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Endocrinology Vol. 139, No. 12 5103-5108
Copyright © 1998 by The Endocrine Society

ARTICLES

Nongenomic Mechanisms of Glucocorticoid Inhibition of Nicotine-Induced Calcium Influx in PC12 Cells: Involvement of Protein Kinase C1

Jian Qiu, Li-guang Lou, Xiu-ying Huang, Shu-jie Lou, Gang Pei and Yi-zhang Chen

Neuroscience Research Institute, Second Military Medical University (J.Q., S.-J.L., Y.-z.C.); and the Key Laboratory of Neurobiology, Shanghai Institute of Physiology (J.Q.), the Shanghai Institute of Cell Biology (L.-g.L., G.P.), and the Beijing Institute of Developmental Biology (X.-y.H.), Chinese Academy of Sciences, Shanghai 200433, China

Address all correspondence and requests for reprints to: Dr. Y. Z. Chen, Neuroscience Research Institute, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, China.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nongenomic mechanisms of corticosterone (B) inhibition of nicotine (Nic)-induced calcium influx were investigated in PC12 cells. Corticosterone could rapidly inhibit the Ca2+ influx induced by Nic, and BSA-conjugated B had a similar inhibitory effect. The inhibition of Nic-induced Ca2+ influx by B could be mimicked by protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and reversed by PKC inhibitors, chelerythrine chloride and Gö6976. When PC12 cells were pretreated with pertussis toxin, the inhibitory effect of B on Nic-induced Ca2+ influx was blocked. Both B and BSA-conjugated B could activate PKC activity, with the maximal responses at 10-9 and 10-7 M at 37 C, respectively. The dose-response curve was bell shaped. At 25 C, however, the dose-response curve considerably shifted to the right, and B was most potent at 10-5 M. The time course showed that PKC activity was highest at 5 min of B’s action. The results suggest that B might act via putative membrane receptors and inhibit the Ca2+ influx induced by Nic through the pertussis toxin-sensitive G protein-PKC pathway and that PKC plays an important role in the mechanisms of glucocorticoid nongenomic action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN RECENT years, the intracellular mechanism of glucocorticoid’s rapid nongenomic action has attracted more and more attention (1, 2, 3, 4). It has been observed in our laboratory that glucocorticoid affects the membrane potential of excitable cells such as neurons within minutes or seconds (1). Corticosterone (B) inhibits the release of arginine vasopressin from rat hypothalamic slices and the secretion of catecholamine in rat adrenal medullary chromaffin cells (5, 6).

In all types of cells, intracellular calcium, as a second messenger, plays an important role in many physiological activities including contraction, neurite outgrowth, and neurotransmitter and hormone secretion. There is an increasing body of data concerning the rapid effects of glucocorticoid on the intracellular calcium concentration ([Ca2+]i). B inhibits calcium inflow in rat pancreatic islets (7). Cortisol rapidly reduces 45Ca2+ accumulation in the cichlid fish pituitary in vitro (8) and inhibits calcium currents in guinea pig hippocampal CA1 neurons via G protein-coupled activation of protein kinase C (9). Glucocorticoid suppressed intracellular calcium release in adenohypophyseal cells (10) and enhanced 45Ca2+ uptake induced by 70 mM K+ in prepared brain synaptosomes (11). Recently in our laboratory, it has been demonstrated that corticosterone inhibits the increase in [Ca2+]i induced by acetylcholine (ACh) and high potassium in rat adrenal medullary chromaffin cells (6) and inhibits the Ca2+ influx induced by bradykinin while not affecting intracellular Ca2+ release in PC12 cells (12).

The mechanism of the rapid effects of glucocorticoid on intracellular calcium is not clear. In the present study, we analyzed how glucocorticoid affects nicotine (Nic)-induced Ca2+ influx and further investigated the signal transduction pathway of glucocorticoid’s rapid nongenomic action, especially G protein and protein kinase C (PKC) that may be involved. Our results have shown that glucocorticoid inhibits the Ca2+ influx induced by Nic through a putative membrane receptor-mediated, pertussis toxin (PTX)-sensitive G protein-PKC pathway and for the first time directly demonstrated that PKC plays an important role in the nongenomic action by PKC activity assay.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Pluronic F-127, fura-2/AM, EGTA, pertussis toxin (PTX), Gö6976, chelerythrine chloride (chelery), phorbol 12-myristate 13-acetate (PMA), ionomycin calcium salt, and phenylmethylsulfonylfluoride were obtained from Calbiochem (La Jolla, CA). B 21-sulfate, (±)nicotine, BSA (fatty acid free), B 21-hemisuccinate:BSA, verapamil hydrochloride (Vp), {omega}-conotoxin GVIA ({omega}-CTX), and ß-mercaptoethanol were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of analytical grade.

