Cholinergic-Induced Ca²⁺ Elevation in Rat Lacrimal Gland Acini Is Negatively Modulated by PKCδ and PKCε

Abstract

purpose. To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca²⁺ elevation in lacrimal gland acini.

methods. Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca²⁺ ([Ca²⁺]_i) were measured using fura-2 as a fluorescent probe.

results. Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca²⁺]_i transient and the plateau of [Ca²⁺]_i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca²⁺-independent PKC isoforms PKCδ and -ε, but not the Ca²⁺-dependent isoform PKCα substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca²⁺ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca²⁺, suggesting that PKC inhibits the influx of Ca²⁺. PMA completely inhibited the cholinergic agonist-induced plateau of[ Ca²⁺]_i. PMA and calyculin A decreased both the [Ca²⁺]_i transient and the plateau of[ Ca²⁺]_i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca²⁺.

conclusions. In the lacrimal gland, agonist-induced changes in[ Ca²⁺]_i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.

Lacrimal gland protein secretion is mainly under the control of cholinergic muscarinic receptors. ^{1 2} The lacrimal gland muscarinic receptors of the M₃ subtype are coupled to the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, ³ generating two second-messenger molecules, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). ⁴ IP₃ interacts with specific receptors present on the endoplasmic reticulum to liberate Ca²⁺ in the cytosol. ⁴ The free Ca²⁺ in conjunction with calmodulin then activates Ca²⁺ and calmodulin-dependent protein kinases. DAG interacts with and activates protein kinase C (PKC). ^{5 6} Activation of these kinases leads to the phosphorylation of specific substrates thought to trigger lacrimal gland protein secretion. 

It is well documented that cholinergic agonists stimulate the release of intracellular Ca²⁺ and influx of Ca²⁺ from the extracellular medium in the lacrimal gland. ^{7 8 9 10} The Ca²⁺ response usually consists of an initial large Ca²⁺ transient (peak) due to IP₃-induced Ca²⁺ release from intracellular stores followed by a sustained plateau of[ Ca²⁺]_i due to influx of Ca²⁺. There is considerable evidence that depletion of IP₃-sensitive intracellular Ca²⁺ stores leads to activation of this secondary Ca²⁺ entry, a process termed by Putney ¹¹ as capacitative Ca²⁺ entry. ^{11 12}  

Thapsigargin, a specific inhibitor of endomembrane Ca²⁺-ATPase activity, inhibits sequestration of Ca²⁺ that has leaked from the intracellular stores, thereby decreasing [Ca²⁺] within the stores. ¹³ It is this decrease in[ Ca²⁺] within the stores that is believed to signal entry of Ca²⁺ into the cells by stimulating capacitative entry of Ca²⁺. ¹¹ Thus, thapsigargin increases[ Ca²⁺]_i without production of IP₃ and can be used to study entry of Ca²⁺ into cells. 

Recent reports have shown that preactivation of PKC results in negative feedback on cholinergic-induced Ca²⁺ release in the lacrimal gland, implicating PKC in the mechanism of desensitization of the cholinergic response. ^{14 15 16} Although it has been shown that preactivation of PKC results in a decreased production of IP₃, the effect of PKC on Ca²⁺ influx in the lacrimal gland has not been investigated. 

PKC is a family of closely related serine and threonine kinases consisting of at least 11 isoforms divided into three groups ¹⁷ : calcium- and phospholipid-dependent protein kinases PKCα, -βI, -βII, and -γ; calcium-independent and phospholipid-dependent protein kinases PKCδ, -ε, -η, -θ, and -μ; and calcium- and phospholipid-independent protein kinases PKCλ/ι and -ζ. All PKC isoforms except PKCμ have a pseudosubstrate sequence in their N-terminal part that is thought to interact with the catalytic domain to keep the enzyme inactive in resting cells. ^{18 19}  

