Departments of 1 Physiological Science and 2 Anesthesiology, University of California, Los Angeles, California 90095
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ABSTRACT |
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We have examined the
interaction between muscarinic and 2-adrenergic receptor
activation on inositol phosphate (IP) formation in the nonpigmented
cells of the ciliary body epithelium (NPE cells) of the rabbit. We have
compared these changes with those previously observed in the
intracellular free Ca2+ concentration. Whereas muscarinic
receptor activation causes an increase in intracellular
Ca2+ and IP formation, activation of
2-receptors does not significantly increase either
intracellular Ca2+ or IPs over basal levels. However,
simultaneous activation of muscarinic and
2-adrenergic
receptors with the specific agonists carbachol and UK-14304 produces
massive Ca2+ increases and results in a synergistic
increase in IP formation. This synergistic IP formation is inhibited by
both muscarinic and
2-adrenergic receptor antagonists as
well as by pertussis toxin and an inhibitor of phospholipase C. IP
formation is predominantly independent of intracellular
Ca2+, because it is decreased but not prevented by blocking
the entry of Ca2+ with LaCl3 or chelating
intracellular Ca2+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca2+ resulting from
simultaneous activation of muscarinic and
2-adrenergic receptors.
muscarinic receptor; 2-adrenergic receptor; calcium; synergism; ciliary epithelium
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INTRODUCTION |
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ACTIVATION OF MANY G protein-coupled receptors results in an increase in the concentration of intracellular Ca2+. For some of these receptors, this increase results from the stimulation of phospholipase C (PLC), which causes the breakdown of phosphatidylinositol 4,5-bisphosphate into the intracellular messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). It is the binding of IP3 to its receptor that causes release of Ca2+ from intracellular stores (1, 35).
Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) can have a significant physiological effects in many cell types. In the cells of the bilayered ciliary body epithelium, the tissue within the eye that is responsible for secretion of the aqueous humor, these changes can be produced by a variety of pharmacological agents that also affect aqueous secretion (see Refs. 23 and 39 for review). Spatial and temporal Ca2+ signaling within the single epithelial layer and across the bilayer has been reported (19, 31). In addition, multiple receptor activation can modulate the levels of intracellular messengers and produce large synergistic increases in Ca2+ (13, 14, 31, 43).
A synergistic increase in the [Ca2+]i occurs
in the nonpigmented cell layer of the rabbit ciliary body epithelium
when muscarinic receptors and 2-adrenergic,
P1 purinergic, or somatostatin receptors are activated
simultaneously. This increase is much larger than that produced by the
activation of any of these receptors alone (13, 14, 31,
43). Of the five types of muscarinic receptor classified to
date, three (M1, M3, and M5) have
been shown to be linked to the PLC-IP3/DAG pathway. Both
M1 and M3 receptors have been shown to be
present in the ciliary epithelium on the basis of pharmacological
(11, 25, 40), immunologic (16), and molecular
biological techniques (18, 20). These subtypes are coupled
to the family of Gq proteins, which activate PLC-
(32, 42). A1-purinergic, somatostatin, and
2-adrenergic receptors in the ciliary epithelium are all
thought to be coupled to G proteins of the Gi/o subclass,
which are sensitive to pertussis toxin (PTX) (26, 38, 41).
The Gi/o proteins act by inhibiting adenylyl cyclase,
resulting in a decrease in cAMP. However, in many cell types,
Gi/o-coupled receptors also have been shown to activate PLC-
in a cAMP-independent manner (see Refs. 9 and 34 for review).
The purpose of this study was to determine how these signal
transduction cascades interact to produce such a large Ca2+
increase with multiple-receptor activation. In particular, we examined
agonist-induced changes in IP3, measured in the form of
total inositol phosphate (IP) accumulation, to determine whether the
simultaneous activation of muscarinic and 2-adrenergic
receptors would result in the synergistic formation of IPs. Portions of this work have been published in abstract form (5, 6).