Cell culture
PC12 cells were the generous gift of Dr. K. Kuba (Department of Physiology, Saga Medical School, Saga, Japan). PC12 cells were grown in DMEM with high glucose (Life Technologies, Grand Island, NY) supplemented with 5% heat-inactivated FBS and 10% heat-inactivated horse serum and incubated in a humidified atmosphere containing 5% CO2 in air at 37 C. Flasks used to culture PC12 cells had been coated with mouse tail collagen to facilitate cell adherence to the surface of the substrate.

[Ca2+]i measurements
PC12 cells grown on a 30-mm diameter dish with 70–80% confluence were washed three times with physiological saline solution (PSS; containing 125 mmol/liter NaCl, 5.6 mmol/liter KCl, 2 mmol/liter CaCl2, 1.2 mmol/liter MgSO4·7H2O, 1.2 mmol/liter NaH2PO4·2H2O, 10 mmol/liter HEPES, and 10 mmol/liter glucose, pH 7.4). Cells were harvested by gently streaming PSS on the surface of the monolayers and then dissociated to yield single cell by passing cells through a pipette. They were suspended in 2 ml PSS containing 0.004% (wt/vol) pluronic, then loaded with 5 µmol/liter fura-2/AM for 40 min at 37 C. At the end of the loading period they were rinsed with PSS and resuspended in 2 ml PSS. Cells were kept at 25 C in the dark and were used within 2 h.

At the beginning of [Ca2+]i determination, 60 µl loaded cell suspension were added to glass coverslips coated with collagen and incubated in an incubator for 15 min at 37 C, then the coverslip was placed in a thermostatically ring chamber holding 300 µl incubation fluid. All drugs were added in volumes of less than 3 µl. All experiments were performed at room temperature (25 C) to minimize dye leakage.

The system used in our laboratory for calcium imaging and [Ca2+]i measurement was the MiraCal Imaging System supplied by Life Science Resources Ltd. (Cambridge, UK). It consisted of a Nikon Diaphot 200 inverted fluorescence microscope coupled to a MiraCal 1000TE low light level CCD camera and a computer station. Images were captured and quantitatively analyzed by the MiraCal version 2.3 software program (Life Sciences Resources). The peak excitation wavelength of the fura-2 shift from 380 to 340 nm upon Ca2+ binding and the ratio of emission intensities in each wavelength provide a measure of free [Ca2+]i. Changes in [Ca2+]i in the PC12 cells were imaged through a Nikon CF-fluor 10x or 20x objective (Melville, NY) and the CCD camera by calculating the ratio of fura-2 fluorescence at 510 nm, excited by UV light alternately at 340 and 380 nm. The light source was a 75-watt xenon and dual monochromater system. The intensity of the UV light was reduced by neutral density filters. Pairs of 340- and 380-nm images were obtained at 3.0-sec intervals and were corrected for background. Ratio images were calculated by computer, and the free [Ca2+]i was calculated using the well known ratio equation (13).

PKC activity assay
PC12 cells were resuspended in serum-free DMEM. After drug treatment, cells were washed twice with PBS and homogenized on ice in lysis buffer (25 mM Tris, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM ß-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride). Homogenates were centrifuged at 86,000 x g for 30 min at 4 C. The pellet was resuspended in lysis buffer containing 0.5% Triton X-100 and homogenized again. The supernatant containing the solubilized membranes was used as the membrane fraction (14). PKC activity in the membrane fractions was measured using the SignaTECT PKC assay system from Promega Corp. (Madison, WI) according to the manufacturer’s instructions. Protein concentrations were determined using the Bio-Rad protein assay kit with BSA as the reference standard (15).

