Stimulation of L-type Ca²⁺ channels by inositol pentakis- and hexakisphosphates in rat vascular smooth muscle cells

Abstract

The electrophysiological effects of d-myo-inositol 1,3,4,5,6-pentakisphosphate (InsP₅) and d-myo-inositol hexakisphosphate (InsP₆), which represent the main cellular inositol polyphosphates, were studied on L-type Ca²⁺ channels in single myocytes of rat portal vein. Intracellular infusion of InsP₅ (up to 50 μm) or 10 μm InsP₆ had no action on Ba²⁺ current, whereas 50 μm InsP₆ or 10 μm InsP₅ plus 10 μm InsP₆ (InsP_5,6) stimulated the inward current. The stimulatory effect of InsP_5,6 was also obtained in external Ca²⁺-containing solution. The stimulated Ba²⁺ current retained the properties of L-type Ba²⁺ current and was oxodipine sensitive. PKC inhibitors Ro 32-0432 (up to 500 nm), GF109203X (5 μm) or calphostin C (100 nm) abolished the InsP_5,6-induced stimulation. Neither the PKA inhibitor H89 (1 μm) nor the protein phosphatase inhibitors okadaic acid (500 nm) or cypermethrin (1 μm) prevented or mimicked the InsP_5,6-induced stimulation of Ba²⁺ current. However, InsP₅ or InsP₆ could mimic some effects of protein phosphatase inhibitor so as to extend after washing-out forskolin the stimulatory effects of the adenylyl cyclase activator on Ba²⁺ current. These results indicate that InsP₅ and InsP₆ may act as intracellular messengers in modulating L-type Ca²⁺ channel activity and so could be implicated in mediator-induced contractions of vascular smooth muscle cells.

Voltage-gated Ca²⁺ channels are key proteins in the regulation of Ca²⁺ homeostasis and contraction in rat portal vein myocytes. The activity of these channels is regulated by membrane depolarization but modulation by protein kinases is essential (Keef et al. 2001).

In rat portal vein, one of the most potent vasoconstrictor agents, angiotensin II (AII), increases the intracellular Ca²⁺ concentration by stimulating L-type Ca²⁺ channels. The signalling pathway leading to this cellular response involves the Gα₁₃β₁γ₃ protein-coupled AT_1A receptor, a phosphoinositide 3-kinase γ (PI3Kγ) and a protein kinase C (PKC) (Macrez et al. 1997; Viard et al. 1999; Quignard et al. 2001). However, different pathways also regulate vascular L-type Ca²⁺ channels. Stimulation of β-adrenergic receptors or adenylyl cyclase induces an increase in the amplitude of the L-type Ba²⁺ current via a protein kinase A (PKA)-dependent pathway (Viard et al. 2001).

Intracellular messengers generated by phospholipase C (PLC) may modulate L-type Ca²⁺ channels, such as, for example, diacylglycerol, which can activate PKC and then Ca²⁺ channels (Touyz & Schiffrin, 2000). Inositol-1,4,5-trisphosphate (InsP₃) produced by PLC leads to the generation of other inositol polyphosphates (InsPs), such as inositol tetrakis- (InsP₄), pentakis- (InsP₅) and hexakisphosphates (InsP₆) through the action of several phosphatases and kinases (York et al. 2001; Verbsky et al. 2002). The possibility that InsPs other than InsP₃ act as intracellular signalling molecules has been suggested in several cell types. For instance, InsP₅ and InsP₆ concentrations may be modulated by various stimuli such as glucose in β-pancreatic cells (Larsson et al. 1997), depolarization in neurons (Sasakawa et al. 1993), abscisic acid in vegetable cells (Lemtiri-Chlieh et al. 2000) or AII in chromaffin cells (Sasakawa et al. 1990). Several specific inositol polyphosphate binding sites have been revealed in cell organelles such as c2b domain-containing protein and AP-2 protein; these binding sites are different from the InsP₃ binding site (Theibert et al. 1992; Llinas et al. 1994; Rowley et al. 1996). InsP₆ has been reported to be involved in the intracellular export of messenger RNA (York et al. 1999), proliferation of cells, stimulation of insulin secretion (Efanov et al. 1997) and regulation of ionic channels by various signalling pathways. In neurons, InsP₆ stimulates L-type Ca²⁺ channels via the adenylyl cyclase pathway (Yang et al. 2001). On the other hand, in β-pancreatic cells, InsP₆ stimulates L-type Ca²⁺ channels only via the ability of InsP₆ to inhibit serine/threonine phosphatases (Larsson et al. 1997). InsP₅ may mimic the effect of InsP₆ (with a lower potency) or it may have a specific effect on neuronal ionic currents (Sawada et al. 1989). High levels of InsP₅ and InsP₆ have been reported in a smooth muscle cell line (Safrany & Shears, 1998). However, in portal vein smooth muscle cells, in which a PLC activity has been detected (Macrez et al. 1999), no information is available on a possible role of highly phosphorylated InsPs, alone or in combination, on voltage-gated Ca²⁺ channels.

