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(Circulation Research. 2003;92:765.)
© 2003 American Heart Association, Inc.

Cellular Biology

A Novel, Voltage-Dependent Nonselective Cation Current Activated by Insulin in Guinea Pig Isolated Ventricular Myocytes

Yin Hua Zhang, Jules C. Hancox

From the Department of Physiology & Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, UK. Present affiliation for Dr Zhang is Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Correspondence to Dr Jules Hancox, Department of Physiology & Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK. E-mail jules.Hancox{at}bristol.ac.uk

	Abstract

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Insulin regulates cardiac metabolism and function by targeting metabolic proteins or voltage-gated ion channels. This study provides evidence for a novel, voltage-dependent, nonselective cation channel (NSCC) in the heart. Under voltage clamp at 37°C and with major known conductances blocked, insulin (1 nmol/L to 1 µmol/L) activated an outwardly rectifying current (I_insulin) in guinea pig ventricular myocytes. I_insulin could be carried by Cs⁺, K⁺, Li⁺, and Na⁺ ions but not by NMDG⁺. It was inhibited by the NSCC blockers gadolinium and SKF96365 but not flufenamic acid. I_insulin was largely blocked by the insulin receptor tyrosine kinase inhibitor HNMPA-(AM)₃ and by the phospholipase C inhibitor U73122 but not by its inactive analogue U73433. Staurosporine, a potent blocker of protein kinase C, did not prevent the activation of I_insulin. Application of an analogue of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, mimicked the effect of insulin. This activated an outwardly rectifying NSCC that could be carried by Cs⁺, K⁺, Li⁺, or Na⁺ and that was blocked by gadolinium but not by flufenamic acid or staurosporine. We conclude that the intracellular pathway leading to activation of this novel cardiac NSCC involves phospholipase C, is protein kinase C–independent, and may depend on direct channel activation by diacylglycerol.

Key Words: cardiac myocytes • diacylglycerol • insulin • nonselective cation current

	Introduction

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Isulin is an essential metabolic hormoneR1-126844 ^1,2 that influences cardiac metabolism, the inotropic state of the heart,R3-126844 R4-126844 ^3–5 cardiac protection,⁶ hypertrophy,⁷ and cardiomyopathy in patients with diabetes mellitus.⁸ Insulin exerts its biological effects through the insulin receptor, an intrinsic tyrosine kinase, and its downstream signal transduction pathways.R1-126844 R2-126844 ^1,2,6 Recently it has been reported that agents that increase tyrosine phosphorylation can activate nonselective cation channels (NSCCs) in smooth muscle cells.⁹ However, functional expression of tyrosine kinase–activated NSCC in cardiac myocytes has not yet been reported.

In the present study we provide evidence that insulin can activate a novel NSCC in isolated ventricular myocytes. This NSCC is distinct from other NSCCs in the heart,R10-126844 R11-126844 ^10–12 such as background NSCC and stretch-activated NSCC,¹⁰ which are voltage-independent. In contrast, the insulin-activated NSCC (I_insulin) exhibits outward rectification. The ion selectivity of this current and the signal transduction mechanism involved in its activation are also described.

	Materials and Methods

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Ventricular myocytes from male guinea pigs (400 to 600 g) were isolated as described previously.¹³ Whole-cell patch-clamp measurements were made at 37°C. Detailed methodological information is supplied in the online data supplement, available at http://www.circresaha.org.

	Results and Discussion

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Bath application of insulin (1 µmol/L) progressively increased both outward and inward current components over 20 to 180 seconds, after which the response magnitude gradually decreased to a steady-state level with time (Figures 1A and 1B, n=15). I_insulin did not show instantaneous activation on step depolarization to +80 mV but showed time dependence in its development (Figure 1A). This time dependence of current activation is reminiscent of that recently reported for a ligand-gated vanilloid receptor channel.¹⁴ Plots of the peak outward and inward current densities against time (Figure 1B) show that the effect of insulin on outward current was more prominent than on inward current. With symmetrical pipette and external Cs⁺, the current-voltage (I-V) relationship for I_insulin showed prominent outward rectification and a reversal potential (E_rev) close to 0 mV (Figure 1A, inset, E_rev, -0.13±1.71 mV, n=9). Given that major known ionic current components had been blocked (see the online data supplement), the identity of I_insulin was investigated further. Its cation selectivity was determined by altering the dominant pipette cation without altering the nature of the dominant permeant anion (chloride). I_insulin could be observed when pipette Cs⁺ was replaced by K⁺, Li⁺, or Na⁺ (P>0.1 ANOVA, with Bonferroni post-hoc test) but not when NMDG⁺ was substituted for Cs⁺ (P<0.01). The peak outward densities of I_insulin for the various cations are shown in Figure 1C. Collectively, these data suggest that I_insulin was an outwardly rectifying NSCC. I_insulin magnitude was concentration-dependent over the insulin concentration range of 1 nmol/L to 1 µmol/L (Figure 1D).