In a recent study, we synthesized and N-myristoylated three peptides derived from the pseudosubstrate sequences of PKCα, -δ, and -ε, three PKC isoforms present in lacrimal gland acini. ²⁰ Using these peptides, we showed that PKCα, -δ, and -ε isoforms are differentially involved in agonist-induced protein secretion. ²¹  

Materials and Methods

Phorbol 12-myristate 13-acetate (PMA), thapsigargin, and calyculin A were from Alexis (San Diego, CA), and collagenase type CLS III was from Worthington Biochemical (Freehold, NJ). Carbamylcholine chloride (carbachol), staurosporine, and sulfinpyrazone were from Sigma (St. Louis, MO). Fura-2 tetra-acetoxymethyl esters and Pluronic F127 were obtained from Molecular Probes (Eugene, OR). All reagents were of the highest purity available. 

Preparation of Lacrimal Gland Acini

All experiments conformed to the guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Both exorbital lacrimal glands were removed from male Wistar rats (125–200 g body weight) that had been anesthetized with CO₂ for 1 minute and then decapitated. Dispersed acini were isolated by collagenase digestion, as described previously. ²² Lacrimal glands were trimmed of fatty and connective tissue and fragmented into small pieces 2 to 3 mm in diameter. The pieces were then washed at 37°C in Krebs–Ringer bicarbonate (KRB) buffer (containing, in mM, 119 NaCl, 4.8 KCl, 1 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, and 25 NaHCO₃), supplemented with 10 mM Hepes, 5.5 mM glucose, and 0.5% bovine serum albumin (BSA; KRB-Hepes; pH 7.4). Lacrimal gland acini were prepared by incubating tissue pieces with collagenase (CLS III, 150 U/ml) in 10 ml KRB-Hepes buffer for 30 minutes at 37°C under a stream of 95% O₂-5% CO₂. Lacrimal lobules were subjected to gentle pipetting 10 times at regular time intervals through tips of decreasing diameter. The preparation was then filtered through nylon mesh (150-μm pore size) and the acini pelleted by centrifugation (50g, 2 minutes). The pellet was washed twice by centrifugation (50g, 2 minutes) through a 4% BSA solution made in KRB-Hepes buffer. The dispersed acini were allowed to recover for 30 minutes in 5 ml fresh KRB-Hepes buffer containing 0.5% BSA. 

Measurement of [Ca²⁺]_i

Acini were incubated in KRB-Hepes buffer containing 0.5% BSA, 0.5 μM fura-2 tetra-acetoxymethyl ester, 10% Pluronic F127, and 250 μM sulfinpyrazone for 60 minutes at 22°C. For the experiments involving the myristoylated PKC pseudosubstrate-derived peptides, acini were incubated first at 37°C for 30 minutes with the peptides and then transferred to 22°C, and fura-2 was added as has been described. For the experiments involving pretreatment with PMA or calyculin A, acini were loaded with fura-2, and then PMA (500 nM) or calyculin A (100 nM) was added 10 minutes before the end of the incubation period. Where indicated, staurosporine (1 μM) was added 10 minutes before addition of PMA. The cells were then washed with KRB-Hepes buffer containing 250 μM sulfinpyrazone, and fluorescence was measured at 22°C. Fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm, as previously described. ²³ To calculate [Ca²⁺], 5.6 mM EGTA, 7.5 mM Tris-HCl (pH 7.5), and 1% Triton X-100 were added at the end of the reaction to obtain minimum fluorescence. Maximum fluorescence was determined by the addition of 14.5 mM CaCl₂. The dissociation constant of 135 nM for fura-2 at 22°C was used to calculate [Ca²⁺] by the ratio method. ²³  

PKC Pseudosubstrate-Derived Peptide Synthesis

Myristoylated PKC pseudosubstrate-derived peptides were synthesized by butyloxycarbonyl strategy on resin (MHBA; Novabiochem, Meudon, France) using an automated synthesizer (model 430A; Applied Biosystems; Foster City, CA). Protocols and reagents were used as recommended by the manufacturer. Myristic acid was coupled to the peptide using dicyclohexylcarbodiimide hydroxybenzotriazole. Peptides were purified by reversed-phase high-performance liquid chromatography (HPLC) on a preparative column (30 × 0.9 cm, C₄; Vydac, Hesperia, CA) using a trifluoroacetic acid-acetonitrile solvent system. Peptide integrity was monitored by amino acid analysis and mass spectrometry. 