In this study, we show that, InsP₅ plus InsP₆ stimulate L-type Ca²⁺ channels of vascular smooth muscle cells. As the production of InsP₅ and InsP₆ could be physiologically modulated, these InsPs may play a role in the stimulating effect of mediators on Ca²⁺ channels.

METHODS

The investigation conforms with the European Community and French guiding principles in the care and use of animals. Authorization to perform animal experiments was obtained from the French Ministère de l'Agriculture et de la Pêche

Cell preparation

Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously (Morel et al. 1996). Wistar rats (150–160 g) were killed by cervical dislocation. The portal vein was cut into several pieces and incubated for 10 min in low Ca²⁺ (40 μm) physiological solution (mm: 130 NaCl, 5.6 KCl, 1 MgCl₂, 10 Hepes, 11 glucose, pH 7.4) and then 0.8 mg ml⁻¹ collagenase, 0.25 mg ml⁻¹ pronase E and 1 mg ml⁻¹ bovine serum albumin were added at 37 °C for 20 min. The solution was then removed, and the pieces of vein were incubated again in a fresh enzyme solution at 37 °C for 20 min. The tissues were then placed in an enzyme-free solution and triturated using a fire-polished Pasteur pipette to release cells. Cells were seeded at a density of about 10³ cells mm⁻² on glass slides in physiological solution and used within the next 24 h. A7R5 smooth muscle cells were cultivated as described by Seki et al. (1999). A7R5 cells were maintained under 5 % CO₂ at 37 °C in Dulbecco's modified Eagle's medium (DMEM) with 10 % fetal bovine serum, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 2 mm glutamine. After the cells were grown to confluence, they were trypsinized by treatment for 1 min with 0.05 % trypsin. After centrifugation to wash out the trypsin, cells were seeded on a Petri dish in culture medium. For electrophysiological recordings, cells were seeded at a density of about 10³ cells mm⁻² on glass slides in culture medium containing 1 % fetal bovine serum and then recorded within the next 24 h.

Electrophysiological recordings

Voltage-clamp and membrane current recordings were made with a standard patch-clamp technique using a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed with patch pipettes having resistances of 2–5 MΩ. Membrane potential and current records were stored and analysed using a PC (pCLAMP system, Axon Instruments, Union City, CA, USA). Capacitive transient and linear leakage currents were subtracted using a 4 subpulse protocol according to the pCLAMP setup. Cell capacitance was determined in each cell tested by imposing 10 mV hyperpolarizing and depolarizing steps from the holding potential (−50 mV) and analysing the amplitude and time course of capacitive current. The time constant of the current decay (τ, seconds) was estimated with a mono-exponential function according to the pCLAMP setup, and capacitance was calculated according to the function: capacitance (C, farads) = τ/R. The resistance (R, ohms) was estimated from the amplitude of the capacitive current. Current density was expressed as the maximum amplitude of the current (for a depolarizing step to 0 mV) per capacitance unit (pA pF⁻¹). When cells were their own controls, results were expressed as a percentage of current stimulation. Kinetics of inactivation of the current (for a depolarizing step to 0 mV, holding potential of −50 mV) were calculated with a mono-exponential function (f(t) = Aexp(−t/τ) +C) according to pCLAMP setup. Steady-state inactivation experiments were performed with a double pulse protocol. A test pulse to +10 mV was preceded by a prepulse of 10 s duration and of variable amplitude (from −80 to 10 mV). Steady-state inactivation curves were fitted using a Boltzmann equation.