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MATERIALS AND METHODS |
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Tissue preparation. Nonpigmented cell layers were isolated from 2- to 3-wk-old pigmented rabbits as described previously (7, 12). For IP measurements, tissue from 1.5-2 eyes was plated into each well of a multiwell dish (1 well/replicate) in 1.0 ml of culture medium. The medium consisted of inositol-free NCTC-135 medium (GIBCO, Rockville, MD) supplemented with 2 µCi myo-[3H]inositol, 5% fetal bovine serum (Hyclone Labs, Logan, UT), 3 mM L-glutamine, 100 µg/ml kanamycin, 50 µg/ml gentamicin, 0.52 µM cholesterol, 1.9 µM ATP, 5 µM O-phosphoethanolamine, 5 µM ethanolamine, 150 µM sodium pyruvate, 3.3 µM ribose, 1 µM FeSO4, 0.49 µM adenine, and 0.79 mg/ml bovine serum albumin. For Ca2+ imaging, additional pieces of nonpigmented cell layer were plated onto laminin-coated glass coverslips. The medium was supplemented with 0.1 µM myo-inositol, a concentration equivalent to the concentration of radioactive inositol used in IP measurements. All cultures were held at 37°C and in 5% CO2 in air. In some cases, a piece of nonpigmented cell layer was removed from the multiwell dish prepared for IP measurements and used for Ca2+ imaging. No differences in responses were seen in tissues prepared in this way from those directly plated into a petri dish.
Ca2+ imaging. To measure changes in [Ca2+]i, nonpigmented cell layers were incubated in 5 µM fura 2-acetoxymethyl ester (fura 2-AM; Molecular Probes, Eugene, OR) in culture medium at 37°C for 60-90 min. The explants were washed in HEPES-buffered Ringer solution, and responses to drug applications were recorded as described previously (8). For each tissue, the fluorescent signal from several areas was measured, and the Ca2+ concentration was calculated after calibration. Because resting levels varied among areas, the percentage of drug-induced increase above the resting level also was determined. The average Ca2+ concentration and percent increase were then calculated for each tissue. Data are given as the means ± SE of several pieces of tissue, with the number of tissues indicated in the text.
IP measurements. Nonpigmented cell layers were held in culture for 48-72 h to allow for sufficient incorporation of the radioactive label. The labeled tissues were then stimulated and the IPs separated according to the method of Zhu et al. (45) with minor modifications, as follows. The tissues were washed three times in HEPES-buffered Ringer (see Ref. 13 for formulation) and incubated for 30 min in 0.5 ml of Ringer at 37°C. LiCl (10 µl at 1 M) was then added, and the tissues were incubated for an additional 20 min. The agonists to be tested were then added in an additional 0.5 ml of Ringer, and the tissues were allowed to incubate for 30 min. At the end of this time, the solution was quickly removed, and the reaction terminated by the addition of 0.75 ml of ice-cold 20 mM formic acid. When blockers were used, they were added 10 min before the agonists except where a different time is noted. For the study of the time course of IP production, the samples were frozen with liquid N2 immediately after the addition of formic acid and allowed to thaw on ice.
After 2 h on ice, the IPs produced were separated from myo-inositol by ion-exchange chromatography. The cell extracts were loaded into columns containing 1 ml of AG1-X8 resin (formate form), immediately followed by 6 ml of 40 mM NH4OH, pH 9.0, and the eluates containing myo-inositol were collected. The columns were then washed with 12 ml of 40 mM ammonium formate. Inositol 4-monophosphate (IP1), inositol 1,4-bisphosphate (IP2), and IP3 were eluted with 0.2 M, 0.4 M, and 2 M ammonium formate-0.1 M formic acid solution, respectively. For most of the experiments, the total IPs accumulated during the 30-min incubation period were eluted in one step with 2 M ammonium formate-0.1M formic acid. The counts from the total or individual IP fractions were divided by the sum of the counts from the IP fractions plus the myo-inositol fraction. In each experiment, each drug tested was run in either duplicate or triplicate (1 well of a multiwell culture dish per replicate). Data are given as means ± SE, averaged over the number of measurements indicated in the text. The number of experiments is also given. Statistical analysis was performed using unpaired Student's t-test (P < 0.05 was considered significant).Materials.