Data Presentation and Statistical Analysis

Where appropriate, data are expressed as means ± SEM. The data were statistically analyzed using Student’s t-test for paired values. P < 0.05 was considered to be significant. 

Results

In the present study, we showed that preactivation of PKC by the phorbol ester PMA or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A decreased both the[ Ca²⁺]_i transient and the plateau of [Ca²⁺]_i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA, further implicating PKC. Inhibition of the Ca²⁺-independent PKC isoforms, PKCδ and -ε, but not the Ca²⁺-dependent isoform PKCα, by synthetic peptides derived from the pseudosubstrate sequence of each isoform, substantially reversed the inhibitory effect of PMA on cholinergic-induced Ca²⁺ elevation. The effect of PMA was obtained only in the presence of extracellular Ca²⁺, suggesting that PKC inhibits the influx of Ca²⁺. PMA and calyculin A decreased both the[ Ca²⁺]_i transient and the plateau of [Ca²⁺]_i induced by thapsigargin, further supporting the idea that PKC modulates entry of Ca²⁺. We concluded that in the lacrimal gland, agonist-induced changes in[ Ca²⁺]_i are negatively regulated by phosphorylation by PKC. 

Effect of Preactivation of PKC on Carbachol-Induced Changes in[ Ca²⁺]_i

It is well documented that cholinergic agonists increase[ Ca²⁺]_i in the lacrimal gland. The response consists of an initial large Ca²⁺ transient (peak) due to Ca²⁺ release from intracellular stores, followed by a sustained plateau of[ Ca²⁺]_i due to influx of Ca²⁺. ^{24 25} Figure 1A shows a typical trace depicting the effect of increasing concentrations of carbachol on Ca²⁺ release from fura-2–loaded lacrimal gland acinar cells. The release of Ca²⁺ is dependent on the concentration of carbachol, with a maximum release reached at 10⁻⁴ M carbachol. Figure 1 shows the effect of increasing concentrations of carbachol on the Ca²⁺ transient (peak, Fig. 1B ) and the plateau of[ Ca²⁺]_i (Fig. 1C) . Peak and plateau values of Ca²⁺ show a similar dose dependency with a median effective concentration (EC₅₀) of approximately 5 μM carbachol. Preincubation of acini with PMA (500 nM) to activate PKC resulted in a dramatic inhibition of carbachol-induced Ca²⁺ release (Fig. 1A) . PMA pretreatment resulted in a significant inhibition of both the[ Ca²⁺]_i transient induced by carbachol concentrations of 10⁻⁶, 10⁻⁵, and 10⁻⁴ M (Fig. 1B) , as well as an inhibition of the plateau of[ Ca²⁺]_i, but only at carbachol concentrations of 10⁻⁶ and 10⁻⁵ (Fig. 1C) . These results confirm previous reports and show that both the release of Ca²⁺ from intracellular stores and the influx of Ca²⁺ are negatively modulated by PKC. 

Effect of Staurosporine and Myristoylated PKC Pseudosubstrate-Derived Peptides on PMA-Induced Decrease of Cholinergic-Induced Increase in [Ca²⁺]_i

To determine whether the effect of PMA was mediated by PKC, we used staurosporine, a reported inhibitor of PKC. ²⁶ When fura-2–loaded acini were preincubated in the presence of staurosporine before addition of PMA, the carbachol-induced[ Ca²⁺]_i increase was no longer inhibited (Fig. 2) . Staurosporine had no effect on cholinergic-induced changes in[ Ca²⁺]_i, whereas it reversed the effect of PMA on both the transient and plateau of[ Ca²⁺]_i (Fig. 2) . These results provide additional support for the hypothesis that the effect of PMA on receptor-induced changes in[ Ca²⁺]_i is mediated by PKC. 