The extracellular solution contained (mm): 130 NaCl, 5.6 KCl, 1 MgCl₂, 11 glucose, 10 Hepes, 5 BaCl₂, pH 7.4 with NaOH. The basic pipette solution contained (mm): 130 CsCl, 10 EGTA, 5 ATPNa₂, 2 MgCl₂, BSA (0.1 %), 10 Hepes, pH 7.3 with CsOH. In some experiments, barium was replaced by 2 mm CaCl₂ and the intracellular concentration of EGTA was reduced to 10 μm in the presence of 5 μm CaCl₂ (free intracellular Ca²⁺ concentration around 50 nm). Ins(1,3,4,5,6)P₅ (InsP₅), InsP₆ or a mixture of both (InsP_5,6) were added to the pipette solution. The Ins(1,3,4,5,6)P₅ isoform was chosen as this isoform was the precursor for InsP₆. Phorbol 12,13-dibutyrate (PDBu) or forskolin was applied on the cells via pressure ejection from a glass pipette. Inhibitors of protein kinases A and C and of protein phosphatases were preincubated with the cells for at least 15 min before patch-clamp experiments. All experiments were performed at 30 ± 2 °C.

Chemicals

Phorbol 12,13-dibutyrate (PDBu) was from LC Laboratories (Woburn, MA, USA). H89 (N-(2-((bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide), forskolin and Ro 32-0432 (2-(8-(dimethylamino)methyl)-6,7,8,9-tetrahydropyrido(1,2-a)indol-3-yl)-3-(1-methyl-1H-indol-3-yl)maleimide) were from Calbiochem (Meudon, France). GF109203X ((1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl) maleimide) was a gift from Glaxo (Les Ulis, France). All other chemicals were purchased from Sigma (St Louis, MO, USA). H89, Ro 32-0432, GF109203X, calphostin, cypermethrin, okadaic acid, PDBu and forskolin were diluted from a stock solution in dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 1/10 000 (except for forskolin, 1/1000) and had no effect on Ca²⁺ channel current (Mironneau et al. 1991).

Statistics

All values are given as means ± s.e.m. Statistical analysis was performed using Student's t test and P values < 0.05 were considered statistically significant.

RESULTS

All experiments and measurements of membrane current were performed at least 5 min after the breakthrough into the whole-cell patch-clamp configuration to allow intracellular diffusion of the pipette solution and stabilization of the Ba²⁺ current. Ba²⁺ current densities and kinetics of inactivation were calculated for depolarizing steps to 0 mV from a holding potential of −50 mV. The mean capacitance of the cells was 19.10 ± 1.18 pF (n = 106).

Intracellular application of 10, 20 or 50 μm InsP₅ alone did not change the density of the basal Ba²⁺ current (Fig. 1A and B). Neither the kinetics of inactivation (time constant: 86.7 ± 4.9 ms, n = 12, in the presence of InsP₅ compared with 91.5 ± 4.1 ms, n = 30, in control conditions) nor the time course of Ba²⁺ currents recorded for different depolarizing steps (Fig. 1A) were affected by intracellular application of 10 μm InsP₅. In addition, the steady-state inactivation curves were superimposed (potential for half-inactivation: −31.3 ± 1.1 mV, n = 5, in the presence of InsP₅ compared with −29.9 ± 0.7 mV, n = 6, in control conditions).

Figure 1. Effects of InsP₅ and InsP₆ on basal Ba²⁺ current in vascular myocytes.

A, typical Ba²⁺ currents recorded in control conditions and in the intracellular presence of 10 μm InsP₅ or 50 μm InsP₆ (depolarizing steps to −30, −20, 0 and 20 mV from a holding potential of −50 mV). B, Ba²⁺ current density measured with a control pipette solution and with a pipette solution containing different concentrations of InsP₅ or InsP₆. Holding potential −50 mV, depolarizing steps to 0 mV. Currents were recorded 5 min after the breakthrough into the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. * Significantly different from the value obtained in control conditions (P < 0.05).

In contrast, intracellular applications of InsP₆ dose-dependently increased the current density, with a significant effect at 50 μm (Fig. 1A and B). In the intracellular presence of 10 and 50 μm InsP₆, the kinetics of inactivation were 91.9 ± 5.2 ms (n = 12) and 93.1 ± 7.5 ms (n = 5), respectively; they were not altered compared with control conditions (91.5 ± 4.1 ms, n = 30). The time course of Ba²⁺ currents recorded for different depolarizing steps (Fig. 1A), and the steady-state inactivation curve (potential for half-inactivation: −33.4 ± 1.8 mV, n = 3, for 10 μm InsP₆ and −32.1 ± 2.1 mV, n = 3, for 50 μm InsP₆) were not statistically affected as compared with control conditions (−29.9 ± 0.7 mV, n = 6).

