Phosphatidylinositol 4,5-Bisphosphate Is Acting as a Signal Molecule in alpha 1-Adrenergic Pathway via the Modulation of Acetylcholine-activated K+ Channels in Mouse Atrial Myocytes*

Hana Cho, Gi-Byoung NamDagger , Suk Ho Lee, Yung E. Earm, and Won-Kyung Ho§

From the National Research Laboratory for Cellular Signalling and Department of Physiology & Biophysics, Seoul National University College of Medicine, 28 Yonkeun-Dong, Chongno-Ku, Seoul 110-799, Korea and the Dagger  Department of Internal Medicine, Asan Medical Center, Seoul 138-140, Korea

Received for publication, June 5, 2000, and in revised form, August 18, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have investigated the effect of alpha 1-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K+ currents (IKACh). IKACh was recorded in mouse atrial myocytes using the patch clamp technique. IKACh was activated by 10 µM ACh and the current decreased by 44.27 ± 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 ± 2.22% (n = 9), indicating the presence of PE-induced desensitization. IKACh was fully recovered from desensitization after a 6-min washout. PE-induced desensitization of IKACh was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP2) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization of IKACh in the presence of PE was further increased (97.25 ± 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of IKACh at the second exposure after washout was reduced to 19.65 ± 2.61% (n = 6) of the preceding level. These data suggest that the KACh channel is modulated by PE through PLC stimulation and depletion of PIP2.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatidylinositol 4,5-bisphosphate (PIP2)1 is well known as a central molecule in the phosphoinositide cycle, by serving as the precursor of important signaling molecules such as inositol trisphosphate (IP3), diacylglycerol, or phosphatidylinositol 3,4,5-trisphosphate. Recently, it was shown that PIP2 is not just a precursor, but also exerts a direct role in the regulation of various ion transporters, such as Na+/Ca2+ exchanger (1), IP3 receptor Ca2+ channel (2), Na+-activated nonselective cation channel (3), and several inwardly rectifying K+ channels including ROMK1 (4, 5), IRK1 (4, 6), KATP channels (1, 4, 7-9), and G protein-gated inward rectifying K+ (GIRK) channels (4, 10). The underlying mechanism of PIP2 action was investigated in detail for GIRK, and it was shown that the activation of GIRK channels by Gbeta gamma depends on the presence of PIP2 (4, 6, 10-12). This result may imply that PIP2 is a final regulator molecule for GIRK channel activity and that Gbeta gamma exerts its effect by strengthening the interaction of the channel with PIP2.

The KACh channel in cardiac myocytes is believed to be heterotetrameric complex formed by GIRK1 and GIRK4. Slowing of heart rate by vagal stimulation is known to be mediated by the activation of KACh channels, which leads to hyperpolarization of membrane potentials in pacemaking cells and in atrial myocytes (13-17). It is also well known that the effect of vagal stimulation fades gradually (vagal escape), due to desensitization of KACh currents (18-22). Molecular mechanisms of KACh activation and desensitization have been subjects of intense researches for more than a decade. However, it is not yet clear whether the recent view on the mechanism of GIRK channel regulation is valid for native KACh channels. It has been generally believed that direct binding of Gbeta gamma to the channel results in activation (23, 24), but the role of PIP2 in this process is still controversial. In rat atrial myocytes, exogenously applied PIP2 and other phospholipid were reported to block agonist-mediated KACh channel activation (25).

The aim of the present study is to investigate the role of PIP2 in normal signaling pathway for the regulation of native KACh channels. Considering that PIP2 content in the membrane can be controlled by the PLC-linked receptor (26, 27), we used mouse atrial myocytes that possess both PLC-linked receptors, such as alpha 1-adrenergic receptor and KACh channels. The results of the present study show that the alpha 1-adrenergic agonist accelerates desensitization of the KACh channel, possibly through the depletion of the PIP2 pool in plasma membrane, supporting the hypothesis that PIP2 is acting as a signal molecule in the regulation of KACh channels through alpha 1-adrenergic pathway.


    EXPERIMNTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Isolation-- The isolation of single atrial myocytes from mice was performed as described by Harrison et al. (28) with minor modifications. Mice were killed by cervical dislocation, and the heart was quickly removed. The heart was cannulated by a 24-gauge needle and then retrogradely perfused via the aorta on a Langendorff apparatus. During coronary perfusion all perfusates were maintained at 37 °C and equilibrated with 100% O2. Initially the heart was perfused with normal Tyrode solution for 2-3 min to clear the blood. The heart was then perfused with Ca2+-free solution for 2 min. Finally the heart was perfused with enzyme solution for 14-16 min. Enzyme solution contains 0.14 mg ml-1 collagenase (Sigma Type 5) in Ca2+-free solution. After perfusion with enzyme solution, the atria were separated from the ventricles and chopped into small pieces. Single cells were dissociated in high K+, low Cl- storage medium from these small pieces using blunt-tip glass pipette. Cells were stored at 4 °C until use.

Materials and Solutions-- Normal Tyrode solution contained (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 10 glucose, 5 HEPES, titrated to pH 7.4 with NaOH. Ca2+-free solution contained (mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 10 glucose, 5 HEPES, titrated to pH 7.4 with NaOH. The high K+, low Cl- storage medium contained (mM): 70 KOH, 40 KCl, 50 L-glutamic acid, 20 taurine, 20 KH2PO4, 3 MgCl2, 10 glucose, 10 HEPES, 0.5 EGTA. Pipette solution contained (mM): 140 KCl, 10 HEPES, 1 MgCl2, 5 EGTA, titrated to pH 7.2 with KOH. ACh chloride (Sigma), phenylephrine (Sigma), and neomycin (Biomol) were dissolved in deionized water to make a stock solution and stored at -20 °C. On the day of experiments one aliquot was thawed and used. Calphostin C (Biomol) and wortmannin (WMN; Biomol) were first dissolved in dimethyl sulfoxide (Me2SO) as a stock solution and then used at the final concentration in the solution. All experiments were conducted at 35 ± 1 °C. In the presence of ACh, 10 µM glibenclamide was applied to inhibit the ATP-sensitive K+ channel. Cells were superfused with solution by gravity at ~5 ml/min. Approximately 30 s were required to change completely the bath contents.

Voltage Clamp Recording and Analysis-- Membrane currents were recorded in nystatin-perforated patch configuration using an Axopatch-1C amplifier (Axon Instruments). Nystatin forms voltage-insensitive ion pores in the membrane patch that are somewhat selective for cations over anions but are impermeant to Ca2+ and other multivalent ions or molecules >0.8 nm in diameter (29). This method, therefore, minimizes dialysis of intracellular constituents with the internal pipette solution. Nystatin was dissolved in Me2SO at a concentration of 50 mg/ml and then added to the internal pipette solution to yield a final nystatin concentration of 200 µg/ml. The patch pipettes were pulled from borosilicate capillaries (Clark Electromedical Instruments, Pangbourne, United Kingdom) using a Narishige puller (PP-83; Narishige, Tokyo, Japan). We used patch pipettes with a resistance of 2-3 megaohms when filled with the above pipette solutions. The electrical signals were displayed during the experiments on an oscilloscope (Tektronix, TDS 210) and a chart recorder (Gould). Data were digitized with pClamp software 5.7.1 (Axon Instruments) at a sampling rate of 1-2 kHz and filtered at 5 kHz. Voltage clamp and data acquisitions were performed by a digital interface (Digidata 1200, Axon Instruments) coupled to an IBM-compatible computer using pClamp software 5.7.1 (Axon Instruments) at a sampling rate of 1-2 kHz and filtered at 5 kHz.

Statistics and Presentation of Data-- The results in the text and in the figures are presented as means ± S.E., n = number of cells tested. Statistical analyses were performed using the Student's t test. The difference between two groups was considered to be significant when p < 0.01 and not significant when p > 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation and Desensitization of IKACh-- Acetylcholine-activated K+ current (IKACh) was activated by adding 10 µM acetylcholine (ACh) to the bath solution, while the cell was voltage-clamped at the holding potential of -40 mV (Fig. 1A). Upon the application of ACh, a rapid increase in outward current was observed. Despite the continuous presence of ACh, the activation of IKACh was not sustained at its peak, but the amplitude of IKACh decreased slowly. We regarded this decrease as a result of ACh-induced desensitization of IKACh. The ACh-induced desensitization was recovered after washout of ACh, so that the amplitude of IKACh at the second exposure to ACh in a 6-min interval was similar to that at the first exposure. In subsequent experiments, such a paired application of ACh was used for investigating the effect of various signal molecules on regulation of KACh channel, regarding the IKACh at the first response as the control.