In another set of experiments we investigated which PKC isoform mediates the inhibitory effect of PMA on receptor-induced changes in[ Ca²⁺]_i by using myristoylated PKC pseudosubstrate-derived peptides. ²¹ We had previously showed that none of the peptides alone has any effect on carbachol-induced Ca²⁺ release. ²¹ The results shown in Figure 2 show that myristoylated (myr)-PKCδ and myr-PKCε, but not myr-PKCα, reversed the effect of PMA on[ Ca²⁺]_i transient. The effect of PMA on the plateau of[ Ca²⁺]_i was reversed only by myr-PKCδ. These results suggest that it is PKCδ and -ε, but not -α, that exert the negative feedback on cholinergic-induced[ Ca²⁺]_i elevation in the lacrimal gland. 

Role of Extracellular Ca²⁺ in the Effect of PMA on Carbachol-Induced Changes in [Ca²⁺]_i

Bird et al. ²⁷ showed that although preactivation of PKC may decrease the IP₃ content in the lacrimal gland, this treatment had no effect on Ca²⁺ release induced by microinjected IP₃. Thus, PKC does not affect the IP₃ receptor. This suggested to us that PKC may affect the Ca²⁺ response by modulating its entry across the plasma membrane, especially because the plateau [Ca²⁺]_i was affected by PMA to the same extent as the peak[ Ca²⁺]_i. To test this hypothesis, we studied the effect of extracellular Ca²⁺ ([Ca²⁺]_o) omission (by omitting [Ca²⁺]_o and adding EGTA, a Ca²⁺ chelator) on carbachol-induced changes in[ Ca²⁺]_i in untreated and PMA-pretreated lacrimal gland acini. Omission of [Ca²⁺]_o plus addition of 2 mM, but not 1 mM, EGTA resulted in an effective decrease of carbachol-induced [Ca²⁺]_i release (data not shown). Thus, we used 2 mM EGTA to chelate[ Ca²⁺]_o and studied the effect of PMA pretreatment on carbachol-induced [Ca²⁺]_i release. As shown in Figure 3A (solid line), omission of [Ca²⁺]_o severely impaired the release of Ca²⁺ induced by carbachol under all concentrations tested (compare with Fig. 1A ). Both the peak and plateau values of Ca²⁺ were similarly affected by this treatment (compare Figs. 3B and 3C with Figs. 1B and 1C ). In the absence of[ Ca²⁺]_o, preactivation of PKC by PMA no longer had an effect on carbachol-induced[ Ca²⁺]_i release (Fig. 3) . This is in contrast to the carbachol-induced [Ca²⁺]_i release in the presence of [Ca²⁺]_o, which was dramatically inhibited by PMA. 

To provide further evidence that preactivation of PKC inhibits cholinergic-induced influx of Ca²⁺, PMA was added during the plateau of [Ca²⁺]_i. The cholinergic-induced plateau of [Ca²⁺]_i is completely dependent on [Ca²⁺]_o and is due to an influx of Ca²⁺ across the plasma membrane. ^{11 12} Thus, if preactivation of PKC affects this entry of [Ca²⁺]_o, then addition of PMA during the plateau of [Ca²⁺]_i should inhibit this phase of the Ca²⁺ response. Figure 4 shows that PMA (500 nM) rapidly and completely inhibited the cholinergic-induced plateau of [Ca²⁺]_i. These results suggest that, under normal conditions, PKC inhibits both the influx of Ca²⁺ and the release of intracellular Ca²⁺ by IP₃. 

