Masanori Sunagawa

ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells

ATP-sensitive K+(KATP) channels are therapeutic targets for several diseases, including angina, hypertension, and diabetes. This is because stimulation of KATP channels is thought to produce vasorelaxation and myocardial protection against ischemia, whereas inhibition facilitates insulin secretion. It is well known that native KATP channels are inhibited by ATP and sulfonylurea (SU) compounds and stimulated by nucleotide diphosphates and K+channel-opening drugs (KCOs). Although these characteristics can be shared with KATP channels in different tissues, differences in properties among pancreatic, cardiac, and vascular smooth muscle (VSM) cells do exist in terms of the actions produced by such regulators. Recent molecular biology and electrophysiological studies have provided useful information toward the better understanding of KATPchannels. For example, native KATPchannels appear to be a complex of a regulatory protein containing the SU-binding site [sulfonylurea receptor (SUR)] and an inward-rectifying K+ channel (Kir) serving as a pore-forming subunit. Three isoforms of SUR (SUR1, SUR2A, and SUR2B) have been cloned and found to have two nucleotide-binding folds (NBFs). It seems that these NBFs play an essential role in conferring the MgADP and KCO sensitivity to the channel, whereas the Kir channel subunit itself possesses the ATP-sensing mechanism as an intrinsic property. The molecular structure of KATPchannels is thought to be a heteromultimeric (tetrameric) assembly of these complexes: Kir6.2 with SUR1 (SUR1/Kir6.2, pancreatic type), Kir6.2 with SUR2A (SUR2A/Kir6.2, cardiac type), and Kir6.1 with SUR2B (SUR2B/Kir6.1, VSM type) [i.e., (SUR/Kir6. x)4]. It remains to be determined what are the molecular connections between the SUR and Kir subunits that enable this unique complex to work as a functional KATP channel.

Levosimendan, a novel Ca2+ sensitizer, activates the glibenclamide-sensitive K+ channel in rat arterial myocytes

The electrophysiological effect of levosimendan, a novel Ca(2+)-sensitizing positive inotropic agent and vasodilator, was examined on rat mesenteric arterial myocytes using the patch clamp technique. Resting potential was significantly hyperpolarized with levosimendan, with an EC50 of 2.9 microM and maximal effect (19.5 +/- 3.5 mV; n = 12) at 10 microM. Levosimendan (10 microM) significantly increased the whole-cell outward current. The currents intersected close to the calculated EK (-84 mV), suggesting that the activated current was a K+ current. Hyperpolarization and stimulation of K+ current by levosimendan were not prevented by 30 microM H-7 (a non-specific inhibitor of protein kinases) and 100 nM charybdotoxin (a blocker of Ca(2+)-activated K+ channels), but were abolished by 10 microM glibenclamide. In single-channel current recording in open cell-attached patches, two types of K+ channels were observed having conductances of 26 and 154 pS. The 154 pS channels were not affected by levosimendan and glibenclamide. The 26 pS channels were evoked in one-fourth of the patches when 10 microM levosimendan (and 0.1 mM UDP) was added (at -60 mV) and channel activity was abolished by glibenclamide. The mean open probability of the 26 pS channels was 0.094 +/- 0.017 (n = 9), and the mean open time (at -60 mV) was 6.6 ms in the presence of UDP and levosimendan. Although significant hyperpolarization (4.7 +/- 1.5 mV, n = 8) was observed at 1 microM levosimendan, the same concentration did not affect Ca2+ channel currents (n = 10). In summary, levosimendan hyperpolarized the arterial myocytes, probably through activation of a glibenclamide-sensitive K+ channel. This mechanism may contribute to the vasodilating action of levosimendan.

Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002

Abstractu2002Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca2+. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60c-src, and PKC have been reported to stimulate the Ca2+ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca2+ channels. The Ca2+-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application of 50 nM AngII significantly increased the amplitude of Ba2+ currents through the voltage-gated Ca2+ channels (IBa) by 34.5±9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 µM), a selective inhibitor of Tyr-PK, AngII failed to stimulate IBa (n=5). AngII stimulation of IBa was also prevented by (5 µM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 µM), an inhibitor of PKC, did not prevent the effect of AngII on IBa (n=6). These results suggest that AngII may stimulate the Ca2+ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C.

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells

Abstractu2002To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5′-triphosphate (ATP)-sensitive K+ (KATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal KATP currents (at 0 mV) induced by metabolic inhibition were 48.9±2.8 pA/pF in control (n=48), 34.3±3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5±3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na+ current and inward-rectifying K+ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of KATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional KATP channels. Furthermore, in four out of seven control cells tested, outward K+ currents were stimulated by diazoxide, which is a potent K+ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional KATP channels. The larger population of KATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2.

Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels

Abstractu2002Two actin filament-depolymerizing agents, DNase I and cytochalasin D, were used to examine the involvement of the cytoskeleton in the functional interaction between the sulfonylurea receptor (SUR) and the ATP-sensitive K+ (KATP) channels. Isolated rat ventricular cardiomyocytes were studied using open cell-attached patches for single-channel recording. Bath application of DNase I (100 μg/ml) or cytochalasin D (10 μM) stimulated the KATP channel activities (in presence of 30 μM ATP), and these channels became resistant to inhibition by tolbutamide (0.5 mM). After exposure to tolbutamide, the relative NPo value was 0.09 ± 0.02 in control patches in absence of actin disrupters, and 0.67 ± 0.22* or 0.65 ± 0.10*, respectively, in cells treated with DNase I or cytochalasine D (*P < 0.05 vs. control). The inhibitory action of glibenclamide (10 μM) on the KATP channels was also attenuated by DNase I. Thus, the disruption of the actin cytoskeleton attenuates the ability of SUR to inhibit the opening of KATP channels.

Intracellular Application of Calmidazolium Increases Ca2+ Current through Activation of Protein Kinase A in Cultured Vascular Smooth Muscle Cells

In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak IBa (Ca2+ channel using 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of –60 mV. To test the effect of CaM on IBa, 1 µM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]i). The amplitude of maximally activated IBa was –4.3 ± 0.5 pA/pF (n = 13) for control and –8.1 ± 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of IBa was not abolished when 5 µM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (–9.5 ± 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal IBa. On the other hand, the CMZ stimulation of IBa was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca2+/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated IBa: control, –4.6 ± 0.4 pA/pF (n = 10); MIBMX, –9.6 ± 1.2 (n = 8), and CMZ, –7.9 ± 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated IBa by +42%: before, –3.7 ± 0.7 pA/pF; after, –5.2 ± 1.0 (n = 6). In conclusion, Ca2+ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of IBa might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.

Carbachol inhibition of Ca2+ currents in ventricular cells obtained from neonatal and adult rats

We investigated the postnatal developmental changes produced by the muscarinic receptor agonist, carbachol, on the L-type Ca2+ current (ICa(L)) in neonatal (aged 5 to 7 days) and adult (aged 2 to 5 months) rat ventricular cells by using the whole-cell voltage clamp technique. Carbachol inhibited the isoproterenol-stimulated ICa(L). The maximal inhibition was 89.3 +/- 4.8% (n = 5) in neonatal cells and 17.7 +/- 7.7% (n = 9) in adult cells. Carbachol inhibited the forskolin-stimulated ICa(L) to almost same extent as the isoproterenol-stimulated ICa(L). In the cells pretreated with pertussis toxin, carbachol failed to inhibit the isoproterenol-stimulated ICa(L), indicating that carbachol produced its effect via a pertussis toxin-sensitive G-protein pathway. The effects of carbachol in adult cells became more pronounced, increasing from 17.7% to 54.8% (n = 11), with the addition of the synthetic inhibitory G-protein alpha subunit (Gi alpha) (1 microM) to the reaction. Conversely, the alpha subunit of another pertussis toxin-sensitive synthetic G-protein (G(o) alpha, 1 microM) failed to mimic the effect of Gi alpha. These results suggest that, in rat ventricular cells, (1) the action of carbachol on ICa(L) showed a marked decrease during development; (2) the decrease in the effect of carbachol in adult cells is in part due to a decrease in the activity of pertussis toxin-sensitive G protein, especially Gi alpha.

CHAPTER 9 – Electrogenesis of the Resting Potential

This chapter focuses on the resting potential of the cells of the heart and vascular smooth muscle of the coronary vessels. The resting potential has very important effects on the action potential (rate of rise and duration), propagation of excitation, and automaticity. Factors that determine intracellular ion concentrations in myocardial cells include the Na–K-coupled pump, the Ca–Na exchange reaction, and a sarcolemmal Ca pump. The Na–K pump enzyme, the Na,K-ATPase, requires both Na + and K + for activity and transport 3Na + ions outward and usually 2 K + ions inward per ATP hydrolyzed. Cardiac glycosides are specific blockers of this transport ATPase. The Na–K pump is not directly related to excitability, but only indirectly related by its role in maintaining Na + and K + concentration gradients. The carrier-mediated Ca/Na exchange reaction may be driven by the Na + electrochemical gradient. The energy for transporting out internal Ca 2+ by this mechanism comes from the Na,K-ATPase. The Ca/Na exchange reaction exchanges one internal Ca 2+ ion for three external Na + ions while working in the forward mode in cells at rest. During the AP depolarization, the energetics causes the Ca/Na exchanger to operate in reverse mode, allowing Ca 2+ influx. The key factor that determines the resting E m in the absence of any electrogenic pump potential contributions and for fixed ionic distributions, is the relative permeability of the various ions, particularly of K + and Na + .