Statistical analysis
The statistical significance of differences between the groups was assessed by Student’s t test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of B on Nic-induced increase in [Ca2+]i
As shown in Fig. 1Go, B could rapidly inhibit the Ca2+ influx induced by Nic. As the concentration of B increased, the inhibition of B on the increase in [Ca2+]i caused by Nic was also gradually enhanced. The IC50 was 0.61 ± 0.19 µM.



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  		Figure 1. Effect of B on Nic-induced increase in [Ca2+]i. A, Cells were preincubated with B at the various concentrations indicated at 25 C for 5 min and then were stimulated with Nic (100 µM). [Ca2+]i was determined as described in the text. Data are presented as the mean ± SE (n = 20 cells/data point); they represent the maximal calcium concentration over basal or resting values. The IC50 value for B to block the Nic-induced increase in [Ca2+]i was 0.61 ± 0.19 µM. B and C, Two representative real traces in A for 10-10 and 10-6 M B, respectively.

 
Effects of B, B-conjugated BSA (B-BSA), PKC inhibitors, activator, and blockers of voltage-gated Ca2+ channels on Nic-induced increase in [Ca2+]i
As shown in Fig. 2Go, after incubation with Ca2+ channel blockers (Vp and co-CTX), the increase in [Ca2+]i induced by Nic was significantly blocked, suggesting that the Nic-induced increase in [Ca2+]i is primarily through Ca2+ influx. The inhibitory effect of B-BSA on Ca2+ influx induced by Nic was the same as the effect of free B. This suggested that B might act via putative membrane receptors. Furthermore, PKC activator (PMA) could mimic whereas PKC inhibitors, chelery and Gö6976, could reverse the inhibitory effect of B. These indicated that Ca2+-dependent PKCs are probably involved in the suppressive effect of B on calcium influx induced by Nic.



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  		Figure 2. Effects of B, B-BSA, PKC inhibitors, activator, and blockers of voltage-gated Ca2+ channels on the Nic-induced increase in [Ca2+]i. PC12 cells were treated with drugs, then [Ca2+]i was determined as described in the text. PKC inhibitors (chelery, 10 µM; Gö6976, 0.1 µM), activator (PMA, 100 nM), and blockers of voltage-gated Ca2+ channels (Vp, 20 µM; {omega}-CTX, 500 nM) were given 10 min before Nic, B (1 µM and B-BSA (1 µM) were given 5 min before Nic (100 µM). Data are presented as the mean ± SE (n = 20 cells/data point); they represent the maximal calcium concentration over basal or resting values. **, P < 0.01 vs. the Nic group, respectively.

 
Effect of B on Nic-induced increase in [Ca2+]i when pretreated with PTX
To clarify the molecular mechanism of the nongenomic effect of glucocorticoid suppression on Ca2+ influx induced by Nic, we studied the involvement of G protein, which is known to modulate many functions of receptor-mediated cellular events, including activation of PKC activity and/or gating of ion channels. As shown in Fig. 3Go, after pretreating PC12 cells with PTX (100 ng/ml) for 24 h, no noticeable inhibitory effect of B on Ca2+ influx induced by Nic could be observed. This indicated that the action of B may be through PTX-sensitive G protein.



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  		Figure 3. Effect of B on the Nic-induced increase in [Ca2+]i in PTX-pretreated PC12 cells. Cells were pretreated with or without PTX (100 ng/ml) for 24 h, then preincubated with or without B (1 µM) at 25 C for 5 min, and Nic was added (100 µM). [Ca2+]i was determined as described in the text. Data represent the mean ± SE (n = 20 cells/data point); they represent the maximal calcium concentration over basal or resting values. P > 0.05 vs. the control group.

 
Effects of B and B-BSA on PKC activity in PC12 cells
It is now generally accepted that activation of PKC is associated with translocation of the enzyme (16). To determine whether B could activate PKC in resting PC12 cells, we measured PKC activity in the membrane fractions. As shown in Fig. 4Go, B could activate PKC activity, and the PKC inhibitor chelery could completely block the activation by B.