The efficiency of InsP₅ and InsP₆ on Ca²⁺ channel activity was then tested by adding both InsP₅ and InsP₆ (InsP_5,6). Figure 2A illustrates the time course of Ba²⁺ currents evoked in the presence and absence of InsP_5,6 after the breakthrough into the whole-cell recording mode. InsP_5,6 (10 μm) induced a time-dependent increase of the Ba²⁺ current reaching a steady-state after 3 min of infusion (Fig. 2B). The stimulatory effect of intracellular InsP_5,6 increased from 1 μm to 10 μm (Fig. 3C) whereas external application of 10 μm InsP_5,6 was ineffective (n = 7). With 2 mm extracellular Ca²⁺ and a low intracellular Ca²⁺ buffering, the Ca²⁺ current density increased from 3.4 ± 0.4 pA pF⁻¹ in control conditions to 4.4 ± 0.4 pA pF⁻¹ in the intracellular presence of 10 μm InsP_5,6. It is noteworthy that the variation of the Ba²⁺ current density was not linked to an increase of the leak current as the seal resistance (more than 1 GΩ) did not change during intracellular application of InsP_5,6 (n = 12). Furthermore, both basal Ba²⁺ currents and InsP_5,6-induced Ba²⁺ currents were totally inhibited by the Ca²⁺ channel antagonist oxodipine (1 μm) (Fig. 2A). In the presence of 10 μm InsP_5,6, the kinetics of inactivation of the Ba²⁺ current (time constant: 95.9 ± 5.2 ms, n = 25, compared with 91.5 ± 4.1 ms, n = 30, in control conditions), the current-voltage relationships (Fig. 2C) and the normalized steady-state inactivation curves (half-inactivation potential: −28.5 ± 0.7 mV, n = 7, compared with −29.9 ± 0.7 mV, n = 6, in control conditions) were also not statistically altered.

Figure 2. Effects of InsP_5,6 on basal Ba²⁺ current.

A, typical Ba²⁺ currents recorded in control conditions and in the intracellular presence of 10 μm InsP₅ + 10 μm InsP₆ (InsP_5,6), before (a) or after (b) application of the calcium channel antagonist oxodipine (1 μm). B, time course of Ba²⁺ current amplitude in cells dialysed with a control pipette solution (•) or with a pipette solution containing 10 μm InsP_5,6 (○). Zero time corresponds to the breakthrough into the whole-cell recording mode. Holding potential −50 mV, depolarizing steps to 0 mV. C, current–voltage relationships obtained from a holding potential of −50 mV in cells dialysed with a control pipette solution (•) or a pipette solution containing 10 μm InsP_5,6 (○). Curves were normalized to −1 for step depolarizations to 0 mV. Currents were recorded 5 min after the breakthrough into the whole-cell patch-clamp configuration. Data are means ± s.e.m., with the number of experiments indicated in parentheses.

Figure 3. Effects of protein kinase inhibitors on InsP-modulated Ba²⁺ current.

A, effects of PKC inhibitors (Ro 32-0432 and GF109203X) and PKA inhibitor (H89) on basal Ba²⁺ current density. Cells were dialysed with a control pipette solution. B, effects of PKC inhibitor (Ro 32-0432) on InsP₆-mediated stimulation of Ba²⁺ current density. Cells were dialysed with a pipette solution containing 50 μm InsP₆. C, effects of PKC inhibitors (Ro 32-0432 and GF109203X) and PKA inhibitor (H89) on InsP_5,6-mediated stimulation of Ba²⁺ current density. Cells were dialysed with a pipette solution containing 10 μm InsP₅+ 10 μm InsP₆. Holding potential −50 mV, depolarizing steps to 0 mV. Current density was measured 5 min after the breakthrough into the whole-cell patch-clamp configuration. Cells were incubated in the presence of inhibitors at least 15 min prior to the recording. Data are means and s.e.m., with the number of experiments indicated in parentheses. * Values significantly different from those obtained in control conditions (P < 0.05).

