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Endocrinology Vol. 138, No. 5 2033-2042
Copyright © 1997 by The Endocrine Society

Articles

Insulin and Insulin-Like Growth Factor-I Inhibit the L-Type Calcium Channel Current in Rat Pinealocytes1

Constance L. Chik2, Bing Li2, Edward Karpinski and Anthony K. Ho

Department of Medicine (C.L.C.) and Department of Physiology (B.L., E.K., A.K.H.), University of Alberta, Edmonton, Alberta, Canada

Address all correspondence and requests for reprints to: C.L. Chik, Room 733 MSB, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tyrosine phosphorylation has recently been shown to modulate ion channel activity. In the present study, the effect of growth factors on the L-type Ca2+ channel current in rat pinealocytes was investigated using the whole cell version of the patch clamp technique. Both insulin and insulin-like growth factor-I (IGF-I) inhibited the L-type Ca2+ channel current. This inhibition was dependent on concentration, with median effective concentration (EC50) values of 60 nM for insulin and 0.14 nM for IGF-I. Heat-inactivated insulin or IGF-I had no effect on the L-type Ca2+ channel current. The presence of anti-IGF-I receptor antibodies blocked the inhibitory effect of IGF-I on the L-type Ca2+ channel current. Two other growth factors, nerve growth factor and epidermal growth factor, had no effect on this current. The effects of insulin and IGF-I were blocked by lavendustin A, a tyrosine kinase inhibitor. Calphostin C, a protein kinase C inhibitor, attenuated the effect of insulin and IGF-I, whereas wortmannin, a phosphatidylinositol 3-kinase inhibitor, was ineffective. These observations indicate that insulin and IGF-I inhibit the L-type Ca2+ channel current in rat pinealocytes, and that tyrosine phosphorylation is involved in these effects of insulin and IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GROWTH FACTORS such as insulin, insulin-like growth factors (IGFs), epidermal growth factor (EGF), and nerve growth factor (NGF), as well as their specific receptors, have been identified in the central nervous system (1, 2, 3). The effects of growth factors on developing neurons are well established (4). In addition to long-term effects such as growth and differentiation, several acute effects of growth factors have been demonstrated in dividing cells. These include pH changes, membrane hyperpolarization, increased intracellular Ca2+ levels, and the activation of enzymes, including phospholipase C-{gamma} and phosphatidylinositol 3-kinase (PI3-K) (5, 6). A recently recognized acute effect of growth factors is their ability to regulate various ion channel activity including the L-type Ca2+ channel (L-channel) (7, 8, 9, 10, 11). Tyrosine phosphorylation is involved in this regulation (12), because the effects of growth factors are blocked by tyrosine kinase inhibitors (7, 10, 11).

Using tyrosine kinase inhibitors, we recently showed that tyrosine phosphorylation plays an important role in the regulation of pineal function (13, 14). The specific growth factors involved in these effects in the pineal, however, remain unknown. The IGFs are of particular interest because of the presence of IGF receptors and the high levels of IGF messenger RNA present in the pineal gland during development (15, 16, 17). The observation that IGF-I has a modulating effect on L-channels in two different cell lines of neural and neuroendocrine origin (7, 10) suggests that these channels may be modulated by IGF-I in the rat pinealocyte. The L-channels in rat pinealocytes have been characterized (18, 19) and in the chick pineal gland, these channels are important in the induction of N-acetyltransferase and melatonin synthesis (20, 21).

The purpose of this study was to determine 1) the effects of insulin and IGF-I on the L-channel current; 2) whether other growth factors such as EGF and NGF have an effect on the L-channel current; and 3) the mechanisms through which growth factors regulate these channels. We found that the L-channel current in rat pinealocytes was inhibited by insulin and IGF-I, and that EGF and NGF had no effect. Furthermore, the effects of insulin and IGF-I were blocked by a specific tyrosine kinase inhibitor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Insulin, 4ß phorbol 12-myristate 13-acetate (PMA), and nifedipine were obtained from Sigma Chemical Corp. (St. Louis, MO). Bay K 8644, calphostin C, lavendustin A, lavendustin B, wortmannin, and anti-IGF-I receptor antibodies were obtained from Calbiochem Corp (San Diego, CA). IGF-I was a gift from Dr. R. Couch (Department of Pediatrics, University of Alberta), and NGF was a gift from Dr. K. Siminoski (Edmonton Endocrine Centre). Cs2-aspartate was prepared by Dr. H.J. Liu (Department of Chemistry, University of Alberta). All other chemicals were of the purest grades available and were obtained commercially. [125I]cAMP and [125I]cGMP were obtained from ICN ImmunoBiologicals (Lisle, IL), and antibodies for the RIA of cAMP and cGMP were gifts from Dr. A. Baukal (National Institute of Child Health and Human Development, NIH, Bethesda, MD).