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Fig. 1.   Characteristics of ACh-activated K+ current recorded with nystatin-perforated whole cell clamp technique. A, chart recordings of the whole-cell current at the holding potential of -40 mV. The dotted line indicates zero current level. The application of 10 µM ACh is indicated by the horizontal bar above the trace. ACh was applied for 4 min, two times in a 6-min interval. The vertical deflections of current trace are the responses to voltage ramps. B, I-V relationships obtained at the points indicated by a-d in A. C, the I-V curves for net IKACh at peak and at 4 min in ACh. Data were calculated from data in B. The reversal potential was approx  -83 mV.

Characteristics of IKACh activation and desensitization were further investigated from the current-voltage (I-V) curves. I-V curves were obtained from the current response induced by voltage ramps between +60 and -120 mV (at a speed of ± 0.6 V s-1) from the holding potential of -40 mV. The ramps were applied before ACh application (a), at peak (b), 4 min in ACh (c), and washout of ACh (d), as indicated in Fig. 1A. Corresponding I-V curves were plotted in Fig. 1B: the reversal potentials were shifted slightly to negative potentials toward K+ equilibrium potential, and the shape of I-V curves was changed by ACh. The degree of inward rectification was less strong in the presence of ACh (b and c) compared with that in control (a). A strong inward rectification in "a" is considered to be typical for inward rectifying K+ currents, IRK (30). The I-V curves for net IKACh were obtained by subtracting the control curve (a) from the I-V curves in the presence of ACh, as shown in Fig. 1C: "b-a" represents IKACh at peak (IKACh, peak), and "c-a" represents IKACh at 4 min in ACh (IKACh, 4 min). The shape of inward rectification and the reversal potential of two curves were not different, indicating that the decrease in current amplitude during exposure to ACh occurs uniformly over the voltage range tested.

Desensitization of IKACh Is Accelerated by alpha 1-Adrenergic Agonist-- In Fig. 2A, 100 µM phenylephrine (PE) was applied together at the second exposure to ACh. From the continuous recording of current trace at -40 mV, it was noticed that the process of desensitization was markedly accelerated by PE, resulting in a greater reduction of IKACh after the same period (Fig. 2A). But, the amplitude of IKACh at peak was not significantly affected by PE. We regarded this increased desensitization by PE as PE-induced desensitization. The effect of PE on IKACh desensitization was well illustrated in I-V curves: the difference between IKACh, peak (b'-a') and IKACh, 4 min (c'-a') in the presence of PE was significantly greater compared with the control (Fig. 2, B and C). But PE did not affect the degree of inward rectification and the reversal potential, indicating that PE modulates IKACh itself, rather than modulating other current systems. PE-induced desensitization was also reversible after a 6-min recovery period, and the third exposure to ACh elicited an outward current with a similar peak amplitude and desensitization (Fig. 2A).



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Fig. 2.   Effect of PE on IKACh. A, the IKACh was activated by the perfusion of 10 µM ACh. The applications of ACh and 100 µM PE are indicated by the horizontal bars above the trace. B, the I-V curves for net IKACh at peak and at 4 min in ACh during control ACh exposure. C, I-V curves of net IKACh during PE exposure. D, summary of the amplitude of IKACh at peak and 4 min in control and PE-treated cell. IKACh was measured at -40 mV. The numbers in parentheses indicate numbers of cells.

The data were summarized in Fig. 2D. The amplitude of IKACh was measured at -40 mV to minimize the possible contamination of IRK and voltage-activated K+ currents. The amplitude of IKACh at peak was not significantly different between control (775.90 ± 90.29 pA, n = 12) and PE (644.96 ± 94.21 pA, n = 9). However, the amplitude of IKACh at 4 min in ACh was significantly smaller in PE, indicating that desensitization was increased by PE. When the extent of desensitization was determined as a proportion of the current decrease during 4 min, it was 44.27 ± 2.38% (n = 12) in control and increased significantly to 69.34 ± 2.22% (n = 9) by PE.

