Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells

Marianne C. Cilluffo1, Evette Esqueda2, and Nasser A. Farahbakhsh1

Departments of 1 Physiological Science and 2 Anesthesiology, University of California, Los Angeles, California 90095


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have examined the interaction between muscarinic and alpha 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 alpha 2-receptors does not significantly increase either intracellular Ca2+ or IPs over basal levels. However, simultaneous activation of muscarinic and alpha 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 alpha 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 alpha 2-adrenergic receptors.

muscarinic receptor; alpha 2-adrenergic receptor; calcium; synergism; ciliary epithelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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-beta (32, 42). A1-purinergic, somatostatin, and alpha 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-beta 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 alpha 2-adrenergic receptors would result in the synergistic formation of IPs. Portions of this work have been published in abstract form (5, 6).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-([17beta -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 2-adrenergic receptor subtypes, because the synergistic response can be produced by the muscarinic receptor agonist carbachol and the specific alpha 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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Fig. 1.   Changes in intracellular Ca2+ (A) and inositol phosphate (IP) formation (B and C) as a result of stimulation with muscarinic and alpha 2-adrenergic agonists, alone and in combination. A: the muscarinic agonist carbachol (50 µM, carb) caused an approximate tripling of the resting Ca2+ concentration. The response to 0.5 µM of the specific alpha 2-adrenergic agonist UK-14304 (UK) was small. However, when combined (carb+UK), the Ca2+ concentration rose >10-fold. B: total IPs, normalized to the sum of the myo-inositol and IP fractions, were measured after a 30-min incubation without any drug (basal) or with 50 µM carbachol, 0.5 µM UK, or their combination (see MATERIALS AND METHODS). On average, the IPs produced by UK applied alone were the same as basal formation. However, the combination of carbachol and UK produced significantly greater IP formation than carbachol alone. *Significantly different (P < 0.001). C: IP formation as a function of carbachol concentration in the absence (dashed line) and presence (solid line) of 0.5 µM UK. Values are means ± SE for each concentration, calculated from at least 3 experiments. Over the concentration range from 50 µM to 10 mM carbachol, UK caused synergistic IP formation. However, the EC50 remained unchanged.


                              
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Table 1.   Changes in intracellular Ca2+ concentration in response to drug applications

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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Fig. 2.   Time course of IP3 (A), IP2 (B), IP1 (C), and total IP accumulation (D) in nonpigmented cell layer explants. IPs varied only slightly in control tissues (black-triangle). Time-dependent changes in all forms of IPs could be observed in response to 10 mM carbachol () and the combination of carbachol and 0.5 µM UK ().

Effect of receptor agonists and antagonists. Synergistic IP formation occurs when carbachol is combined with an alpha 2-adrenergic receptor agonist, but not with agonists of other adrenergic receptor subtypes. Substitution of UK-14304 with the alpha 1-adrenergic agonist methoxamine or the beta -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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Fig. 3.   The synergistic increase in IPs only occurs when carbachol is combined with an alpha 2-adrenergic agonist. Carbachol (100 µM) plus UK (1 µM) produced a synergistic increase in IPs compared with carbachol alone. However, 10 µM of the alpha 1-adrenergic agonist methoxamine (mxa) or 1 µM of the beta -adrenergic agonist isoproterenol (iso) produced very little IP accumulation compared with basal levels. Neither methoxamine nor isoproterenol acted with carbachol to produce a synergistic increase in IPs. Dagger Not significantly different (P > 0.05).

Changes in the [Ca2+]i produced by activation of acetylcholine and epinephrine receptors in the nonpigmented cell layer are inhibited by blocking either muscarinic or alpha 2-adrenergic receptors with specific antagonists (8, 13, 27) (see Table 1). We therefore examined IP formation in the presence of these antagonists. The effect of the muscarinic antagonist atropine on IP formation is shown in Fig. 4A. Atropine had no effect on the basal activity or the response to UK-14304. However, nearly all of the IP accumulation observed in response to carbachol alone or in combination with UK-14304 was prevented in the presence of 1 µM atropine. In two experiments, the response to carbachol in the presence of atropine was reduced such that it was not significantly different from basal levels (P > 0.05). Atropine had the same effect on the response to the combination of carbachol and UK-14304.


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Fig. 4.   Both muscarinic and alpha 2-adrenergic receptor blockers decrease IP accumulation. A: application of 1 µM atropine, a muscarinic antagonist, has a profound effect on IP formation. In the presence of atropine, the IPs formed as a result of stimulation with either carbachol or the combination of carbachol and UK were not significantly greater than basal levels, or each other (Dagger P > 0.05). B: an alpha 2-adrenergic antagonist, yohimbine (1 µM), significantly reduced the IPs produced by the combination of carbachol and UK (*P < 0.05). However, at the concentration used it did not significantly affect the response to application of either drug alone.