Carbachol, methoxamine, yohimbine, isoproterenol, PTX,
5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6- quinoxalinamine
(UK-14304), and
1-[6-([17-3-methoxyestra-1,3,5(10)trien-17-yl]amino)hexyl]-1H-pyrrole-2-dione (U-73122) were purchased from RBI (Natick, MA). All drugs were prepared
as concentrated stock solutions and stored at
20°C until use, with
the exception of PTX, which was stored at 4°C. UK-14304 and U-73122
were dissolved in DMSO. Fura 2-AM and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM were purchased from Molecular Probes and dissolved in DMSO
on the day of the experiment. The final concentrations of DMSO used
were 0.1% and 1%, respectively. Vehicle controls were run in all
experiments. myo-[3H]inositol was purchased
from NEN (Boston, MA). All other chemicals were purchased from Sigma
Chemical (St. Louis, MO).
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RESULTS |
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In most cells, activation of PLC leads to an increase in the
[Ca2+]i. To assess the level of PLC activity
produced by stimulation of muscarinic and 2-adrenergic
receptors in the nonpigmented cell layer of the ciliary body epithelium
of the rabbit, we made measurements of both
[Ca2+]i and IPs.
In the nonpigmented layer, simultaneous application of acetylcholine
and epinephrine results in a synergistic increase in the
[Ca2+]i (8, 13, 30). This
response is a result of the activation of muscarinic cholinergic and
2-adrenergic receptor subtypes, because the synergistic
response can be produced by the muscarinic receptor agonist carbachol
and the specific
2-adrenergic receptor agonist UK-14304
(13). A representative response recorded from a
nonpigmented cell layer is shown in Fig.
1A. On average, from a resting
[Ca2+]i of 99 ± 3 nM (n = 58), 50 µM carbachol caused an increase in the
[Ca2+]i of 225 ± 21% over basal levels
(n = 39), whereas 0.5 µM UK-14304 caused an
insignificant increase compared with vehicle control (n = 23, P > 0.05). However, the combination of carbachol
and UK-14304 caused an increase in the
[Ca2+]i of 1,922 ± 288%
(n = 26). The absolute values for the average Ca2+ responses produced by carbachol, UK-14304, and the
combined drugs are shown in Table 1.
These responses are similar to those for acetylcholine and UK-14304 as
previously reported in other ciliary body epithelial preparations by us
and others (8, 13, 30).
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To confirm that the increase in the [Ca2+]i was due to IP formation, we measured the total IPs accumulated after a 30-min stimulation of the tissues with the agonists. As shown in Fig. 1B, the basal activity of these cells resulted in 31 ± 1% of the total counts (the sum of the myo-inositol and IP fractions) in the IP fraction (n = 66 in 25 experiments). Stimulation of the tissue with 50 µM carbachol caused an increase in the mean IP formation to 49 ± 2% of total counts (n = 33 in 13 experiments), representing a 55% increase over the basal level. UK-14304, applied at 0.5 µM, had no significant effect on IP formation compared with the basal level, resulting in 34 ± 1% of the total counts in the form of IPs (P > 0.05; n = 25 in 10 experiments). However, the combination of carbachol and UK-14304 caused an increase in the IP fraction to 66 ± 2% of the total counts (n = 27 in 10 experiments), or an 111% increase over the basal level. This is, on average, twice the response to carbachol alone.
The effect of carbachol concentration, in the presence and absence of 0.5 µM UK-14304, on IP formation is shown in Fig. 1C. Both carbachol and carbachol plus UK-14304 caused dose-dependent increases in IP accumulation that saturated at 1 mM. However, the maximal response to the combined drugs (Vmax: 78 ± 2% total IPs) was significantly larger than that produced by carbachol alone (Vmax: 64 ± 2% total IPs). In the range of concentrations examined from 50 µM to 10 mM, the carbachol-induced IP formation was significantly increased by the presence of 0.5 µM UK-14304 (Fig. 1C). At 0.1 and 10 µM, IP formation resulting from carbachol stimulation was not significantly different from IP formation in response to the combined drugs. The EC50 values for carbachol in the absence and presence of UK-14304 were similar (26 ± 9 and 25 ± 7 µM, respectively).