Cell preparations
Male Sprague-Dawley rats (150 g) were decapitated after cervical dislocation. Pinealocytes were then prepared by trypsinization as described previously (22). The cells were suspended in DMEM containing 10% FCS and maintained overnight at 37 C in a mixture of 95% air and 5% CO2.

Ca2+ channel current recordings were obtained using the whole cell version of the patch clamp technique (23). Patch electrodes were pulled from borosilicate glass capillary tubes (outer diameter = 1.2 mm, inner diameter = 0.9 mm, FHC, Brunswick, ME) and heat polished. They were filled with a solution containing (in mM): 70 Cs2-aspartate, 20 HEPES, 11 EGTA, 1 CaCl2, 5 MgCl2.6H2O, 5 glucose, 5 ATP-Na2, and 5 K-succinate. Creatine phosphokinase (50 U/ml) and phospho-creatine-Na2 (20 mM) were added to the pipette solution to reduce current run down. The bath solution contained (in mM): 105 Tris Cl, 0.8 MgCl2.6H2O, 5.4 KCl, 20 BaCl2, 0.02 tetrodotoxin, and 10 HEPES. Ba2+ (20 mM) was used as the charge carrier. All solutions were filtered (0.22 µm) before use. The osmolarity was adjusted to 320 mosmol and the pH to 7.4. The membrane currents were measured using an Axon (Axopatch, ß = 1) whole cell patch clamp amplifier (Axon Instruments, Foster City, CA). The data were sampled using pClamp software (pClamp 5.5) and a Digidata 1200 analog-to-digital interface (Axon Instruments). Analysis was performed using pClamp software. To generate current-voltage (I-V) relationships, 250 msec depolarizing test pulses of increasing amplitude were applied at a frequency of 0.3 Hz. On-line leakage subtraction was implemented using the P/2 protocol in pClamp software. At a holding potential of -50 mV hyperpolarizing pulses did not activate any currents, and identical results were obtained with the P/2 or P/4 protocol. The experiments were performed at room temperature (20–22 C).

Pineal cells were evaluated for current run down before they were used for experiments. After the whole cell configuration was established, the current amplitude increased for 2–3 min due to inhibition of the outward K+ current by intracellular Cs+. When the current reached its peak amplitude, it was monitored for an additional 5 min to estimate the run down rate. In 90% of cells, the initial run down rate was <5%, and a stable current could be recorded for the next 30 min. In 10% of cells, the initial run down rate was >5%. These cells tended to continue to run down and were not used for experiments. If the initial run down rate was less than 5%, the drugs were added into the bath solution after 5 min.

To determine steady state activation, the normalized conductance of L-channels in the absence or presence of insulin or IGF-I was estimated from the equation G = I/(VT - Vrev), where G = normalized conductance, I = current, VT = test potential, and Vrev = potential at the intersection of the I-V relationship before and after 2 mM La3+. The data points were least squares fitted to a Boltzman function G = Gmax(1/1 + exp(V1/2-Vx)k), where G = normalized conductance, Gmax = maximal conductance, V1/2 = voltage at which half of the channels are activated, Vx = test voltage, and k = slope. Tail currents were measured by depolarizing the cell from a holding potential of -60 to -10 mV with repolarization to -40 mV. The depolarizing pulse length was set at 30 msec, and data were sampled at 10 KHz to obtain good resolution of the fast (~1 msec) current tails. For the analysis of tail currents, the first 350 µsec of the tail current was omitted to eliminate imperfect capacitive transient cancellation.