Effect of PMA and Calyculin A on Thapsigargin-Induced Changes in[ Ca²⁺]_i

Thapsigargin inhibits sequestration of Ca²⁺ that has leaked from the intracellular stores, and this is believed to signal entry of Ca²⁺ into the cells. ¹³ Thus, thapsigargin increases[ Ca²⁺]_i without production of IP₃. To test the hypothesis that PMA, in addition to decreasing agonist-induced production of IP₃, inhibits entry of Ca²⁺, we used thapsigargin. Figure 5A shows that pretreatment with PMA (500 nM) inhibited the thapsigargin (1 μM)-induced [Ca²⁺]_i increase. Both the peak and the plateau[ Ca²⁺]_i induced by thapsigargin were significantly decreased by PMA (Fig. 5B) . To determine whether the effect of PKC was mediated by phosphorylation, we tested the effect of calyculin A, an inhibitor of PP1/2A ²⁸ on the thapsigargin- and carbachol-induced increase of[ Ca²⁺]_i. As shown in Figure 5A , pretreatment with calyculin A reproduced the effect of PMA and decreased the thapsigargin (1 μM)-induced[ Ca²⁺]_i increase. Both the peak and the plateau[ Ca²⁺]_i, induced by thapsigargin were decreased by calyculin A (Fig. 5B) . Similarly, calyculin A inhibited the carbachol-induced[ Ca²⁺]_i increase (Table 1) . Both the peak and the plateau[ Ca²⁺]_i were inhibited (Table 1) . 

These results suggest that PKC modulates the cholinergic Ca²⁺ response at two levels: decreased production of IP₃ resulting in a decreased peak of[ Ca²⁺]_i and inhibition of Ca²⁺ entry resulting in a decreased plateau of[ Ca²⁺]_i. They also suggest that the effect of PKC is through phosphorylation of a target protein that is sensitive to PP1/2A. 

Discussion

Cholinergic agonists stimulate the release of intracellular Ca²⁺ and influx of Ca²⁺ in the lacrimal gland. ^{7 8 9 10} The Ca²⁺ response usually consists of an initial large Ca²⁺ transient (peak) due to Ca²⁺ release from intracellular stores, followed by a sustained small plateau of[ Ca²⁺]_i due to influx of Ca²⁺. Recent reports have shown that preactivation of PKC results in a negative feedback on cholinergic-induced Ca²⁺ release in the lacrimal gland. ^{14 15 16} It has also been reported that preactivation of PKC results in a negative feedback on the production of the Ca²⁺ mobilizing agent IP₃, explaining the decreased Ca²⁺ response observed after such a treatment. ^{15 16} However, Bird et al. ²⁷ have shown that although preactivation of PKC may decrease the IP₃ content in the lacrimal gland, this treatment has no effect on microinjected IP₃-induced Ca²⁺ release, suggesting that the IP₃ receptor is not the target for PKC. The results of the present investigation show that PKC and PP1/2A may also inhibit the cholinergic response by modulating the influx of Ca²⁺, either through a direct mechanism (i.e., modulation of the membrane Ca²⁺ channel) or indirectly by preventing capacitative entry of Ca²⁺. 