The time-dependent formation of total and individual inositol
polyphosphates is shown in Fig. 2. In
control tissues, the accumulation of the individual IPs varied little
with time. However, in tissues treated with 10 mM carbachol alone and
in combination with 0.5 µM UK-14304, a time-dependent change could be
seen in all forms of IPs. A high dose (10 mM) of carbachol was used to
elicit maximum IPs for these experiments. Within 30 s of
stimulation, an accumulation of IP3 above basal levels was
observed in response to the combination of carbachol and 0.5 µM
UK-14304, and this accumulation peaked at 1 min (24 ± 12% above
basal, n = 11 in 4 experiments, P > 0.05) (Fig. 2A). The IP3 formation in response
to carbachol alone was not detectable above basal levels at 30 s
but reached a maximum at 1 min of stimulation (17 ± 14% above
basal, P > 0.05). The accumulation of
IP2 was apparent within 30 s of stimulation and reached a maximal level at 3 min for both carbachol and the combination (Fig. 2B). IP1 formation increased nearly
linearly in response to both carbachol and the combined drugs (Fig.
2C). Figure 2D shows the sum of the individual IP
fractions, expressed as total IPs formed. The combination of carbachol
and UK-14304 produced statistically more total IPs than control at each
time point examined (P < 0.05). Within 30 s, an
~36 ± 6% increase above basal levels was observed
(n = 12 in 4 experiments), which climbed to 58 ± 8% (n = 11 in 4 experiments), 135 ± 11%
(n = 6 in 2 experiments), and 242 ± 9%
(n = 6 in 2 experiments) over basal levels at 1, 3, and
10 min of stimulation, respectively. Carbachol alone did not produce
significantly more total IPs than control until 1 min of stimulation
(P < 0.05, 15 ± 8% over basal level). Total IPs
continued to accumulate in response to carbachol to 72 ± 18% and 138 ± 10% above basal levels at 3 and 10 min, respectively.
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Effect of receptor agonists and antagonists.
Synergistic IP formation occurs when carbachol is combined with an
2-adrenergic receptor agonist, but not with agonists of other adrenergic receptor subtypes. Substitution of UK-14304 with the
1-adrenergic agonist methoxamine or the
-adrenergic
agonist isoproterenol, in combination with carbachol, results in IP
formation that is no larger than that produced by carbachol alone (Fig. 3). The combination of 10 µM
methoxamine and 100 µM carbachol produced an increase in the mean IPs
of 87% over basal levels (n = 6 in 2 experiments),
which is not significantly different from the response to 100 µM
carbachol alone (81% increase on average, n = 12 in 5 experiments). Carbachol and 1 µM isoproterenol decreased the IP
formation compared with carbachol, resulting in only a 73% increase
over basal level (n = 6 in 2 experiments). The IP formation as a result of application of either methoxamine or isoproterenol alone was not significantly greater than basal levels. These results correlate well with changes in
[Ca2+]i in response to these drugs that we
have previously reported (8, 13).
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Effect of signaling blockers.
In the ciliary body epithelium, 2-adrenergic receptors
have been shown to be coupled to a G protein of the Gi/o
subclass (21). In addition, we have shown that other
agonists that activate Gi/o proteins can substitute for
UK-14304 in producing a synergistic Ca2+ response when
combined with acetylcholine (14, 43). We therefore examined the effect of an inhibitor of this class of G proteins, PTX
(38), on IP formation and Ca2+ increases. The
results from these experiments are shown in Fig. 5 and Table 1. A 4-h preincubation of the
tissues with 1 µg/ml PTX significantly decreased the synergistic IP
formation normally produced by carbachol and UK-14304 incubation from
61% to 50% over basal levels (P < 0.05). In three
experiments, the response to the combined agonists in the presence of
PTX was 100 ± 2% of the carbachol response in the presence of
PTX. PTX had only a slight effect on basal IP formation that was not
statistically significant (P > 0.05). The effects of
PTX on the IP formation resulting from carbachol or UK-14304
stimulation also were not significant (P > 0.05) (Fig.