View larger version (29K): [in this window] [in a new window]   Figure 1. Insulin activates a voltage-dependent NSCC. A, Representative current traces showing the time-dependent activation of Iinsulin. Inset shows the current in symmetrical Cs+ conditions. B, Current density of peak outward and inward Iinsulin (these typically occurred at between +65 and +78 mV and over a plateau range between -69 and -114 mV, respectively). C, Current densities (pA/pF) for maximal outward Iinsulin with different internal monovalent cations: Cs+, n=21; Li+, n=8; K+, n=10; Na+, n=7; and NMDG+, n=4. D, Concentration-dependent activation of maximal outward Iinsulin. E, Effects of NSCC blockers on maximal outward Iinsulin (Gd, n=5, *P<0.01 against control; SKF96365, n=10, *P<0.01; and flufenamic acid, n=6, P>0.1). F, Maximal outward Iinsulin densities with HNMPA-(AM)3 (n=8, *P<0.01 against control; U73122, n=10, *P<0.01; U73433, n=6, P>0.1; and staurosporine, n=8, P>0.1).

Voltage-dependent NSCCs in tissues other than the heartR14-126844 ^14,15 exhibit differential sensitivities to pharmacological NSCC blockers. For example, the vascular ₁-adrenoceptor–activated cation channel (of which the transient receptor potential homologue TRPC6 is an essential component) is blocked by gadolinium (Gd) and SKF96365 but enhanced by flufenamic acid (FFA), whereas TRPC3 and TRPC7 can be inhibited by FFA.¹⁶ Therefore, we studied the sensitivities of I_insulin to these 3 blockers. As summarized in Figure 1E, Gd (100 µmol/L) significantly attenuated I_insulin and so did SKF96365 (10 µmol/L). However, FFA (100 µmol/L) did not affect I_insulin. Collectively, the observations described in Figures 1A through 1E indicate that insulin activated a novel voltage-dependent NSCC that differs from NSCCs reported to date from the heart.R10-126844 R11-126844 ^10–12

Activation of the insulin receptor (an intrinsic tyrosine kinase) induces the activation of phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol (DAG)R1-126844 R2-126844 ^1,2,17; inositol 1,4,5-trisphosphate and DAG then act as second-messenger molecules to mobilize intracellular calcium and activate protein kinase C (PKC), respectively. The involvement of these signal-transduction pathways in the activation of I_insulin was studied. Pretreatment of cells with hydroxy-2-naphthalenyl-methyl phosphonic acid tris-acetoxy-methyl ester [HNMPA-(AM)₃; 1 mmol/L for >15 minutes] followed by the application of insulin with HNMPA-(AM)₃ abolished the activation of I_insulin (Figure 1F). A specific PLC inhibitor, U73122 (10 µmol/L), significantly inhibited activation of I_insulin, whereas an inactive analogue of U73122, U73433 (10 µmol/L), did not affect the response, suggesting that a PLC-dependent pathway is involved in the activation of I_insulin. A potent inhibitor of PKC, staurosporine (100 nmol/L),¹⁸ did not prevent the activation of I_insulin (Figure 1F). These data suggest that mobilization of PKC is not obligatory for the activation of I_insulin (additional supporting results are described in the online data supplement).