The mechanisms responsible for the stimulatory effect of InsP₆ and InsP_5,6 were then investigated. As InsPs could modulate PKA or PKC activity, inhibitors of these kinases were tested. As described previously, Ba²⁺ currents of vascular myocytes were stimulated by at least two different pathways, i.e. a PKA pathway (Viard et al. 2001) and a PKC pathway (Macrez-Leprêtre et al. 1996). Neither the PKA inhibitor H89 (500 nm, Chijiwa et al. 1990) nor the PKC inhibitors Ro 32-0432 (40 nm or 500 nm, Wilkinson et al. 1993) and GF109203X (Toullec et al. 1991) at 5 μm significantly altered the basal current density (Fig. 3A). Stimulations of Ba²⁺ current by 5 μm forskolin or 100 nm PDBu were inhibited by H89 (500 nm) or Ro 32-0432 (500 nm), respectively (Fig. 4A and B). In the presence of H89 (Fig. 3C), the InsP_5,6-induced stimulation of the Ba²⁺ current was not statistically altered. However, in the presence of 40 or 500 nm Ro 32-0432, or 5 μm GF109203X, the Ba²⁺ current density measured in the presence of 10 μm InsP_5,6 was reduced to the control level (Fig. 3C). In the presence of a different type of PKC inhibitor, calphostin C (100 nm, activated by UV illumination, Kobayashi et al. 1989), the InsP_5,6-induced stimulation of the Ba²⁺ currents was also abolished (n = 8). Similar results were obtained in the presence of 50 μm InsP₆. As illustrated in Fig. 3B, the InsP₆-induced stimulation of the Ba²⁺ current density was suppressed in the presence of 500 nm Ro 32-0432.

Figure 4. Effects of protein kinase stimulators and InsPs on Ba²⁺ current.

A, typical current traces before (a) and after (b) external application of forskolin (5 μm) in control conditions and in the internal presence of InsP_5,6 (10 μm InsP₅ + 10 μm InsP₆). Right panel, percentage of stimulation of the Ba²⁺ current induced by the adenylyl cyclase activator forskolin in cells infused with a control pipette solution or solutions containing 10 μm InsP₅, InsP₆ or InsP_5,6 (alone or in the presence of Ro 32-0432). B, typical current traces before (a) and after (b) external application of PDBu (100 nm) in control conditions and in the internal presence of InsP_5,6. Right panel, percentage of stimulation of the Ba²⁺ current induced by the PKC stimulator PDBu in cells infused with a control pipette solution or solutions containing 10 μm InsP₅, InsP₆ or InsP_5,6. Note that the amplitudes of the current before application of the stimulating substances were quite different (see Figs 1 and 2). Holding potential −50 mV, depolarizing steps to 0 mV. Current density was measured 5 min after the breakthrough into the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. * Significantly different from values obtained in control conditions (P < 0.05).

The cumulative effects of InsP₅, InsP₆ and InsP_5,6 on the PKA/PKC signalling pathways were then tested by using forskolin and PDBu. Forskolin (5 μm) stimulated the peak Ba²⁺ current in control conditions (Fig. 4A). The drug was externally applied 5 min after the rupture of the patch and the maximum stimulatory effect was obtained 1–3 min after the beginning of the application. In the intracellular presence of InsP₅, InsP₆ or InsP_5,6 (10 μm each), forskolin was still able to increase the Ba²⁺ current amplitude (Fig. 4A). It could be noted that Ro 32-0432 did not alter the forskolin-induced response in these conditions (Fig. 4A). Intracellular infusion of InsP₅ or InsP₆ (10 μm) did not affect or slightly increased the PDBu-mediated stimulation of the Ba²⁺ currents (Fig. 4B). In contrast, the stimulation of the Ba²⁺ current by PDBu was statistically reduced in the presence of InsP_5,6 as compared with control conditions (Fig. 4B).