PLC, but Not PKC, Is Involved in the Increased Desensitization by PE-- To elucidate the mechanisms for PE-induced desensitization, we blocked each step of the signal transduction pathway related with PE. When PKC inhibitor, calphostin C (2.5 µM), was pretreated before the application of PE at the second exposure to ACh, acceleration of IKACh desensitization by PE was still observed (Fig. 3A). To focus on the change in desensitization, I-V curves for desensitized current were plotted in Fig. 3B. It was noticed that desensitized current in the presence of PE and calphostin C (b'-c') was almost completely overlapped by that in the presence of PE only (b-c). The extent of desensitization in the presence of PE and calphostin C was 79.20 ± 4.46% (n = 4), indicating no significant difference from that in the presence of PE only (69.34 ± 2.22%). Furthermore phorbol 12-myristate 13-acetate (100 nM), a specific PKC activator, did not mimic the effect of PE on the desensitization of IKACh (n = 5, data not shown). The amplitude of IKACh at peak was affected neither by calphostin C nor by phorbol 12-myristate 13-acetate.



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Fig. 3.   Effect of calphostin C and neomycin on PE effect. PE (100 µM) was applied together with ACh (10 µM), resulting in acceleration of desensitization of IKACh. Application of drugs are indicated by horizontal bars. A, calphostin C (100 µM) was pretreated 2 min before the second application of ACh and PE. B, difference I-V curve from data in A, corresponding to the decrease in current amplitude during 4-min exposure to PE and ACh in the absence (b-c) and in the presence of calphostin C (b'-c'). C, neomycin (500 µM) was pretreated before the second application of ACh and PE. D, difference I-V relation from data in C, corresponding to the decrease in current during 4-min exposure to PE and ACh in the absence (b-c) or presence of neomycin (b'-c'). E, summary of the extent of desensitization in various conditions. The numbers in parentheses indicate numbers of cells. * indicates p < 0.01.

We then tested the effect of PLC inhibitor, neomycin. When neomycin (500 µM) was pretreated before the application of PE at the second exposure to ACh, the increase in desensitization by PE was no longer observed (Fig. 3C). This finding suggests that PE-induced desensitization of IKACh is antagonized by neomycin. The I-V curve for desensitized current in the presence of neomycin and PE (b'-c') was significantly smaller than that in the presence of PE (b-a). The extent of desensitization in the presence of PE and neomycin was 48.79 ± 3.95% (n = 9), showing a significant difference from the extent of desensitization in the presence of PE (69.34 ± 2.22%), but not different from the control (44.27 ± 2.38%). Neomycin itself did not significantly affect the activation of IKACh and ACh-induced desensitization (IKACh, peak: 759.20 ± 93.43 pA; desensitization: 48.65 ± 3.76%, n = 7).

The extent of desensitization obtained in various conditions was summarized in Fig. 3E. Based on these results, it is suggested that PE-induced desensitization of IKACh is the result of the activation of PLC, but not through the activation of PKC.

Effect of the Depletion of PIP2 Pool by Wortmannin on IKACh-- We postulated that PLC involvement is related with PIP2, and this possibility was tested by using WMN (an inhibitor of PI 3-kinase and PI 4-kinase). It was reported that WMN inhibits the replenishment of PIP2 after the depletion of PIP2 by the receptor-mediated activation of PLC (27). Therefore, we examined whether the inhibition of PIP2 replenishment by WMN affects PE-induced desensitization of KACh current and its recovery. In Fig. 4A, we first applied ACh and PE simultaneously to induce the increased desensitization of IKACh and confirmed the full recovery of IKACh from desensitization after a 6-min washout with normal Tyrode solution. Then the same series of experiment was performed in the presence of 100 µM WMN (Fig. 4B). PE-induced desensitization of IKACh was greatly accelerated by WMN, and IKACh, 4 min became very smaller. The extent of desensitization in the presence of WMN was 97.25 ± 7.63% (n = 6), and this value was significantly greater than 64.22 ± 5.70% (n = 7) in the absence of WMN. In contrast, WMN alone without PE did not significantly affect the activation and desensitization of IKACh (IKACh, peak: 603.14 ± 80.63 pA; desensitization: 45.84 ± 4.07%, n = 6, data not shown). These results suggest that blockade of PIP2 synthesis by WMN facilitated PE-induced desensitization, but not ACh-induced desensitization.



