlated
with changes in the mass of PKC-
, but not PKC-
. A similar
correlation was observed for the mass of PKC-
and the potentiation
of secretion by the neuropeptide bombesin. Thus PKC-
and PKC-
and/or -µ are likely targets of phorbol esters or DAG in the
stimulation of insulin secretion.
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METHODS |
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Culturing of clonal pancreatic -cell line.
HIT cells, subclone T-15, were a gift from Dr. A. E. Boyd III.
HIT-T15 cells were cultured in T-75 plastic culture flasks with RPMI
1640 medium containing 50 mU/ml penicillin and 50 µg/ml streptomycin
(Sigma), 10% FCS, 10
7 M selenious acid, and 10 µg/ml
glutathione (14). Cells were harvested by rinsing in
Ca2+- and Mg2+-free Hanks' buffer (Sigma)
followed by either exposure to 0.02% EDTA for 10 min or trypsinization
at 37°C in 0.005% trypsin (GIBCO). Cells used were from
passages 72-85.
Secretion and insulin assays. HIT-T15 cells were plated in 24-well plates and used 2-4 days after passaging for secretion assays. Cells were washed two times in Krebs-Ringer bicarbonate (KRB) with 1.8 mM CaCl2, buffered with HEPES at pH 7.4, and then preincubated with KRB containing 0.05% fatty acid-free BSA for 30 min at 37°C. The buffer was removed, and the cells were incubated for 30 min under the conditions cited in each figure. Alternatively, after the hour of PMA washout (see next section), cells were incubated for 30 min without any preincubation. In either case, the experiments were stopped by cooling the plates on ice and removing an aliquot for determination of insulin. Samples were assayed for insulin by RIA using the double-antibody assay protocol for rat insulin distributed by Linco Research.
Washout of PMA after PKC downregulation. The time course of PMA washout was established using [3H]PMA to determine its efflux rate with increasing concentrations of BSA from cells grown in 24-well plates. After downregulation, cells were washed with KRB alone and either solubilized and counted for total counts per minute (cpm; time 0) or incubated for various times with KRB containing increasing concentrations of BSA. Efflux was expressed as the percentage of counts that were found in the media vs. the total cpm found in the tissue at time 0. Cells were solubilized with KRB containing 25 mM NaOH and 0.1% Triton X-100, and radioactivity was measured using liquid scintillation counting. These extracts included [3H]PMA contained within the cells and bound to the plate itself. In these initial experiments, no correction was made for [3H]PMA binding to plastic.
After this initial experiment, the removal of PMA was optimized by using 6- or 12-well plates. Once the optimal concentration of BSA was determined, HIT cells were grown in 12-well plates and downregulated using standard RPMI 1640 supplemented with 10% FCS and 200 nM PMA forPreparation of cell samples and SDS-PAGE. Cells were extracted in ice-cold 50 mM Tris · HCl, pH 7.5, containing 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM 2-mercaptoethanol, and 25 µg/ml each of leupeptin and aprotinin. Cells were collected from three replicate wells with 300 µl extraction buffer by scraping and sonicated on ice. Sonication was done with a Branson Cell Disrupter using a micro-tip at a power level of two using two 6-s pulses at 50% duty cycle. The mixture was spun for 10 min at 700 g to remove cellular debris and nuclei. The protein content of these homogenates was determined using Bio-Rad reagent. SDS-PAGE was performed according to the method of Laemmli (16).
Western blotting. Transfer of protein to nitrocellulose paper was done electrophoretically using a semidry apparatus from Owl Scientific (Cambridge, MA). A transfer buffer of Tris · HCl, pH 7.4, containing SDS and methanol was used, and the transfer was performed with constant current at room temperature. Blots were probed with isozyme-specific polyclonal antibodies as outlined by the various suppliers (GIBCO Life Sciences, Santa Cruz Biochemicals, and Transduction Laboratories). The secondary antibody was a goat anti-rabbit IgG conjugated to horseradish peroxidase purchased from Boehringer Mannheim. The specificity of the interaction was assessed by using the isozyme-specific blocking peptide provided. Visualization of the binding of the horseradish peroxidase-conjugated secondary antibody was achieved using the enhanced chemiluminescense (ECL) kit from Amersham.