One possible mechanism by which the activation of PKC or the inhibition of PP1/2A may inhibit agonist-induced changes in[ Ca²⁺]_i would be the stimulation of Ca²⁺ uptake by the stores by modifying the activity of the Ca²⁺-ATPase present in the endomembranes, thereby decreasing the[ Ca²⁺]_i. Increased loading of intracellular Ca²⁺ stores would lead to an inhibition of Ca²⁺ influx. Indeed, it has been shown that PKC stimulates Ca²⁺ uptake into the Ca²⁺ stores of platelets by increasing the activity of the Ca²⁺-ATPase pump. ^{29 30} The mechanism by which PKC modulates the activity of this pump is not known, but it has been suggested to occur through a mechanism similar to the one used by the cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA). In platelets, PKA is thought to increase the activity of the Ca²⁺-ATPase pump through phosphorylation of an accessory protein, which has been identified as rap1, a low-molecular-weight guanosine triphosphate (GTP)–binding protein of the ras superfamily of proteins. ³¹ This is of interest because Bird and Putney ³² showed that in the lacrimal gland, [Ca²⁺]_i mobilization may be controlled through a GTP-binding protein, probably one of the low-molecular-weight family. PKC or PP1/2A may also increase the activity of Ca²⁺-ATPase in the plasma membrane to stimulate efflux of Ca²⁺. Alternatively, PKC may prevent capacitative entry of Ca²⁺ by inhibition of IP₃ production through a negative feedback on phospholipase C activity. A reduced amount of IP₃ would result in a reduced increase in [Ca²⁺]_i and therefore a decreased state of emptiness of the intracellular stores, which would result in decreased influx of Ca²⁺ from the extracellular milieu. It is well accepted that the filling state of the intracellular Ca²⁺ stores regulates entry of Ca²⁺ across the plasma membrane through the capacitative model first proposed by Putney. ¹¹ The same mechanism could apply in the case of thapsigargin. A recent study showed that the thapsigargin-induced increase in[ Ca²⁺]_i in lacrimal gland acini is not simply through a passive leak of Ca²⁺ from the intracellular stores, but also depends on a basal level of IP₃, and this effect can be blocked by heparin, an IP₃ antagonist. ³³  

It is worth noting that neither the activation of PKC nor the inhibition of PP1/2A completely inhibited[ Ca²⁺]_i mobilization induced by carbachol or thapsigargin. This implies that[ Ca²⁺]_i mobilization is under a complex control that is especially crucial in the case of an exocrine gland such as the lacrimal gland where Ca²⁺ is necessary to trigger exocytosis. 

Using staurosporine, we showed that the inhibitory action of PMA on the cholinergic-induced increase in[ Ca²⁺]_i is mediated by PKC because staurosporine completely reversed the effect of PMA. Staurosporine alone had no effect on carbachol-induced[ Ca²⁺]_i changes, which is in agreement with the findings of Bird et al. ²⁷ In another set of experiments, we used myristoylated PKC pseudosubstrate-derived peptides to determine which PKC isoform was implicated in the negative feedback on cholinergic-induced increase in[ Ca²⁺]_i. We found that inhibition of PKCδ and -ε, but not -α, partially reversed the effect of PMA. It is worth noting that it is the two Ca²⁺-independent isoforms, PKCδ and -ε that seem to be involved. That a complete reversal of the PMA effect could not be obtained with the myristoylated PKC peptides may be because of the temperature used. Indeed, in secretion experiments in which these peptides were first tested, ²¹ we used a temperature of 37°C and a preincubation period of 60 minutes. In the Ca²⁺ experiments, we used a shorter preincubation period (30 minutes) at 37°C, and the cells were then transferred to a lower temperature (22°C) for fura-2 loading. It is possible that the entry of the peptides into the cells or the efficiency of interaction of these peptides with PKC is temperature sensitive. Nevertheless, our data suggest that PKCδ and -ε, but not -α, exert a negative feedback on cholinergic-induced[ Ca²⁺]_i elevation in the lacrimal gland. 

In summary, we conclude that activation of PKC or inhibition of PP1/2A results in a negative feedback on cholinergic- and thapsigargin-induced[ Ca²⁺]_i changes. We suggest that the site of action of PKC-PP1/2A may include the plasma membrane Ca²⁺ channels in the inhibition of entry of Ca²⁺ and that PKCδ and -ε, but not -α, are implicated in the negative feedback on cholinergic-induced[ Ca²⁺]_i elevation in the lacrimal gland. 

 Supported by National Institutes of Health Grant EY06177 (DAD) and a Fight For Sight Prevent Blindness America research grant (DZ).