Data are presented as the mean ± SEM percentages of control values. At least three different cell preparations were used for each study. The pretreatment I-V relationship was plotted and used as a control. The effects of the drugs were monitored continuously using depolarizing pulses at a frequency of 0.03 Hz except when generating I-V relationships. The paired t test was used for comparison between values of the control and those obtained after drug administration. In the case of multiple comparisons, ANOVA in conjunction with the Newman-Keuls test was applied. Hill plots were used to determine median effective concentration (EC50) values. Statistical significance was set at P < 0.05.

cAMP and cGMP assays
cAMP and cGMP measurements were made on samples of cells (50,000 cells/500 µl) treated with various agents for 15 min; the RIA method of measurement has been described in detail (24, 25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of insulin and IGF-I on L-type Ca2+ channel current in rat pinealocytes
The only voltage-dependent Ca2+ channel current found in dissociated pinealocytes is the dihydropyridine-sensitive Ca2+ channel current (L-channel current) (18, 19, 26, 27). When Ba2+ (20 mM) was used as the charge carrier, and the current was activated by depolarizing the cell from a holding potential of -50 to -10 mV, an inward current was measured (Fig. 1AGo). Bay K 8644 (1 µM), an L-channel agonist, increased this current 3-fold, and nifedipine (10 µM), an L-channel antagonist, completely blocked this current (Fig. 1Go). Bay K 8644 shifted the peak of the L-channel current towards more negative potentials (Fig. 1BGo); this is consistent with the dihydropyridine agonist causing an increase in channel mean open time.



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  		Figure 1. L-channel current in rat pinealocytes. A, L-channel current (20 mM Ba2+ charge carrier) was activated by depolarizing cell from a holding potential of -50 to -10 mV. Bay K 8644 (1 µM) increased current, and nifedipine (10 µM) almost completely blocked this current in presence of Bay K 8644. I-V relationships obtained from same cell are shown on the right. B, Combined data from a group of cells using same experimental protocol as described in A are shown. Number of experiments are shown in parentheses above histograms. *, P < 0.05 compared with control current. Additional details are given in Materials and Methods.

 
The effect of insulin on the L-channel current is shown in Fig. 2Go. Insulin (1 µM) decreased the peak amplitude of the L-channel current in a pinealocyte by about 42% (Fig. 2AGo). It did not change the inactivation kinetics or shift the peak inward current along its voltage axis, as shown in the I-V relationship (Fig. 2BGo). The inhibition of the L-channel current by insulin was concentration-dependent (Fig. 2CGo) with an EC50 value of 60 nM. No inhibition was seen with insulin (1 nM); there was a 10% inhibition with 10 nM and a 54% inhibition with 10 µM. Heat-inactivated insulin had no significant effect on the L-channel current (Fig. 2CGo, inset).



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  		Figure 2. Effect of insulin on L-channel current in rat pinealocytes. A, Representative tracing of L-channel current activated by depolarizing pinealocyte from -50 to -0 mV in absence (•) or presence ({circ}) of insulin (1 µM). B, Combined I-V relationship from a group of cells in absence (•) or presence ({circ}) of insulin (1 µM) (n = 5). C, Effect of insulin on L-channel current as a function of concentration. Data were normalized as described in Materials and Methods. Inset, Comparison of effects of insulin (INS) and heat-inactivated insulin (iINS) (1 µM). Number of experiments are shown in parentheses. *, P < 0.05 compared with control current.

 
IGF-I also reduced the amplitude of the L-channel current (Fig. 3AGo). IGF-I (10 nM) decreased the peak current amplitude by about 45% in this experiment. The effect of IGF-I was similar to that of insulin: there was a decrease in the current amplitude but there was no change in the inactivation kinetics or shift in the peak inward current along the voltage axis as shown in the I-V relationship (Fig. 3BGo). The effect of IGF-I on the L-channel current was concentration dependent, with an EC50 value of 0.14 nM (Fig. 3CGo). A 10% inhibition was seen with IGF-I (10 pM) and a 38% inhibition with 0.1 µM. Heat-inactivated IGF-I had no significant effect on the L-channel current (Fig. 3CGo, inset).