Measurement of intracellular free Ca2+. Cytosolic free Ca2+ was measured in suspensions of cells loaded with the Ca2+ indicator fura 2 by use of the ratios method, as described previously (6).
Measurement of extracellular free Ca2+. Free Ca2+ in RPMI 1640 used during PMA-induced downregulation of PKC isoforms was measured in the presence or absence of 425 µM EGTA. Measurements were made using an Orion Ca2+ selectrode (Boston, MA) attached to an amplifier from the Biomedical Instrumentation Group (University of Pennsylvania School of Medicine) and standards provided by World Precision Instruments (Sarasota, FL).
Monitoring changes in membrane potential. Changes in cellular membrane potential were monitored using the cell-permeant indicator bis-oxonol (excitation, 540 nm; emission, 590 nm), as previously outlined (30).
Monitoring changes in cellular NADH. The changes in cellular NADH levels were monitored by measuring the change in its intrinsic fluorescence using excitation and emission wavelengths of 340 and 460 nm, respectively (4). These changes were expressed as a percentage of the full scale determined from a minimum seen with the uncoupling agent FCCP (50 nM) and the maximum obtained using the respiratory blockers antimycin A (0.5 µM) and oligomycin (2.5 µg/ml).
Scanning and densitometry of SDS-PAGE. Films exposed to ECL were scanned, and the image were captured using an Agfa Studiostar flatbed scanner in conjunction with PhotoLook for Windows (version 2.09). The digitized image was analyzed using Scion Image for Windows (version 4.1), which is derived from NIH Image for Macintosh. Comparison of band density was done only within a single film, reflecting samples run on the same gel.
Statistics. The data are reported as means and SE of individual observations. Significance was assessed by Student's t-test and considered significant at P values <0.05. Multiple comparisons were examined using a one-way ANOVA with a Student-Newman-Keuls test done post hoc. Differences were considered significant at P values <0.05.
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RESULTS |
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Enhanced secretion seen after overnight PMA treatment and PKC
downregulation.
Basal, glucose-stimulated, and KCl-induced insulin secretion was not
only preserved but was enhanced in clonal -cells (HIT-T15) after
overnight PMA-induced downregulation of PKC (Fig.
1). Like glucose, 30 mM KCl-induced
depolarization of the
-cell stimulates secretion by activating
L-type and other voltage-dependent Ca2+ channels and raises
[Ca2+]i (30). The enhanced
secretory response to KCl in clonal
-cells seen here (2-fold) was
similar to the 2.5-fold stimulation previously described in islets
cultured overnight in the presence of phorbol ester (31).
However, in this clonal cell line, unlike islets, KCl elicited a
greater secretory response than glucose. The inactive phorbol ester
phorbol 12,13-didecanoate (4
-PDD) was compared with its active
stereoisomer 4
-PDD and PMA. Only prolonged exposure to 4
-PDD and
PMA resulted in the enhanced response seen with downregulation and was
characterized by the lack of response to the acute addition of PMA
(data not shown).
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Effect of preserving insulin content.
Because of the stimulatory effect of PMA on insulin secretion, insulin
content decreases during overnight PMA treatment. To minimize the loss
of insulin content during downregulation and to ensure that control and
downregulated cells were well matched, extracellular
[Ca2+] was reduced in the culture medium to blunt insulin
secretion. Results in Table 1 demonstrate
that the addition of EGTA to the medium reduced Ca2+ levels
from 875 to 155 µM and preserved -cell insulin content during
chronic PMA treatment and PKC downregulation. In contrast, the insulin
content of
-cells chronically treated in the presence of normal
levels of extracellular Ca2+ was reduced by 29% compared
with controls. Comparison of secretion from
-cells having normal and
reduced insulin content demonstrated that preservation of insulin
content increased the secretory responses (Fig.