Phosphatase activity can also be modulated by InsPs. As shown in Fig. 5A, okadaic acid (inhibitor of phosphatase (PPase) 1, PPase 2A and PPase 3 at a concentration of 500 nm, Cohen, 1989) or cypermethrin (1 μm; inhibitor of PPase 2B, Enan & Matsumura, 1992) (alone or together) did not stimulate the basal Ba²⁺ current density and so did not mimic the effect of InsP₆ or InsP_5,6. Furthermore, okadaic acid or okadaic acid plus cypermethrin did not reduce the InsP_5,6-stimulated current density (Fig. 5B). However, InsPs could mimic some effects of the phosphatase inhibitors on Ca²⁺ channels. Cypermethrin did not alter the forskolin-mediated stimulation of the current (Fig. 6A) whereas in the presence of okadaic acid, the stimulation of the current was larger than in control conditions (Fig. 6A), indicating that phosphatase activity was present when the channel was previously phosphorylated. Furthermore, okadaic acid modified the time course of the effect of forskolin. In control conditions, stimulation of the Ba²⁺ current by forskolin (5 μm) was maximal within 3–4 min. After withdrawal of forskolin, the current returned to its control value within 10–14 min (Fig. 6B). In the presence of okadaic acid, the forskolin-induced increase in Ba²⁺ current persisted after washout of forskolin, even after 15 min (Fig. 6B and C). In the presence of InsP₅, InsP₆ or InsP_5,6, the time courses of recovery of the Ba²⁺ current were slowed down (Fig. 6B and C) and the effect of forskolin was still present after 10 min of washout. In comparison, effects of InsP_5,6 were tested in the vascular smooth muscle cell line A7R5. Infusion of InsP_5,6 (10 μm) did not change the current density (control: 3.3 ± 0.4 pA pF⁻¹, n = 9; in the presence of InsP_5,6: 3.5 ± 0.7 pA pF⁻¹, n = 6), and furthermore PDBu was unable to stimulate the Ba²⁺ current (−4.9 ± 0.2 %, n = 5).

Figure 5. Effects of phosphatase inhibitors on Ba²⁺ current density.

A, effects of PPases 1, 2A and 3 inhibitor okadaic acid and PPase 2B inhibitor cypermethrin on basal Ba²⁺ current density. Cells were dialysed with a control pipette solution. B, effects of the same inhibitors on the InsP_5,6-mediated stimulation of the Ba²⁺ current density. Cells were dialysed with a solution containing 10 μm InsP_5,6. Holding potential −50 mV, depolarizing steps to 0 mV. Current density was measured 5 min after the breakthrough into the whole-cell patch-clamp configuration. Cells were incubated in the presence of inhibitors at least 15 min prior to the recording. Data are means and s.e.m., with the number of experiments indicated in parentheses.

Figure 6. Effects of okadaic acid and InsPs on the time course of forskolin-mediated stimulation of Ba²⁺ current.

A, percentage of stimulation of the Ba²⁺ current induced by the adenylyl cyclase activator forskolin in the presence of the protein phosphatase inhibitors okadaic acid and cypermethrin. Cells were dialysed with a control pipette solution. B, typical time course of normalized Ba²⁺ current amplitude in cells dialysed with a control pipette solution (•), in the external presence of okadaic acid (□) or with a pipette solution containing 10 μm InsP_5,6 (○). Forskolin (5 μm) was applied as indicated by the horizontal bar. C, percentage of residual stimulation of the Ba²⁺ current after 10 min of forskolin washing out in control conditions or in the presence of extracellular okadaic acid (in these two conditions, cells were dialysed with a control pipette solution) or in the intracellular presence of InsP₅, InsP₆ or InsP_5,6. Maximal current after application of forskolin was normalized to 100. Holding potential −50 mV, depolarizing steps to 0 mV. Current density was measured 5 min after the breakthrough into the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. * Values significantly different from those obtained in control conditions (P < 0.05).

DISCUSSION

Our results illustrate a novel pathway of modulation of vascular L-type voltage-gated Ca²⁺ channels, in which InsPs (mainly Ins(1,3,4,5,6)P₅ and InsP₆) play a crucial role in the activation of these channels. These molecules are not simple catabolites of phosphoinosides but appear as intracellular messengers. We show here for the first time that InsPs can stimulate vascular L-type Ca²⁺ channels via a PKC activation pathway.