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  		Figure 3. Effect of IGF-I on L-channel current in rat pinealocytes. A, Representative tracing of L-channel current activated by depolarizing pinealocyte from -50 to -0 mV in absence (•) or presence ({circ}) of IGF-I (10 nM) in a pinealocyte. B, Combined I-V relationship from a group of cells in absence (•) or presence ({circ}) of IGF-I (10 nM) (n = 5). C, Effect of IGF-I on L-channel current as a function of concentration. Data were normalized as described in Materials and Methods. Inset, Comparison of effects of IGF-I (IGF) and heat-inactivated IGF-I (iIGF) (10 nM). Number of experiments are shown in parentheses. *, P < 0.05 compared with control current.

 
The effects of insulin (1 µM) and IGF-I (10 nM) on the L-channel current as a function of time are shown in Fig. 4Go, B and C. The current run down from a group of control cells is shown in Fig. 4AGo for comparison. The onset of the inhibition caused by insulin occurred within 2–4 min, and the maximal inhibition was observed between 12–16 min. The effect of insulin was not reversed by washout (Fig. 4BGo). The time course of the IGF-I effect was similar to that of insulin. However, unlike the effect of insulin, which was minimally reversed by washout, the effect of IGF-I was 80% reversed by washout (Fig. 4CGo).



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  		Figure 4. Effect of insulin and IGF-I on L-channel current in rat pinealocytes as a function of time. A, L-channel current run down in rat pinealocytes. Run down was evaluated as described in Materials and Methods. Peak L-channel current at 1-min intervals were normalized to current 5 min after whole cell configuration was established. Data from a group of cells (n = 6) were plotted as mean ± SEM. B, Effect of insulin (1 µM) on L-channel current as a function of time. Insulin was applied as indicated. Washout was started 17 min after insulin was applied; effect of insulin was not reversed by washout. a, b and c are peak currents at indicated times. (C) effect of IGF-I (10 nM) as a function of time. IGF-I was applied as indicated and maximal effect was observed 15 min later. Washout was started 18 min after IGF-1 was applied; effect of IGF-I was reversed by washout. a, b, and c are peak inward currents at indicated times.

 
Additional parameters used to characterize the effects of insulin and IGF-I on the L-channel current included steady state activation, deactivation tail currents, and voltage dependence. The effect of insulin and IGF-I on steady state activation is shown in Fig. 5Go, A and B, respectively. Steady state activation was calculated from the normalized conductance of L-channels. Insulin (1 µM) and IGF-I (10 nM) did not have a significant effect on the voltage dependence of activation. Deactivation tail currents, which give a more accurate measure of the inward current, were used to establish that insulin and IGF-1 decreased the peak and the tail current amplitude by the same percentage. Figure 5CGo shows the effect of insulin (1 µM) on the tail current amplitude. Insulin decreased the tail current amplitude in parallel with the peak inward current. Similar results were obtained with IGF-I (data not shown). Figure 5DGo shows the inhibition of the L-channel current by insulin (1 µM) as a function of membrane potential. The maximal inhibition caused by insulin occurred at the peak inward current. Therefore, changes in the peak inward current were used to determine the effects of inhibitors and activators for all subsequent studies.



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  		Figure 5. Effect of insulin and IGF-I on L-channel steady state activation, tail current, and voltage dependence. A, Steady state activation as described by normalized conductance in absence (•) or presence ({circ}) of insulin (1 µM). Data points were obtained as described in Materials and Methods. Solid line is a least squares curve fit to Boltzman function for data points in absence of insulin and dashed line in presence of insulin. For solid line, V1/2 = -13.0 mV, K = 7.4 and for dashed line V1/2 = -14.0 mV, K = 7.2 (n = 6). B, Steady state activation as described by normalized conductance in absence (•) or presence ({circ}) of IGF-I (10 nM). Data points were obtained and fitted as described in A. For solid line, V1/2 = -13.0 mV, K = 7.4 and for dashed line V1/2 = -9.5 mV, K = 8.0 (n = 6). C, Effect of insulin (1 µM) on tail currents. Two tail currents were activated by depolarizing cell from a holding potential of -60 to -10 mV. A 350-µsec segment was blanked to remove imperfect transient cancellation. Insulin decreased tail currents in parallel with peak currents. D, Insulin-mediated L-channel inhibition plotted as a function of membrane potential (n = 6).