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  		Figure 4. Effect of PKC inhibitor (chelery) on PKC activity stimulated by B in PC12 cells. Cells were exposed to different drugs (B, 1 nM; PMA, 100 nM) at 37 C for 5 min after preincubation with or without chelery (10 µM) for 15 min, then PKC activity in the membrane fractions was assayed as described in the text. Data represent the mean ± SE of three to five experiments. **, P < 0.01 vs. the B group.

 
The time course showed that the PKC activity was kept at a higher level from 5–15 min of B’s action and was highest at 5 min (Fig. 5Go). This finding explained why the inhibitory effect was most efficient after 5-min preincubation with B min before the addition of Nic.



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  		Figure 5. Time course of activation of PKC by B in PC12 cells. Cells were exposed to B (1 nM) at 37 C for the time intervals indicated, then PKC activity in the membrane fractions was determined as described in the text. Data represent the mean ± SE of three to five experiments.

 
As shown in Fig. 6Go, B and B-BSA activated PKC activity. The dose-response curve was bell shaped. B was most potent at 10-9 M, but B-BSA was most potent at 10-7 M at 37 C. At 25 C, however, the dose-response curve considerably shift to the right, and B was most potent at 10-5 M (Fig. 7Go).



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  		Figure 6. Dose-dependent activation of PKC by B and B-BSA in PC12 cells. Cells were exposed to B or B-BSA at various concentrations at 37 C for 5 min, then PKC activity in the membrane fractions was assayed as described in the text. Data represent the mean ± SE of three to five experiments. ** and ##, P < 0.01 vs. basal.

 


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  		Figure 7. Dose-dependent activation of PKC by B in PC12 cells at 25 and 37 C. Cells were exposed to B at various concentrations for 5 min, then PKC activity in the membrane fractions was assayed as described in the text. Data represent the mean ± SE of three to five experiments. ** and ##, P < 0.01 vs. basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nicotinic ACh receptors are expressed in PC12 cells (17). Nic would be expected to raise [Ca2+]i due to Ca2+ entry predominantly through voltage-dependent channels and additionally through nicotinic receptor-associated channels. After incubation with Vp and co-CTX, the increase in [Ca2+]i induced by Nic was significantly blocked, suggesting that the Nic-induced increase in [Ca2+]i is mainly through its influx. Our results are in accordance with the reports that there are predominantly L-type Ca2+ channels, with N-type Ca2+ channels in the minority, in undifferentiated PC12 cells (18).

The peak free B concentration in rat serum reached 7.5 x 10-7 M during stress (19). The adrenal medulla in mammals is encapsulated by the adrenal cortex, and venous blood from the latter drains through the former in the intraadrenal portal vascular system before entering the general circulation. This vascular system subjects the chromaffin cells of the medulla to extremely high concentrations of adrenal cortical hormones, 2 orders of magnitude higher than those in the peripheral circulation (20). Thus, it is very likely that the glucocorticoid inhibits Nic-induced calcium influx in concentrations within the physiological range.

In a previous study, we have shown the time course of the inhibitory effect of B on the increase in [Ca2+]i induced by high K+ in PC12 cells, with the maximal effect at 5 min (21). In the present experiment, B could rapidly inhibit the Ca2+ influx induced by Nic after preincubation with B for 5 min. In good agreement with the above-mentioned results is the finding that B activated the PKC activity, with the maximal effect also at 5 min. As for B-activated PKC activity, the maximal response was 10-9 M at 37 C, which is 4 orders of magnitude lower than that at 25 C (10-5 M). The mechanism for the rightward shift in dose response at 25 C after B treatment is not yet clear. It is probably due to the influence of temperature itself, as temperature usually greatly alters the activity of enzymes.

In addition to the intracellular glucocorticoid receptor, plasma membranes in several types of cells have binding sites for glucocorticoids (22, 23, 24, 25). In our study, B-BSA could also activate PKC activity. As B-BSA is practically membrane impermeable, our results here provide support for the idea that B might act via putative glucocorticoid membrane receptors. The data in Fig. 6Go show that the maximal effect on PKC activity was at 10-7 M for B-BSA, 2 orders higher than that of free B. This rightward shift is probably caused by the hindrance effect, because BSA is a very big molecule that may hinder, and therefore attenuate, the action of B in B-BSA.