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Fig. 4.   The effect of wortmannin on PE effect. The applications of ACh, PE, and WMN are indicated by the bars above the recording. A, recovery of the PE effect on desensitization of IKACh. B, in the presence of WMN, IKACh was not fully recovered at the second application of ACh 6 min after washout of PE and ACh. C, activation of IKACh when the cell was pretreated with WMN and PE for 3 min prior to ACh. D, summary data of the relative amplitudes (percent) of IKACh, 4 min and IKACh, peak after recovery in respect to IKACh, peak obtained from the experiments A and B. IKACh was measured at -40 mV. The numbers in parentheses indicate numbers of cells.

IKACh, peak was not different, but IKACh, peak was significantly smaller in the presence of WMN, indicating that PE-induced desensitization of IKACh was further accelerated by WMN. The extent of desensitization in the presence of WMN was 97.25 ± 7.63% (n = 6), and it was significantly greater than 64.22 ± 5.70% (n = 7) in the absence of WMN. In contrast, WMN alone without PE did not significantly affect the activation and desensitization of IKACh (IKACh, peak: 603.14 ± 80.63 pA; desensitization: 45.84 ± 4.07%, n = 6, data not shown). These results suggest that blockade of PIP2 synthesis by WMN facilitated PE-induced desensitization, but not ACh-induced desensitization.

The recovery from PE-induced desensitization was also affected by WMN (Fig. 4B). In the continuous presence of WMN, the amplitude of IKACh, peak measured at the second exposure to ACh was only 19.65 ± 2.61% (n = 6) of the preceding level (Fig. 4D), indicating that the recovery from desensitization was significantly inhibited by WMN. WMN also inhibited the recovery from ACh-induced desensitization, but to a lesser extent: IKACh, peak measured at the second exposure to ACh in the absence of PE was 70.92 ± 9.18% of preceding level (n = 6, data not shown). The degree of inhibition is comparable with the magnitude reduction of basal PIP2 levels in unstimulated cells by WMN in this period time (27), suggesting that the incomplete recovery of ACh-induced desensitization was the result of basal reduction of PIP2 level by WMN.

Facilitation of PE effect by WMN was further confirmed in Fig. 4C, where PIP2 pool in the membrane was depleted before the first exposure to ACh by pretreating WMN and PE for 3 min. The amplitude of IKACh, peak was only 178.33 ± 85.36 pA (n = 3), and this value was significantly smaller than IKACh, peak in control (775.90 ± 90.29 pA, n = 12). Above results imply that depletion of PIP2 pool by PE causes a decrease in IKACh and that the increase in desensitization by PE can be explained by the same mechanism.

Effect of PE on IRK-- As well as KACh channel, IRK is known to be regulated by PIP2 (4, 6). We examined whether IRK is affected by the various substances used in the present study. IRK was determined from the I-V curve, as described previously in Fig. 1B. The amplitude was measured at -120 mV, where IRK is large. IRK was not affected either by PE (100 µM) or by WMN (100 µM). However, the addition of PE in the presence of WMN decreased IRK by 31.41 ± 1.93%. These results suggest that the change of PIP2 level induced by normal signal molecule such as PE may not contribute to IRK regulation, although IRK is inhibited when PIP2 level is reduced further down by inhibiting its replenishment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The main question addressed in the present study is whether PIP2 is acting as a signal molecule for the regulation of native KACh channels. The results obtained can be summarized as follows: 1) PE, alpha 1-adrenergic agonist, accelerates desensitization of the IKACh; 2) PE-induced desensitization was inhibited by PLC inhibitor, neomycin, but not by PKC inhibitor, calphostin C; 3) when wortmannin, an inhibitor PI 3-kinase and PI 4-kinase was applied with PE, desensitization of IKACh was further accelerated, and the recovery from desensitization was inhibited. From these results it was suggested that KACh channels are regulated by alpha 1-adrenergic agonist through the depletion of the PIP2 pool in plasma membrane.