3). KCl-induced secretion was used for
this comparison because of its large stimulation of secretion before
and after PKC downregulation (Fig. 1). The increased response obtained
by preserving insulin content was seen under basal conditions, with KCl
alone, or in combination with glucose and forskolin, an agent that
greatly increases cAMP. These results demonstrate that loss of stored insulin decreases the responses to secretagogues and thereby emphasize the importance of matching insulin content so as not to underestimate the effect of chronic PMA treatment on secretion.
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Enhanced secretion after downregulation requires PMA retention.
Because phorbol esters are lipophilic and DAG-sensitive PKC isoforms
remain after downregulation, a possible reason for the enhanced
secretion observed might be the retention of cellular PMA. Therefore,
we compared the secretion from downregulated -cells with preserved
insulin content (Fig. 3, reduced Ca2+) with the response of
control
-cells in which 200 nM PMA was added acutely (Fig. 3, acute
PMA). Although the degree of increase in secretion resulting from
KCl-induced depolarization was large, it was not maximal, as judged by
the further response to the combination of glucose, KCl, and forskolin.
Furthermore, the response to KCl or the combination of secretagogues
was similar in these two groups, suggesting that the enhanced response
to KCl in the overnight-treated cells, shown in Fig. 1, could be
because of retention of cellular PMA. In contrast, basal insulin
secretion in acutely stimulated cells was greater than that seen in
downregulated cells with or without normal insulin content (Fig. 3).
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Readdition of PMA after its washout stimulated insulin secretion.
A functional consequence of PMA-induced downregulation, without
adequate washout, was that subsequent addition of phorbol ester did not
further stimulate secretion (Fig.
5A, PMA retained). The
comparison of control cells and downregulated cells with PMA retained
demonstrated the expected potentiation of both basal secretion and
glucose-stimulated insulin secretion without further stimulation of
secretion by acute PMA. In contrast, when the PMA retained from
overnight incubation was removed rapidly, basal and glucose-stimulated
secretion returned to control levels, and the potentiation of
glucose-stimulated insulin secretion by PMA was possible again.
Readdition of an inactive phorbol ester had no effect on
glucose-stimulated secretion (data not shown). Unexpectedly, with PMA
washout, this repotentiation of glucose-stimulated secretion was
greater than the enhanced secretion observed after PKC downregulation and matched the potentiation by PMA seen in control cells (Fig. 5A).
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Resynthesis of PKC- and PKC-
.
Western blots, paired to the three secretion conditions shown in Fig.
5A, indicated that PKC-
, PKC-
II, and PKC-
were lost with downregulation and did not reappear with PMA removal (Fig. 5B). As expected, PKC-
and -µ were not downregulated.
However, if the blots of PKC-
, PKC-
II, and PKC-
were
overexposed, a small, but detectable, resynthesis of PKC-
and
PKC-
was evident, as seen in the bottom blot of each pair in Fig.
5B. In contrast, PKC-
II was undetectable after
downregulation and did not reappear during PMA washout. Therefore, we
asked if blocking the resynthesis of
and
would affect the
potentiation of insulin secretion by the acute PMA.
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Mechanism responsible for enhanced secretion is distal to changes in [Ca2+]i. First, to assess the influence of chronic PMA treatment and PKC downregulation on metabolism, the ability of glucose to reduce cellular NAD was monitored using the intrinsic fluorescence of NADH in cell suspensions. Changes in the pyridine and flavin nucleotide oxidation state have been shown to be early events in glucose metabolism, preceding changes in O2 consumption and [Ca2+]i (4). Glucose addition caused an equivalent increase in NADH fluorescence in both control and downregulated cells (20% increase over basal levels), indicating that glucose metabolism, as reflected by the redox state, was not affected by PKC downregulation (data not shown).