5A). The Ca2+ responses of nonpigmented cell
explants were similarly affected by PTX. The mean increase in the
[Ca2+]i produced by the combined application
of carbachol and UK-14304 was reduced to the response to carbachol
alone by the presence of PTX (Fig. 5B and Table 1). PTX had
only a slight effect on the resting Ca2+ and the response
to carbachol or UK-14304, reducing them to between 10 and 20% of mean
control levels.
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Effect of Ca2+ on IP formation.
The change in [Ca2+]i seen in response to
simultaneous muscarinic and 2-adrenergic receptor
activation is composed of a transient peak followed by a sustained
level that remains above the baseline. In nonpigmented cells, the peak
is predominantly a result of release from internal stores, whereas the
plateau is a result of Ca2+ entry (8).
Blocking Ca2+ entry with LaCl3 results in a
significant reduction in the sustained phase of the synergistic
Ca2+ response in the intact ciliary epithelium
(13). Because some phospholipases have been shown to be
Ca2+ dependent (17, 22, 28), we measured the
IPs produced in the presence of 10 µM LaCl3 to determine
whether the synergistic IP formation was due to Ca2+
feedback on PLC. The results of these experiments are shown in Fig.
7.
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DISCUSSION |
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In this study, we have examined agonist-induced increases in IP3 formation, measured either as IP3 itself or in the form of total IPs, in the nonpigmented cells of the rabbit ciliary body epithelium. IP3 is the messenger that causes release of Ca2+ from internal stores. However, rapid hydrolysis of IP3 leads to the formation of IP2 and IP1. Therefore, we have used the total IPs generated as a measure of phospholipase activity induced by receptor activation in this tissue.
Our goal was to examine the mechanism that underlies the synergistic
increase in intracellular Ca2+ elicited by multiple
receptor activation. We have demonstrated that simultaneous activation
of a muscarinic receptor and an 2-adrenergic receptor
causes a synergistic increase in IP formation. The muscarinic receptor
involved in this response activates PLC, resulting in an increase in IP
accumulation of ~55% over basal levels (Fig. 1B). This
result is similar to that previously reported for human (11,
40) and rabbit (25) ciliary epithelial cells. When the muscarinic receptor is stimulated along with the
2-adrenergic receptor, it causes a more than twofold
increase in total IPs over the levels produced by muscarinic
stimulation alone (measured at 30 min).
This difference in IP formation is even greater at times more closely corresponding to the peak of the Ca2+ response. Total IPs produced by the combined agonists at 30 s are approximately eight times those produced by carbachol alone and approximately four times the response at 1 min, when the production of IP3 by both single- and multiple-receptor activation is at its maximum as measured by us (Fig. 2). By 3 min, this difference has already fallen to two times the response. Thus, within the first couple of minutes of the response to multiple-receptor activation, both total IPs and [Ca2+]i have increased to similar extents.
Because IP3 is the messenger that releases Ca2+ from internal stores, the question remains as to why we have not observed a large increase in IP3 itself in stimulated tissues at these early times (Fig. 2A). For this, we can offer a couple of explanations. First, it is possible that in a stimulated cell, the local concentration of IP3 near the plasma membrane is high but becomes diluted by the mass of the cytoplasm. The concentration that we measure is therefore much less than exists locally. Second, the IP3 concentration would be dependent on the rate of its formation and degradation. The fact that we have such a large basal IP accumulation (~20% of total counts from 1 to 3 min) and that the distribution of the individual IP forms is fairly constant with time in control tissues (Fig. 2) implies that both mechanisms are constitutively active. It is possible that the enzymes responsible for IP3 degradation (e.g., IP3 5-phosphatase) are sufficiently active to prevent any significant global increase in IP3. Our data support the notion that IP3 is rapidly degraded because of the high levels of IP2 and IP1 that are formed within 30 s by the combined agonists.
Our observation of a synergistic production of IPs is in contrast with a previous report using rabbit ciliary processes in which only additive IP formation was observed in response to maximal doses of both carbachol and norepinephrine (25). In our hands, even maximal doses of carbachol when combined with 0.5 µM UK-14304 produced synergistic increases in IP formation (Fig. 1C). The differences in these observations may be due to several factors such as preincubation duration or specificity of the agonists employed in the assay. However, the primary difference between that study and ours was their use of the bilayered epithelium (or cells derived from both layers) versus the isolated nonpigmented epithelial layer that we employed. Other studies that used only nonpigmented cells isolated from the human ciliary epithelium (11, 40) reported an EC50 for carbachol-stimulated IP formation of 40 µM, which is similar to the value we observed. The EC50 for the bilayered epithelial preparation used by Mallorga et al. (25) was 154 µM.