The predominant alpha 2-adrenergic nature of the synergistic IP response was verified with the use of the specific alpha 2-adrenergic antagonist yohimbine. IP formation in the presence and absence of yohimbine is shown in Fig. 4B. In two experiments, 1 µM yohimbine reduced the mean IP formation resulting from incubation in carbachol plus UK-14304 from 89% to 66% over basal levels. The response to the combination in the presence of yohimbine was significantly smaller than the combined response in its absence (P < 0.05). Yohimbine had no significant effect on the IPs produced by either carbachol or UK-14304 alone.

Effect of signaling blockers. In the ciliary body epithelium, alpha 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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Fig. 5.   Pertussis toxin (PTX), an inhibitor of Gi/o proteins, decreased the synergistic IP accumulation and Ca2+ increase. A: after a 4-h incubation in 1 µg/ml PTX, the total IP levels produced by the combination of 50 µM carbachol and 0.5 µM UK were not significantly larger than those produced by carbachol alone (Dagger P > 0.05). Data are means ± SE averaged over 4 experiments. For the control data sets, the responses were recorded in the presence of 1 mM Na2HPO4 and 5 mM NaCl (vehicle for PTX). B: a representative recording of the time course of intracellular Ca2+ changes in a nonpigmented cell layer after incubation in PTX. The magnitude of the response to carbachol plus UK was nearly the same as the response to carbachol alone.

Because activation of M1 and M3 receptors is reported to stimulate PLC, we also examined the effect of U-73122, a known inhibitor of PLC (3). As shown in Fig. 6A, a 10-min preincubation of the tissues with 5 µM U-73122 significantly reduced the mean IPs produced in response to carbachol plus UK-14304 from 115% to 75% over basal levels. The response to carbachol was similarly reduced from 62% to 34% over basal levels by U-73122. Thus U-73122, at the concentration used in this experiment, reduced the amount of IP production to both carbachol and the combined drugs by 45% and 35%, respectively.


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Fig. 6.   The effect of an inhibitor of phospholipase C (PLC) on IP production and intracellular Ca2+. A: the IPs formed by carbachol or the combination of carbachol and UK were significantly reduced in the presence of 5 µM U-73122. *P < 0.05; **P < 0.05 compared with vehicle alone (0.5% DMSO). Data are averaged over 2 experiments. B: perfusion of a nonpigmented cell explant with 0.5% DMSO for 5 min caused a small increase in resting Ca2+ and then decreased the synergistic response to carbachol plus UK by about one-half. However, the addition of 5 µM U-73122 (U7) caused a near complete inhibition of the synergistic Ca2+ increase. The response to carbachol plus UK could not be recovered.

The effect of U-73122 on the [Ca2+]i was much greater (Table 1). An example is shown in Fig. 6B. U-73122 on its own caused a 1.8-fold increase in resting [Ca2+]i (n = 4 tissues). However, 50% of this increase was due to the effect of the DMSO (0.5% final concentration) used to dissolve the U-73122. U-73122 then blocked 74% of the increase in [Ca2+]i seen in response to the combination of carbachol and UK-14304 (with DMSO), decreasing it from 1,726% to 184% over basal levels. The synergistic Ca2+ response was not recoverable. However, the lack of a response in the presence of U-73122 was not due to depletion of stores, because tissues treated with vehicle only maintained their synergistic increase through multiple applications of carbachol plus UK-14304 (Fig. 6B).

Effect of Ca2+ on IP formation. The change in [Ca2+]i seen in response to simultaneous muscarinic and alpha 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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Fig. 7.   Block of Ca2+ entry with 10 µM LaCl3 decreased the IPs produced but did not prevent the synergism. The presence of La3+ significantly decreased the IPs produced by the combination of carbachol and UK (*P < 0.05). However, La3+ did not prevent the synergistic IP production resulting from carbachol plus UK compared with carbachol alone (dagger P < 0.005).

In three experiments, blocking Ca2+ entry with La3+ had no significant effect on basal IP formation or on the response to UK-14304 (P > 0.05). La3+ reduced the IP formation as a result of carbachol incubation from 45% to 34% over basal levels, although this was not statistically significant. La3+ did have a significant effect on the IP formation resulting from incubation with carbachol plus UK-14304, causing it to fall from 91% to 60% over basal levels. However, in the presence of La3+, the IP formation produced by the combination of carbachol and UK-14304 was still significantly larger than that produced by carbachol alone (Fig. 7).