 Submitted for publication May 14, 1999; revised August 9, 1999; accepted September 17, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

Effect of preactivation of PKC on carbachol-induced[ Ca²⁺]_i changes in lacrimal gland acini. (A) Typical trace of fura-2–loaded acini pretreated with vehicle or PMA (500 nM) for 10 minutes. Carbachol at increasing concentrations (arrows, time of addition) was then added, and changes in [Ca²⁺]_i were measured. Solid line, no pretreatment; dashed line, pretreatment with 500 nM PMA. (B, C) Effect of preactivation of PKC on the [Ca²⁺]_i transient (peak) and plateau induced by carbachol. Data are means ± SEM from five separate experiments similar to the one shown in (A). ○, No treatment; •, pretreatment with 500 nM PMA;* significantly different from no pretreatment.

Figure 2.

 

Effect of staurosporine (St) and the myristoylated (Myr) PKC pseudosubstrate-derived peptides on PMA-induced inhibition of the[ Ca²⁺]_i transient and plateau stimulated with carbachol (Cch). Fura-2–loaded lacrimal gland acini were pretreated with vehicle or PMA (5 × 10⁻⁷ M) for 10 minutes. Staurosporine (1 μM) was added 10 minutes before addition of PMA. The myristoylated pseudosubstrate-derived peptides (10⁻⁷ M) from PKCα (Myr-PKCα), PKCδ (Myr-PKCδ), or PKCε (Myr-PKCε) were added 80 minutes before addition of PMA. Carbachol (10⁻⁵ M) was added, and changes in[ Ca²⁺]_i were measured. Data are means ± SEM from three to seven experiments. *Significantly different from no pretreatment.

Figure 3.

 

Effect of preactivation of PKC on carbachol-induced[ Ca²⁺]_i changes in lacrimal gland acini in the absence of extracellular Ca²⁺ (plus addition of 2 mM EGTA). (A) Typical trace of fura-2–loaded acini pretreated with vehicle or PMA (500 nM) for 10 minutes. Carbachol (Cch) at increasing concentrations (arrows, time of addition) was then added, and changes in [Ca²⁺]_i were measured. Solid line, no pretreatment; dashed line, pretreatment with 500 nM PMA. (B, C) Effect of preactivation of PKC on[ Ca²⁺]_i transient and plateau induced by carbachol. Data are means ± SEM from five separate experiments similar to the one shown in (A). •, No treatment; ○, pretreatment with 500 nM PMA.

Figure 4.

 

Effect of PMA on cholinergic-induced plateau of[ Ca²⁺]_i. Fura-2–loaded lacrimal gland acini were stimulated with carbachol (10⁻⁴ M; arrow, time of addition of carbachol, Cch). Vehicle (solid line) or PMA (5 × 10⁻⁷ M, dotted line) was then added during the plateau of[ Ca²⁺]_i (arrow, time of addition of PMA). Changes in [Ca²⁺]_i were measured. The trace is a mean of three independent experiments.

Figure 5.

 

Effect of preactivation of PKC on thapsigargin (Thap)-induced[ Ca²⁺]_i changes in lacrimal gland acini. Fura-2–loaded lacrimal gland acini were pretreated with vehicle, PMA (5 × 10⁻⁷ M) or calyculin A (10⁻⁷ M), for 10 minutes. Thapsigargin (10⁻⁶ M) was then added (arrow) and changes in [Ca²⁺]_i were measured. (A) Typical trace; (B) effect of PMA and calyculin A (Cal A) on the [Ca²⁺]_i transient and the plateau induced by thapsigargin. Data in (B) are means ± SEM from three separate experiments.* Significantly different from thapsigargin alone.

Table 1.

 

Effect of Calyculin A on Carbachol-Induced[ Ca²⁺]_i Elevation (in nM)

	Peak	Plateau
Carbachol	134 ± 13	76 ± 19
Calyculin A + carbachol	94 ± 16*	46 ± 06

 Data are means ± SEM of three separate experiments. Calyculin A (10⁻⁷ M) was added 10 minutes before addition of carbachol (10⁻⁴ M).

*  Significantly different from carbachol alone.