The activation of the muscarinic receptor/PLC/IP3 pathway is required to produce the synergism, because block of the receptor with atropine or inhibition of the PLC with U-73122 causes a significant reduction in the IPs accumulated in response to both single- and multiple-receptor activation (Figs. 4A and 6). U-73122 was somewhat less effective in blocking the responses in IPs than Ca2+. This may be due to the fact that the response in IPs is measured as an accumulation over 30 min versus the very short duration of the response in [Ca2+]i. The responses in the presence of other blockers (yohimbine or PTX, for example) may be similarly affected (but see DISCUSSION above).
A second receptor type participates in the signal transduction cascade
that produces synergistic increases in Ca2+ and IPs. For
the experiments described in this paper, we have used the
2-adrenergic receptor to examine this interaction,
although preliminary experiments with somatostatin have produced
similar results (unpublished observations). Of the adrenergic receptor subtypes, only
2 appears to participate in producing
synergistic increases in IPs and Ca2+, because specific
1- or
-adrenergic receptor agonists do not substitute
for the
2-agonist (Fig. 3). In addition,
[Ca2+]i (8, 13) and IP (Fig.
4B) responses can be inhibited with the
2-adrenergic antagonist yohimbine.
To date, we have identified three receptors
(2-adrenergic, A1-purinergic, and
somatostatinergic) that can be activated along with the muscarinic
receptor to produce synergistic [Ca2+]i
responses (13, 14, 43). All three of these receptor types are reported to act through a PTX-sensitive G protein, probably Gi (41). Incubation with PTX caused an
inhibition in the accumulated IPs and the intracellular
Ca2+ increase produced by the combined drugs to the levels
produced by carbachol alone. Thus activation of both a PTX-insensitive G protein (possibly a member of the Gq/11 family), via the
muscarinic receptor, and a PTX-sensitive G protein, via a different
receptor type (the
2-adrenergic in this case), is
required to produce the synergism. Gi is most widely
recognized as interacting with adenylyl cyclase to cause a change in
cAMP (36, 37). However, we have been unable to show an
effect of either an inhibitor (SQ-22536) or activator (forskolin) of
adenylyl cyclase or permeant analogs of cAMP on the synergistic
response (unpublished observations). Gerwins and Fredholm
(15) also could not find a role for cAMP in producing
synergistic IP increases in response to adenosine and bradykinin
stimulation in smooth muscle cells, although this has been reported in
other tissues (24).
The experiments with a Ca2+ entry blocker and a Ca2+ chelator suggest that Ca2+ feedback on the PLC is not responsible for the synergistic increase in IP formation and intracellular Ca2+. The presence of BAPTA prevented the increase in the [Ca2+]i normally seen in response to both single- and multiple-receptor stimulation (Fig. 8A). Nonetheless, a synergistic increase in IP accumulation could still be detected (Fig. 8B). Furthermore, the increase in [Ca2+]i in response to carbachol, in the absence of BAPTA, was greater than the response to the combined drugs in its presence. Thus the [Ca2+]i level achieved by carbachol was not sufficient to cause a synergistic increase in IP formation. Therefore, Ca2+ feedback on the PLC could not be responsible for the synergistic IP production. However, these experiments do not exclude the possibility of some Ca2+-dependent component to IP formation, because IP formation was decreased on average in the presence of BAPTA or LaCl3.
The dose response curves (Fig. 1C) demonstrate that
synergistic IP formation was observed over all carbachol concentrations examined >10 µM. UK-14304 did not change the apparent affinity of
the receptor, because the EC50 for carbachol in the
presence and absence of UK-14304 was the same. Instead, it appears that the effect of UK-14304 was to cause an increase in the Vmax
of the response (from 64% to 78%). It is possible that this effect is
a result of the concurrent interaction of the -subunit of Gi/o and G
q with PLC-
[see Quitterer and
Lohse (29)].