The increase of inward current amplitude by InsP₆ or InsP_5,6 was specifically linked to a stimulation of L-type Ca²⁺ channels. The InsP-induced current had the characteristics of the L-type current (similar potentials for threshold, maximum amplitude and half-inactivation, similar kinetics of inactivation and sensitivity to oxodipine). Furthermore, no specific leak current induced by InsPs was observed. These results are in contrast with the observation that prolactin promotes Ca²⁺ entry through voltage-independent Ca²⁺ channels that may be activated by Ins(1,3,4,5)P₄ and InsP₆ (Ratovondrahona et al. 1998). Participation of T-type Ca²⁺ currents, which are present in these cells (Morel et al. 1997), could be also ruled out since T-type Ca²⁺ current was largely inactivated at a holding potential of −50 mV. Similarly, InsP₆ has not been reported to stimulate T-type Ca²⁺ channels in neurons (Yang et al. 2001).

There are multiple targets for InsPs in cells. Firstly, non-specific interactions of InsPs with the membrane due to the charge of the molecule (Shears, 2001) could be ruled out since application of 10 or 20 μm of each InsP had no effect, whereas 10 μm InsP_5,6 induced a stimulation of the Ca²⁺ channels. Secondly, InsPs could chelate Ca²⁺ ions (Luttrell, 1993), and it is well known that the free intracellular Ca²⁺ level may modulate Ca²⁺ current (Neveu et al. 1993). In vascular myocytes, this hypothesis could be discarded since most of the results were obtained with an intracellular solution containing 10 mm EGTA and a Ca²⁺-free, Ba²⁺-containing extracellular solution. However, in more physiological conditions (i.e. 2 mm extracellular Ca²⁺ and low intracellular Ca²⁺ buffering), the InsP-mediated stimulation of Ca²⁺ channels was still observed indicating that Ca²⁺ ions did not act as an inhibitor of this regulation. Thirdly, a direct action of InsP_5,6 on Ca²⁺ channels is certainly unlikely as binding sites of InsP₆ and InsP₅ have not been observed in Ca²⁺ channels (Theibert et al. 1992; Norris et al. 1995).

Activation of PKA by β-adrenergic receptors is known to stimulate L-type Ca²⁺ channels in rat portal vein myocytes. However, the PKA inhibitor H89, which inhibited both PKA- and forskolin-mediated stimulation of the Ba²⁺ current (Viard et al. 2001 and the present study), was unable to inhibit the InsP_5,6-mediated activation of the current. Furthermore, InsPs did not alter the adenylyl cyclase/PKA pathway since the effects of InsP₅, InsP₆ or InsP_5,6 were cumulative with the forskolin-induced stimulation of L-type Ca²⁺ channels. The lack of effect of H89 contrasts with previous observations which have shown that 10 μm InsP₆ may stimulate adenylyl cyclase and L-type Ca²⁺ channels in hippocampal neurons (Yang et al. 2001). However, even if there is an increase in cAMP production in vascular myocytes, this increase is probably not sufficient or not localized near the PKA isoform associated with Ca²⁺ channels (Johnson et al. 1994).