 
The EC50 values of 60 nM for insulin and 0.14 nM for IGF-I suggest that these peptides may be interacting with IGF-I receptors. This was assessed by using anti-IGF-I receptor antibodies. Addition of anti-IGF-I receptor antibodies (1 µg/ml) had no effect on the L-channel current. However, in cells pretreated with anti-IGF-I antibodies for 2 h, IGF-I (0.1 µM) no longer had an effect on the L-channel current (Fig. 6Go).



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  		Figure 6. Effect of anti-IGF-I receptor antibodies on IGF-I-mediated inhibition of L-channel current in rat pinealocytes. A, Cells were pretreated with anti-IGF-I receptor antibodies (1 µg/ml) for 2 h. L-channel current activated by depolarizing pinealocyte from -50 to -10 mV in absence (•) or presence ({circ}) of IGF-I (0.1 µM). B, L-channel current activated by depolarizing pinealocyte from -50 to -10 mV in absence (•) or presence ({circ}) of IGF-I (0.1 µM). C, Data from a group of cells were normalized to control and are shown as mean ± SEM. Number of experiments are shown in parentheses above histograms. Pretreatment of pinealocytes with anti-IGF-I receptor antibodies completely blocked inhibitory effect of IGF-I on L-channel current.

 
Comparisons with other growth factors
To determine the specificity of growth factors on the L-channel current, the effects of NGF and EGF were investigated. Unlike insulin and IGF-I, the effects of high concentrations of NGF (50 ng/ml) (98.1 ± 4.9% of control, n = 5) and EGF (10 nM) (93.2 ± 5.1% of control, n = 5) on the L-channel current was not significant even after 20 min of drug treatment. After treatment with EGF or NGF, the cells remained responsive to insulin (1 µM) (data not shown). Therefore, of the four growth factors tested, only insulin and IGF-1 inhibited the L-channel current.

Involvement of tyrosine phosphorylation
Because insulin and IGF-I are coupled to a family of receptor tyrosine kinases (28), the involvement of tyrosine kinase in their modulating effects on the L-channel current was investigated. Lavendustin A (1 µM), a tyrosine kinase inhibitor (29), was added to the bath solution before treatment with insulin as shown in Fig. 7AGo. Treatment with lavendustin A reduced the L-channel current by 14 ± 6% (Fig. 7AGo). In lavendustin A pretreated cells, the inhibitory effect of insulin (1 µM) was almost completely abolished (8 ± 5% vs. 52.8 ± 3.9% in the presence and absence of lavendustin A) (Fig. 7BGo). The inactive compound, lavendustin B (0.1 mM), did not block the effect of insulin on the L-channel current (data not shown). Figure 7CGo shows the effect of lavendustin A and insulin on the L-channel current in a single cell as a function of time. Similar results were obtained with IGF-I (data not shown). These results suggest that tyrosine phosphorylation is involved in the inhibitory effects of insulin and IGF-I on the L-channel current.



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  		Figure 7. Effect of lavendustin A on insulin-mediated inhibition of L-channel currents in rat pinealocytes. A, Lavendustin A (1 µM) caused a small decrease in L-channel current. B, In cells pretreated with lavendustin A, inhibitory effect of insulin (1 µM) was not significant. Number of experiments are shown in parentheses above histograms. C, Effect of lavendustin A on time course of inhibitory effect of insulin. Pretreatment with lavendustin A (1 µM) significantly blocked inhibitory effect of insulin on L-channel current.

 
Involvement of intracellular mechanisms
Because insulin and IGF-I are known to activate PI3-K (5, 6), the effect of wortmannin, a specific PI3-K inhibitor (30), on the insulin and IGF-I inhibition of the L-channel current was determined. Wortmannin (1 µM), which had no effect on the basal L-channel current, also had no effect on the inhibition by insulin or IGF-I (data not shown).