Because La3+ blocks the entry of Ca2+ but does not prevent the initial large release of Ca2+ from internal stores, we examined IP accumulation after incubating the tissue in the fast Ca2+ chelator BAPTA-AM (Fig. 8). To be sure we were preventing any significant rise in [Ca2+]i, we first examined drug-induced changes in intracellular Ca2+ in tissues incubated in 50 µM BAPTA-AM. A representative recording is shown in Fig. 8A, and average Ca2+ responses are given in Table 1. For the experiments like that shown in Fig. 8A, BAPTA incubation for 1 h virtually eliminated any change in [Ca2+]i in response to carbachol and the combination of carbachol plus UK-14304. The response to the combined drugs in tissues after a 1-h incubation in 1% DMSO without BAPTA was the same as the response recorded before treatment with DMSO (data not shown). Tissues incubated in BAPTA-AM for 3 h and then examined for changes in [Ca2+]i had responses similar to those shown in Fig. 8A (data not shown).


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Fig. 8.   Chelating intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevents the large Ca2+ increase but not the synergistic IP production. A: a representative recording of agonist-induced increases in intracellular Ca2+ in a nonpigmented cell layer before and after incubation in 50 µM BAPTA-AM. The combination of 50 µM carbachol and 0.5 µM UK produced a massive increase in the intracellular Ca2+ that was prevented after a 1-h incubation in BAPTA-AM. B: IPs resulting from carbachol or UK stimulation were not affected by the presence of BAPTA. Synergistic IP production was reduced but was still evident (*P < 0.05).

Figure 8B shows the IPs produced after a 3-h incubation in the presence of 50 µM BAPTA-AM. BAPTA did not significantly affect basal production compared with vehicle control (P > 0.05, n = 5 in 2 experiments). It also did not have any effect on the IPs produced by either carbachol or UK-14304 incubation. BAPTA somewhat reduced the mean IP production resulting from incubation with the combined drugs from 101% to 73% over basal levels. These responses, however, were not statistically different (P > 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 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 alpha 2-adrenergic receptor to examine this interaction, although preliminary experiments with somatostatin have produced similar results (unpublished observations). Of the adrenergic receptor subtypes, only alpha 2 appears to participate in producing synergistic increases in IPs and Ca2+, because specific alpha 1- or beta -adrenergic receptor agonists do not substitute for the alpha 2-agonist (Fig. 3). In addition, [Ca2+]i (8, 13) and IP (Fig. 4B) responses can be inhibited with the alpha 2-adrenergic antagonist yohimbine.

To date, we have identified three receptors (alpha 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 alpha 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 beta gamma -subunit of Gi/o and Galpha q with PLC-beta [see Quitterer and Lohse (29)].

It is possible that the PLC activity is enhanced by both muscarinic and alpha 2-adrenergic stimulation. It is now generally accepted that some PLC-beta isoforms are activated by both the alpha -subunit of Gq (alpha q) and, to a lesser extent, the beta gamma -subunits of Gi proteins (4). It is also known that PLC-beta isoforms have separate binding sites for alpha q- and beta gamma -subunits (22). It was originally proposed that the effects of the beta gamma -subunits on PLC-beta were independent of the stimulatory action of an alpha -subunit (10, 34). It now appears, however, that at least in some cases, the stimulatory effects of the beta gamma -subunits on PLC-beta depend on costimulation with an appropriate alpha -subunit (44). A similar mechanism has also been proposed for the potentiating effect of the beta gamma -subunits on the stimulation of adenylyl cyclase by alpha s (36).

At present, the isoforms of PLC-beta or of the beta - or gamma -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-beta takes place. However, findings reported here, as well as those reported previously (8, 13), have indicated that in the ciliary body epithelium, alpha 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 alpha 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 alpha 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 alpha 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-beta is a GTPase-activating protein (GAP), thus accelerating the hydrolysis of GTP (2). As suggested by Clapham and Neer (10), inhibition of PLC-beta GAP function by Gbeta gamma could lead to prolongation of the Galpha response, thus creating synergism between Galpha and Gbeta gamma . Alternatively, Gbeta gamma subunits from Gi proteins may accelerate receptor-Gq protein interaction by directly combining with Galpha q (29).

In conclusion, we have demonstrated that IPs accumulate synergistically when both muscarinic and alpha 2-adrenergic receptors are stimulated. Our experiments suggest that in the ciliary body epithelium, beta gamma -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-beta , and thus greater IP generation.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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