It is known that undifferentiated PC12 cells contain PKC{alpha}, -{delta}, -{epsilon}, and -{zeta} (26, 27). Our finding that Gö6976, which is a compound that inhibits PKC{alpha} and -ß specifically, could block the effect of B indicated that PKC{alpha} is probably involved in the suppressive effect of B on calcium influx induced by Nic.

Glucocorticoids have been reported to inhibit the ACh-induced current in chromaffin cells and were reported to bind to the specific site on the outer cell membrane, probably on the ACh receptor-coupled channel (28). In our results, the mechanism of the inhibitory action of glucocorticoid may not be channel blocking, because PKC activator (PMA) could mimic the inhibitory effect of the B and PKC inhibitors, chelery and Gö6976, could reverse the inhibitory effect of B. Phosphorylation of AChR has been implicated in regulation of the ligand-gated ion channel. PKC has been shown to enhance receptor desensitization (29, 30). On the other hand, PKC can inhibit two types of calcium currents in GH3 cells (31), and the activity of N-type channels was regulated through phosphorylation by PKC (32). Furthermore, activation of PKC could reduce L-type calcium channel activity in PC12 cells (33, 34, 35) as well as GH3 pituitary cells (36), pituitary AtT-20 cells (37), and cultured embryonic chick dorsal root ganglion neurons (38). Our data suggest at least two possible sites of action for the nongenomic effect of glucocorticoid through phosphorylation by PKC. One is at AChR, and another is at calcium channels.

In Taricha, results from radioligand binding assays demonstrated that [3H]B binding in neuronal membranes is negatively modulated by nonhydrolyzable guanine nucleotide analogs, especially GTP-{gamma}-S (39). Other studies revealed that [3H]B-specific binding was enhanced in a concentration-dependent manner by adding Mg2+ to the assay buffer. These results are consistent with comparable studies for known G protein-coupled receptors and provide evidence that the putative membrane receptor of B is coupled to G protein (40). Furthermore, it has been reported that cortisol inhibits the calcium currents in guinea pig hippocampal CA1 neurons via G protein-coupled activation of PKC (9). Clearly, our data also indicated that the nongenomic action of B may be through a PTX-sensitive G protein-PKC pathway. More importantly our study demonstrates for the first time rapid stimulation of PKC activity by glucocorticoid in PC12 cells.

Previous studies have shown that glucocorticoids could rapidly inhibit the cAMP production and PRL release induced by vasoactive intestinal peptide by acting through specific glucocorticoid receptors in normal rat pituitary cells in culture (41), and cortisol also rapidly reduces PRL release and cAMP in the cichlid fish pituitary in vitro (8). In glucocorticoid inhibition of ACTH secretion and cAMP, the possible involvement of a PTX-sensitive G protein has been indicated in the mouse corticotroph tumor cell line AtT20 (42), but our data suggested that there were no effects of B on resting, forskolin-induced changes in cAMP accumulation and protein kinase A activity (not shown) in PC12 cells. These data indicated that the nongenomic signal transduction mechanisms of glucocorticoid’s rapid action varied in different tissues or cells.

Based on our data, a new model of the rapid nongenomic action of B in PC12 cells is proposed (as summarized in Fig. 8Go). In summary, glucocorticoid might act via putative membrane receptors and inhibit the Ca2+ influx induced by Nic through the PTX-sensitive G protein-PKC pathway in PC12 cells. PKC plays an important role in the mechanism of the nongenomic action of glucocorticoid.



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  		Figure 8. A model of the rapid nongenomic action of B in PC12 cells. The steps that are not well defined are indicated by arrows with dashed lines. R, G protein-coupled B receptor; G, G protein; VOCs, voltage-operated channels; N-AChR, nicotinic ACh receptor; BK, bradykinin; PC12 cells, pheochromocytoma cells.

 


   Acknowledgments
 
We thank Drs. Guo-huang Fan and Zhi-jie Chen (Shanghai Institute of Cell Biology, Shanghai, China) and Chen-guang Wang (Beijing Institute of Developmental Biology, Beijing, China) for their helpful discussions.


   Footnotes
 
1 This work was supported by a research grant from the Natural Science Foundation of China. Back

Received June 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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