Because insulin and IGF-I can activate PKC through phospholipase C-{gamma} (31), PKC may mediate the effect of these growth factors on the L-channel current. This possibility was examined using a specific inhibitor of PKC, calphostin C (32). Calphostin C (0.1 µM) had a small inhibitory effect on the L-channel current (13.7 ± 3.6%) (Fig. 8Go). In calphostin C-pretreated cells, insulin (1 µM) caused an additional 25% reduction in the L-channel current (Fig. 8Go, A and C). Although calphostin C (0.1 µM) was only partially effective in blocking the inhibition by insulin, it completely blocked the inhibition by PMA (0.1 µM) (Fig. 8Go, B and C). PMA (0.1 µM) alone reduced the amplitude of the L-channel current by 37 ± 8% (Fig. 9Go, A and B). In PMA-treated cells, insulin (1 µM) caused an additional inhibition of the L-channel current (Fig. 9Go, A and B). Similar results were obtained with IGF-I (data not shown). These results are consistent with the involvement of PKC in the inhibitory effect of insulin and IGF-I on the L-channel current.



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  		Figure 8. Effect of calphostin C on insulin- and PMA-mediated inhibition of L-channel currents in rat pinealocytes. A, L-channel current was activated by depolarizing cell from -50 to -0 mV. Calphostin C (CAL C, 0.1 µM) caused a small reduction in L-channel current, and insulin (1 µM) caused a further decrease in this current in presence of calphostin C. I-V relationships obtained from same cell are shown on right. B, L-channel current was activated by depolarizing cell from -50 to -10 mV. Calphostin C (0.1 µM) was effective in blocking decrease in L-channel current caused by PMA (0.1 µM). C, Combined data from a group of cells using same experimental protocol as described in A and B. Shown for comparison are effect of insulin (1 µM) and PMA (0.1 µM). Number of experiments are shown in parentheses above histograms. *, P < 0.05 between different treatments.

 


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  		Figure 9. Effect of PMA on insulin-mediated inhibition of L-channel currents in rat pinealocytes. A, L-channel current was activated by depolarizing cell from -50 to -0 mV. PMA (0.1 µM) decreased current, and insulin (1 µM) caused a further decrease in this current in presence of PMA. I-V relationships obtained from same cell are shown on right. B, Combined data from a group of cells using same experimental protocol as described in A are shown. Number of experiments are shown in parentheses above histograms. *, P < 0.05 between different treatments.

 
Cyclic nucleotides are known to modulate the L-channel current in rat pinealocytes (18, 19), and some effects of insulin and IGF-I are mediated by cyclic nucleotides through their actions on phosphodiesterases (33). Therefore the effect of insulin and IGF-I on cAMP and cGMP accumulation was determined. Neither insulin nor IGF-I had an effect on basal cAMP and cGMP accumulation (Table 1Go).


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  		Table 1. Effect of insulin and IGF-I on cAMP and cGMP accumulation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are several observations that suggest both IGF-I and insulin may be important regulators of pineal function. First, messenger RNA for IGF-I has been identified in the pineal gland during development (15) suggesting that IGF-I, in addition to melatonin, may be a product of the pineal gland. Second, IGF-I receptors have been found in the pineal glands (16). Third, a mutation of the insulin receptor in man results in pineal hyperplasia (34). In the present study, we examined the regulatory role of insulin and IGF-I on L-channels in rat pinealocytes. It was found that both insulin and IGF-I had an inhibitory effect on the L-channel current. Because the EC50 value for IGF-I is less than that for insulin in inhibiting the L-channel current and the IGF-I-mediated inhibition is not observed in cells treated with the anti-IGF-I receptor antibody, these peptides are probably interacting with IGF-I receptors that are present in the pineal gland (16). However, unlike the effect of IGF-I, the effect of insulin could not be reversed by washout. Therefore, the presence of insulin receptors in the rat pineal gland could not be excluded. The effects of insulin and IGF-I are specific because not all growth factors had an inhibitory effect on the L-channel current. NGF and EGF had no effect on the L-channel current.