It is possible that the PLC activity is enhanced by both muscarinic and
2-adrenergic stimulation. It is now generally accepted that some PLC-
isoforms are activated by both the
-subunit of Gq (
q) and, to a lesser extent, the
-subunits of Gi proteins (4). It is also
known that PLC-
isoforms have separate binding sites for
q- and
-subunits (22). It was
originally proposed that the effects of the
-subunits on PLC-
were independent of the stimulatory action of an
-subunit (10,
34). It now appears, however, that at least in some cases, the
stimulatory effects of the
-subunits on PLC-
depend on
costimulation with an appropriate
-subunit (44). A
similar mechanism has also been proposed for the potentiating effect of
the
-subunits on the stimulation of adenylyl cyclase by
s (36).
At present, the isoforms of PLC- or of the
- or
-subunits of G
proteins in ciliary body epithelial cells have not been identified.
Furthermore, there is no direct evidence that in this tissue any such
interaction at the level of PLC-
takes place. However, findings
reported here, as well as those reported previously (8,
13), have indicated that in the ciliary body epithelium,
2-adrenergic receptor activation, on its own, fails to
induce a significant rise in the [Ca2+]i
(Fig. 1A) or total IPs (Fig. 1B) above basal
levels. In the presence of carbachol, the
2-agonist
UK-14304 induces synergistic, rather than additive, increases in both
[Ca2+]i and total IPs above the
levels reached in response to carbachol alone. In other words,
increases in [Ca2+]i and total IPs in
response to
2-adrenergic receptor activation are
conditional on the concurrent stimulation of the
Gq/11-linked muscarinic receptor. In addition, IP formation
resulting from both single- and multiple-receptor activation is
decreased to a similar extent by an inhibitor of PLC (Fig. 6). This
raises the possibility that the synergistic cross talk between pathways linked to the muscarinic and
2-adrenergic receptors
takes place at the level of the PLC, as suggested by Zhu and Birnbaumer
(44). However, at this point alternative mechanisms should
also be considered. For example, it has been shown that PLC-
is a
GTPase-activating protein (GAP), thus accelerating the hydrolysis of
GTP (2). As suggested by Clapham and Neer
(10), inhibition of PLC-
GAP function by G
could
lead to prolongation of the G
response, thus creating synergism
between G
and G
. Alternatively, G
subunits from
Gi proteins may accelerate receptor-Gq
protein interaction by directly combining with G
q
(29).
In conclusion, we have demonstrated that IPs accumulate synergistically
when both muscarinic and 2-adrenergic receptors are stimulated. Our experiments suggest that in the ciliary body
epithelium,
-subunits released by the activation of a
Gi/o-coupled receptor may enhance the effect of
Gq on PLC, either directly or through another mechanism.
Alternatively, this effect could be due to an agonist-induced increase
in activity of phosphatidylinositol 4-phosphate 5-kinase, as
demonstrated in neutrophils (33). This would lead to an
increase in the mass of phosphatidylinositol 4,5-bisphosphate available
for hydrolysis by PLC-
, and thus greater IP generation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gordon L. Fain for continued support and comments on the manuscript and Dr. Mariel Birnbaumer for allowing the use of her laboratory, teaching the technique of IP measurements, and providing invaluable discussions. We also thank Alisa Mendez for excellent technical assistance in the tissue culture of the nonpigmented layers and Emily Abe, Patil Armenian, Steven Chen, Connie Gomez, Laura Kim, Babak Mikhak, Annaha Song, and Scott Um for helping with the tissue dissections that made this work possible.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants EY-06969 (to N. A. Farahbakhsh), EY-07568 (to G. L. Fain), and DK-41244 (to M. Birnbaumer).
Address for reprint requests and other correspondence: M. Cilluffo, Dept. of Physiological Science, Life Science 3836, UCLA, PO Box 951527, Los Angeles, CA 90095-1527 (E-mail: mariannc{at}physci.ucla.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. §1734 solely to indicate this fact.
Received 24 September 1999; accepted in final form 21 March 2000.
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