InsP₆ has been previously shown to stimulate PKC (Efanov et al. 1997; Hoy et al. 2002). We observed that PKC inhibitors significantly reduced the InsP_5,6- and InsP₆-induced stimulation of Ca²⁺ channels. Three different types of PKC inhibitors (Ro 32-0432, GF109203X and calphostin) revealed similar inhibitory effects. In the presence of InsP_5,6, PDBu-induced stimulation of Ba²⁺ current was strongly reduced, as expected if PKC was previously stimulated by InsP_5,6. Finally, in A7R5 smooth muscle cells, where PDBu is ineffective on L-type Ca²⁺ channels (Obejero-Paz et al. 1998), InsP_5,6 had no effect. Ro 32-0432 may be considered to be a specific inhibitor of PKC, as this drug did not inhibit basal current density, but it inhibited PDBu-induced stimulation of the current and it did not alter forskolin-induced stimulation of the Ca²⁺ channel. The effect of Ro 32-0432 was observed for a low concentration (40 nm) suggesting that the PKC involved should be a classical PKC. At 40 nm, Ro 32-0432, which binds to PKC at the ATP binding site, totally inhibits the PKCα isoform (IC₅₀: 9 nm) and partially the PKCβ1, β2 and γ isoforms (IC₅₀ between 28 and 37 nm, Wilkinson et al. 1993). At 500 nm, Ro 32-0432 also inhibits the PKCε isoform but does not affect other serine/threonine kinases such as PKA (present study). The involvement of PKC in the InsP-mediated effect is confirmed by calphostin C, which at a concentration of 100 nm inhibits both classical and novel PKC isoforms by competing at the binding site of 1,2-diacylglycerol (DAG; Kobayashi et al. 1989; Rotenberg et al. 1995). A concentration of 5 μm GF109203X was required to inhibit the InsP-mediated effect. At this concentration, the PKC α, β, δ, ε, ζ isoforms but also other serine/threonine kinases such as PKA could be inhibited (Toullec et al. 1991; Martiny-Baron et al. 1993). At 0.1 μm, GF109203X, which has been reported to inhibit the PKCα and β1 isoforms (Martiny-Baron et al. 1993), had no effect on the InsP_5,6-mediated stimulation of the Ca²⁺ channels (unpublished data) suggesting that these PKC isoforms were not involved in the InsP-mediated stimulation of the current. This result contrasts with the stimulation of L-type Ca²⁺ channels by AII which is GF109203X (100 nm) sensitive (Macrez-Leprêtre et al. 1996). Although stimulation of PKC by 10 μm InsP₆ has been reported in other tissues (Efanov et al. 1997; Hoy et al. 2002), 50 μm InsP₆ alone or simultaneous application of 10 μm InsP₅ + 10 μm InsP₆ was required to stimulate Ca²⁺ channels in vascular myocytes. It is unlikely that transformation of 10 μm InsP₅ into InsP₆ or 10 μm InsP₆ into InsP₅ could explain these effects since 20 μm InsP₆ or InsP₅ alone did not show any stimulatory effects on Ca²⁺ channel activity. It can be postulated that the increased efficiency of InsP₆ in the presence of InsP₅ should be linked to the modulation of another target by InsP₅. Indeed, phosphorylation of Ca²⁺ channels by PKC could be enhanced by the inhibition of serine/threonine phosphatase (Herzig & Neumann, 2000) since InsPs have been reported to act as serine/threonine phosphatase inhibitors (Barker & Berggren, 1999). However, in vascular myocytes, inhibitors of protein phosphatases (i.e. okadaic acid, 500 nm, inhibitor of PPases 1, 2A and 3, and cypermethrin, 1 μm, inhibitor of PPase 2B (Cohen, 1989) had no effect on basal Ca²⁺ channel activity, as also reported by Viard et al. (2001). These results are in contrast with the modulating effects of PPase 1 or PPase 2B inhibitors on Ca²⁺ channels in other smooth muscle cells (Obara & Yabu, 1993; Schuhmann et al. 1997), or with the powerful effects of okadaic acid on Ca²⁺ channels in β-pancreatic cells (Efanov et al. 1997). Although phosphatase activity did not regulate basal Ca²⁺ channel activity in vascular myocytes, okadaic acid enhanced and extended the forskolin-induced stimulation of Ba²⁺ current, as described by Perez-Reyes et al. (1994). The InsP_5,6-induced increase of the Ba²⁺ current was only slightly reduced in the presence of inhibitors of protein phosphatases, indicating that the current stimulation was mainly due to an activation of PKC. However, the inhibitory effects of InsPs on protein phosphatases were present. InsPs significantly extended the effect of forskolin after washing out and so mimicked the effects of okadaic acid. Then, InsPs could prolong the stimulating effects of physiological agonists on L-type Ca²⁺ channels. In rat portal vein, AII induces long-lasting contractions which are inhibited by Ca²⁺ channel antagonists (Pelet et al. 1995). Activation of contraction is linked to AII-induced stimulation of vascular L-type Ca²⁺ channels via a PI3K and PKC and release of Ca²⁺ from the intracellular store by opening of ryanodine-sensitive Ca²⁺ channels (rapid pathway; Macrez et al. 1997; Viard et al. 1999). By stimulating a delayed production of InsP₅ and InsP₆ (L. Rakotoarisoa, personal communication), AII may maintain Ca²⁺ channel activity, and this stimulatory effect may account for the prolongation of contraction (slow pathway). Another effect of InsPs could be to extend the effects of physiological agents which stimulate the PKA pathway via inhibition of phosphatases.

In conclusion, the present data indicate that, in vascular smooth muscle cells of rat portal vein, intracellular infusion of InsP₆ or InsP₅ plus InsP₆ produces a stimulation of the L-type Ca²⁺ channel via a PKC-dependent pathway.