Second, the ability of increased extracellular KCl to depolarize the ![]() |
DISCUSSION |
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Downregulation of PKC activity by chronic stimulation has been used in various cell types and by many investigators to provide evidence for the involvement of PKC in cell processes. Downregulation of PKC isozymes is thought to occur via activation and subsequent proteolysis at membrane surfaces and may depend on transactivation by other enzymes (12, 20). The conventional and novel isozymes, because of their sensitivity to phorbol esters, are activated and targeted to membranes where they may be degraded by Ca2+-dependent proteases with prolonged PMA exposure.
Our recent work (29), and data presented here, have shown
that chronic PMA treatment of clonal -cells (HIT) resulted in the
downregulation of three of the seven isoforms expressed. The PKC
isoforms
,
II, and
were undetectable after overnight exposure to PMA and correlated with the loss of PKC activity that was
Ca2+/DAG dependent (29). The nPKC isoforms
and µ were resistant to downregulation. The resistance of PKC-
to
clearance could be specific to the
-cell (clonal or native), for it
is cleared from other cell types after chronic PMA treatment
(12). The presence of PKC-µ was previously reported in
another
-cell line, MIN6, where it was also unaffected by chronic
PMA treatment (26).
The functional result of this chronic PMA treatment was the enhancement
of both KCl- and glucose-stimulated insulin secretion in clonal
-cells (HIT-T15) and rat islets (29, 31). This was
originally suggested to result from a long-lived phosphorylation of
cellular substrates still remaining after downregulation of PKC-
(6, 31). However, the short time to downregulation and the
rapid reversal of this enhancement on PMA removal, within 1 h,
after the overnight PMA treatment are inconsistent with this proposal.
Instead, these data indicate that the enhancement is because of the
continued activation of one or both of the isoforms that were not
downregulated but are PMA sensitive, namely
and µ. Interestingly,
MIN6 cells, which apparently do not contain PKC-
, were reported not
to exhibit enhanced secretion after downregulation (26),
suggesting that the PKC-
isoform is involved in the enhanced secretion exhibited by HIT cells and primary
-cells
(31). Alternatively, a constitutively active PKC
M-fragment produced by downregulation might underpin enhanced
secretion, but this proposal is incompatible with the requirement for
retained PMA, since this species is not activated by phorbol esters. In
addition, PKC-
and -
also remain after downregulation but cannot
be activated by PMA. Thus the involvement of nPKC would be consistent
with earlier studies where downregulation was assessed by the
readdition of phorbol ester or the loss of cPKC isoforms. A non-PKC
target of PMA is also a possibility. One such alternative is the C1
motif-containing presynaptic protein Munc13-1, which is expressed in
neuronal cells and might be consistent with our data. This protein has
been suggested to mediate the enhancement of neurotransmitter release
by phorbol esters and DAG (3, 23).
The results presented here indicate that the enhanced secretion seen with glucose or KCl after chronic PMA treatment does not involve major changes in cell depolarization, Ca2+ handling, or glucose metabolism. Subtle changes in Ca2+ metabolism seen at the single-cell level may have been missed in these studies (1) but are unlikely to account for the enhanced responses observed. Because glucose-stimulated and KCl-induced secretion were similarly affected, it is likely that the enhanced secretion resulted from an alteration in the cellular response to a rise in intracellular Ca2+. Therefore, because this enhancement of secretion is likely the result of activation of nPKC isoforms, the results imply that at least these PKC isoforms affect a step in the stimulus-secretion coupling pathway downstream from the Ca2+ rise.