Recent evidence suggests that DAG can itself act as an intracellular messenger and directly activate voltage-dependent NSCC.R16-126844 R19-126844 ^16,19,20 The potential role for DAG in activating I_insulin was investigated by using a membrane-permanent analogue of DAG, 1-oleoyl-2-sn-acetyl-glycerol (OAG). OAG (50 µmol/L) increased both outward and inward current components in a time-dependent manner (Figures 2A and 2B). With symmetrical Cs⁺, the OAG-activated current (I_OAG) showed an outwardly rectifying I-V relationship (E_rev was +1.34±0.54 mV; n=6; Figure 2A inset). Similar to I_insulin, I_OAG could still be activated when internal Cs⁺ was replaced by Na⁺, Li⁺, or K⁺ but not when a NMDG⁺-rich pipette solution was used (Figure 2C; P<0.01, ANOVA with Bonferroni post hoc test). I_OAG was significantly inhibited by 100 µmol/L Gd but unaltered by flufenamic acid (100 µmol/L; Figure 2D). In addition, staurosporine (100 nmol/L) did not prevent the activation of I_OAG (Figure 2D). These data indicate that OAG activated a voltage-dependent NSCC similar to I_insulin and suggest that the insulin-activated NSCC could be mediated directly through an action of DAG without requiring mobilization of PKC.

View larger version (21K): [in this window] [in a new window]   Figure 2. An analogue of diacylglycerol, OAG activates a NSCC similar to Iinsulin. A, Representative records showing the time-dependent activation of voltage-dependent current by OAG (50 µmol/L). Inset shows the I-V relationship of OAG-activated current with symmetrical Cs+. B, Current density of peak outward and inward IOAG with time. C, Current densities (pA/pF) for maximal outward IOAG with different internal monovalent cations: Cs+, n=10; Li+, n=5; K+, n=5; Na+, n=6; and NMDG+, n=6). D, Similar to Iinsulin, IOAG was not sensitive to FFA (P>0.1) but was largely inhibited by Gd application (n=5, *P<0.01). E, IOAG was not sensitive to staurosporine (100 nmol/L, n=4, P>0.05).

Ligand-gated, voltage-dependent NSCCs have been reported in several tissue types (eg, smooth muscle cells,R16-126844 ^16,20 endothelial cells,²¹ and sensory neurons²²) in which NSCCs may be important in depolarizing the membrane potential or inducing calcium entry.R16-126844 R20-126844 R21-126844 ^16,20–22 Our data provide clear evidence for the existence of a hitherto unreported, voltage-dependent, and ligand-activated NSCC in the heart. Moreover, both I_insulin and I_OAG induced shortening of the AP duration in the presence of staurosporine (Figures 3A through 3C; insulin and OAG shortened APD₉₀ by 26.5±4.2%; OAG, 33.4±1.8%, respectively). The changes of action potential profile by insulin and OAG in the presence of PKC inhibition suggest that I_insulin has the potential to modulate cardiac electrophysiology (see the online data supplement for additional discussion of this issue). Additional investigation of the regulation and potential roles of this novel NSCC is now warranted to understand its contributions to normal and pathological cardiac electrophysiology.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effects of insulin and OAG on ventricular action potentials in the presence of staurosporine. A, In the presence of staurosporine (100 nmol/L), insulin (1 µmol/L) shortened the duration of the action potential. B, Similar to insulin, application of OAG (50 µmol/L) in the presence of staurosporine shortened the duration of action potential. C, Bar charts summarizing the mean shortening of APD90 with insulin and OAG. For insulin this was by 45.2±8.7 ms, n=9, P<0.05, paired t test compared with that in staurosporine alone; for OAG this was by 67.0±3.7 ms, n=12, P<0.05.

	Acknowledgments

 
This work was supported by British Heart Foundation Grants PG/98097 and PG/02/066 and by a Research Career Development Fellowship to J.C.H. from Wellcome Trust. We thank Lesley Arberry for assistance with myocyte isolation and Drs Dave Bates and Andy James for useful discussion.

Received September 5, 2002; revision received February 20, 2003; accepted February 26, 2003.

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 92/7/765    most recent 01.RES.0000065920.64121.FCv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, Y. H. Articles by Hancox, J. C. Search for Related Content PubMed PubMed Citation Articles by Zhang, Y. H. Articles by Hancox, J. C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB LITHIUM COMPOUNDS LITHIUM, ELEMENTAL POTASSIUM SODIUM Related Collections Cell signalling/signal transduction Ion channels/membrane transport Receptor pharmacology