Classical signal molecules produced by the activation of PLC-linked receptors are IP3 and diacylglycerol. The role of IP3 on ion channels has not been reported in cardiac myocytes, but diacylglycerol may possibly contribute to the regulation of ion channels via PKC. Desensitization of KACh channel was known to be caused by phosphorylation of m2 muscarinic receptor (31-34), so it is possible that PKC is involved in the increased desensitization of KACh channel by PE. We tested this possibility, but calphostin C, a PKC-specific inhibitor did not inhibit PE action (desensitization of 79.20 ± 4.46%, Fig. 3, A and E). Furthermore, the effect of PE was not mimicked by direct pharmacological activation of PKC with phorbol 12-myristate 13-acetate. These results support the idea that the mechanism for PE-induced desensitization was the depletion of PIP2 rather than the production of PIP2 metabolites that may inhibit the KACh channel. Although we did not carry out the biochemical measurements of PIP2 concentration in the present experiment, the decrease in PIP2 concentration in the plasma membrane by PLC-linked receptor has been reported previously in Chinese hamster ovary cells and human neuroblastoma cells (26, 27). The involvement of PIP2 in the PE effect was further supported by the finding that the PE-induced desensitization became irreversible when the replenishment of PIP2 was blocked by WMN (Fig. 4B). Furthermore when the PIP2 pool was depleted by preincubation of PE and WMN, activation of the KACh current was reduced (Fig. 4C).

IRK channels, on the other hand, showed a different response. In the case of IRK, PE did not affect the channel activity, although PE with WMN affected the channel activity. These data suggest that interaction of IRK with PIP2 is stronger than that of KACh channel, as suggested previously (4, 6, 35), and that the effect of PIP2 depletion on IRK occurs at much lower concentration. Another possibility that should be tested in future studies is a co-localization of a specific PLC-linked receptor and a specific ion channel. In this view, the PIP2 pool, which is regulated by PE, may not be uniformly distributed over the whole membrane, but localized closely with KACh channels.

It has previously been reported by other studies that several PLC-linked receptor can inhibit KACh channel. Braun et al. (36) reported that the selective alpha 1-adrenergic agonist methoxamine reduced both the IK1 and KACh current in rabbit atrial myocytes. Yamaguchi et al. (37) reported that endothelin-1 and endothelin-3 inhibited KACh current in guinea pig atrial myocytes. Their observation is similar to the effect of PE presented in this paper, but they failed to identify the mechanism of the alpha 1-adrenergic agonist or endothelin induced inhibition. They only demonstrated that these effects were not mediated by PKC or IP3. But it now seems to be very likely that PIP2 is involved in those effects. Recently, channel expression studies have demonstrated that PLC-linked receptors inhibit GIRK1/GIRK4 channels (38) or KATP channels (39), and these effects were mediated by depletion of the PIP2 pool in membrane.

The functional consequence of accelerated desensitization of IKACh by alpha 1-adrenergic receptor may be an early cessation of parasympathetic effect in the continuous presence of ACh. This discovery may be of particular importance, since it provides a novel pathway for sympathetic-parasympathetic interaction. Interaction between sympathetic and parasympathetic system was recognized early in various experimental conditions, and this interaction is also considered to be of clinical importance. However, the precise signal transduction pathways involved in this interaction are not fully understood, except the inhibition of adenylate cyclase by acetylcholine as a mechanism of parasympathetic antagonizism to sympathetic stimulation. To our knowledge, the pathway presented in the present study seems to be the first report of a reciprocal pathway through which sympathetic stimulation can antagonize parasympathetic activity.

In conclusion, alpha 1-adrenergic agonist accelerates the desensitization of KACh channel through the regulation of PIP2 pool, suggesting that the receptor mediated regulation of the PIP2 pool may play an important role in the control of cellular function through the modulation of ion channels.


    FOOTNOTES

* This work was supported by BK21 Human Life Sciences and the Korean Research Foundation (KRF, 1999-015-FP0035).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Seoul National University College of Medicine, 28 Yonkeun-Dong, Chongno-Ku, Seoul 110-799, Korea. Tel.: 82-2-740-8227; Fax: 82-2-763-9667; E-mail: wonkyung@snu.ac.kr.

Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M004826200


    ABBREVIATIONS

The abbreviations used are: PIP2, phophatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; GIRK, G protein-gated inward rectifying K+ current; PLC, phospholipase C; WMN, wortmannin; ACh, acetylcholine; IRK, inward rectifying K+ current(s); PE, phenylephrine; PKC, protein kinase C; PI, phophatidylinositol.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMNTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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