The dependence of the enhanced secretion on the continued presence of
PMA also argues against another previously proposed explanation, that
the enhancement is the result of removal of an inhibitory action from
one of the downregulated PKC isoforms (17). Furthermore,
the enhanced secretion after downregulation was at best equal to the
secretion of control cells acutely stimulated by PMA, when loss of
insulin content was prevented (Fig. 3), indicating that removal of a
negative input cannot be the primary explanation. Examination of the
individual downregulated PKC isoforms did not suggest an inhibitory
action. Thus the positive correlation of PKC- mass with secretion
indicated that this isoform has a positive input. There was no obvious
correlation, either positive or negative, of secretion with
resynthesized PKC-
mass. Whether the third downregulated isoform,
PKC-
II, has either positive or negative actions could not be
ascertained, since it was not resynthesized.
The positive correlation of the potentiation of glucose-stimulated
secretion with resynthesized PKC- mass indicated that this isoform
provided a second mechanism for coupling PMA/DAG to insulin release.
This is consistent with PMA enhancing basal secretion more strongly in
control cells than downregulated cells where PKC-
has been lost
(Fig. 3). Our finding that the mass of PKC-
was limiting for
restimulation of secretion by PMA (Fig. 7) is consistent with earlier
studies showing that GSIS correlated with the translocation of PKC-
to the plasma membrane (8, 9). Perhaps it should be
expected that PMA- and glucose-stimulated secretion would have a
similar correlation with the mass of PKC-
based on the assumption
that PMA and DAG activate the same subset of PKC isoforms. The present
study, however, goes beyond those initial studies in that all of the
isoforms expressed in the
-cells were examined, not just PKC-
.
The correlation seen here with PKC-
was not seen with PKC-
and
could not be tested for PKC-
II, since the latter was not
resynthesized after downregulation within the time frame examined.
Perhaps one of the most striking findings of this series of experiments
was that resynthesis of even a small fraction of the PKC- normally
found in these cells appeared responsible for the recovery of normal
stimulation of secretion by PMA (Fig. 5). Support for the specificity
of this effect is provided by the observation that the resynthesis of
PKC-
under identical conditions did not correlate with potentiation
of secretion by PMA. The fact that PKC-
mass must be reduced by
>95% to be limiting suggests that only a small fraction of this
isoform normally present is catalytically competent or involved in
secretion. If so, this fact raises the possibility that the fraction of
PKC-
involved in secretion is discreetly localized in the cell to
support this function. The physiological relevance of this finding is
seen in data demonstrating an analogous situation for the potentiation
of GSIS by bombesin where retarding the resynthesis of PKC-
with
cycloheximide also prevented restoration of bombesin sensitivity (Fig.
8). Previously, bombesin has been shown to cause a rapid (20-s),
significant, and biphasic rise in both total DAG and inositol
1,4,5-trisphosphate in HIT cells (24). Potentiation of
GSIS by bombesin is more complex than the addition of PMA, since
phorbol esters do not increase intracellular Ca2+ in HIT
cells. However, we have shown here that the ability of bombesin to
increase intracellular Ca2+ and the ability of these cells
to respond to this signal were not impaired by exposure to cycloheximide.
In conclusion, these data with clonal -cells (HIT-T15) are
consistent with a model of stimulated secretion where phorbol esters or
endogenous DAG provide a positive input for secretion by the activation
of both cPKC and nPKC isoforms. After downregulation of PKC-
, the
enhancement of secretion that is seen would be mediated by nPKC
isoforms and depend on the short-lived phosphorylation of a substrate
and the continued presence of PMA. The result of activating nPKC
isoforms would occur distally in stimulus-secretion coupling, perhaps
modulating the Ca2+ sensitivity of exocytosis itself.
Whether the same is true of the action of PKC-
remains to be determined.
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ACKNOWLEDGEMENTS |
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This work was supported by NIDDK Grants DK-50662, DK-35914, and DK-53064.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. C. Yaney, Boston Univ. Medical Center, EBRC, Rm. 810C, 650 Albany St., Boston, MA 02118 (E-mail: gyaney{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 30, 2002;10.1152/ajpendo.00474.2001
Received 21 October 2001; accepted in final form 19 June 2002.
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