The established mechanism of action of insulin and IGF-I is through tyrosine phosphorylation (28). The effect of insulin and IGF-I on the L-channel current observed in this study also appears to be mediated through tyrosine phosphorylation. This conclusion is based on the observation that the inhibitory effects of insulin and IGF-I were abolished in cells pretreated with lavendustin A, a tyrosine kinase inhibitor, whereas the inactive inhibitor, lavendustin B, was ineffective. Our results are in agreement with the known modulating effects of insulin and IGF-I on the L-channel current in GH4C1 (a pituitary tumor cell line) and 108CC15 (a N x G cell line) cells, and in Aplysia neurons (7, 10, 35). The effect of IGF-I on the L-channel current in GH4C1 cells (10) and that of insulin in Aplysia neurons (35) are also blocked by tyrosine kinase inhibitors, indicating the involvement of tyrosine kinase.

In spite of this similarity, there are also distinct and significant differences in the effects of insulin and IGF-I on the L-channel current observed in the present study and in those mentioned above (7, 10, 35). First, unlike the rapid (2–4 min) effect of insulin and IGF-I on the L-channel current in rat pinealocytes, the effects of these growth factors in the N x G cells and in Aplysia neurons are much slower (7, 35). In N x G cells, the effect of IGF-I can only be observed after 1 h, and in Aplysia neurons the effect of insulin is observed after 20–40 min. Second, whereas insulin and IGF-I increase Ca2+ channel activities in the other studies (7, 10, 35), the effect of these two growth factors in rat pinealocytes is inhibitory. Although the reason for these differences in the modulation of the L-channel current by insulin and IGF-I is unclear, the present study clearly demonstrates a rapid, inhibitory effect of insulin and IGF-I on the L-channel current in rat pinealocytes.

In a separate study, PKC has been shown to mediate the effect of IGF-I on the L-channel current in the N x G cells (7). Our present results support the involvement of PKC in the effect of insulin and IGF-I on the L-channel current in rat pinealocytes. This is based on the observation that pretreatment of the cells with a specific PKC inhibitor, calphostin C, attenuated the inhibition of the L-channel current by insulin and IGF-I. The inhibitory effect of insulin and IGF-I was mimicked by PMA, a PKC activator, and the effects of PMA and insulin were not additive.

In addition to PKC, other mechanisms activated by insulin and IGF-I appear to modulate the L-channel because the PKC inhibitor, calphostin C, was only partially effective in blocking the inhibition by insulin and IGF-I, although it was completely effective in blocking the inhibition by PMA. One mechanism activated by these growth factors is PI3-K (5, 6). However, a specific PI3-K inhibitor, wortmannin, did not block the effect of insulin and IGF-I on the L-channel current; this argues against the involvement of PI3-K. A second mechanism examined was phophodiesterase. Insulin and IGF-I, through their action on phosphodiesterases (33), can elevate cAMP and cGMP, two known modulators of L-channels (18, 19, 36). However, neither cAMP nor cGMP is likely to be involved in the effect of insulin and IGF-I on the L-channel current because these two growth factors have no effect on cAMP or cGMP accumulation in rat pinealocytes. Therefore, neither PI3-K nor cyclic nucleotides are involved in the effects of insulin and IGF-I on the L-channel current.

It is of interest to note that both calphostin C and lavendustin A have a small inhibitory effect on the L-channel current in rat pinealocytes. Together with our previous observation that H7, a nonspecific kinase inhibitor, has a small inhibitory effect on this current (18), this result suggests that the basal channel activity is under tonic regulation by protein phosphorylation. In agreement with our results, other studies have shown that the basal activities of several ion channels are regulated by protein phosphorylation (8, 9, 37, 38).

In summary, we found that physiological concentrations of insulin and IGF-I have a rapid inhibitory action on the L-channel current. Tyrosine phosphorylation is involved in these effects of insulin and IGF-I. Furthermore, activation of PKC appears to be one of the mechanisms that mediates these effects of insulin and IGF-I. These results add to the existing evidence that insulin and IGF-I are potential regulators of pineal function.


   Footnotes
 
1 This work was supported by a grant from the Medical Research Council of Canada. Back

2 C.L.C. and B.L. should be regarded as equal first authors. Back

Received October 11, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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