Nicholas Sperelakis

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.

Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc.115, 11799–11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nm) than ruthenium red (IC50 = 6.85 nm) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (K d = 0.34 nm, B max = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nm, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+uptake. Ru360, as high as 10 μm, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μm) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

cGMP-Dependent Protein Kinase Regulation of the L-Type Ca2+ Current in Rat Ventricular Myocytes

Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.

Beneficial effect of taurine in rabbits with chronic congestive heart failure

To examine the effect of daily treatment with taurine on improving the status of congestive heart failure (CHF), we used rabbits with artificially induced aortic regurgitation. Ten rabbits were treated daily with taurine (100 mg/kg by mouth) and eight with guanidinoethyl sulfonate (GES) (100 mg/kg by mouth) immediately after induction of aortic regurgitation. The cumulative mortality rate at 8 weeks in the taurine-treated CHF group was 10% (1 of 10) compared with 53% (16 of 30) in the nontreated CHF group and 75% (6 of 8) in the GES-treated CHF group (p less than 0.05). Although cardiac function (max dP/dt) in CHF rabbits was significantly decreased (p less than 0.001), taurine-treated CHF rabbits maintained the same values as control rabbits. Taurine content of the left ventricular tissue of the CHF rabbits was significantly increased (p less than 0.01). Administration of taurine and GES to control rabbits for 8 weeks affected neither the hemodynamics nor the taurine content of the heart. It was concluded that taurine slowed the rapid progression of heart failure and consequently prolonged life expectancy.

Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells.

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.

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

Abstract Angiotensin 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.

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca2+ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca2+ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate ICa, Ca2+ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca2+ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca2+ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca2+ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of ICa(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated ICa(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca2+ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for ICa(L) stimulation by the β-adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (αs*) of the Gs-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of ICa(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca2+ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca2+ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.

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

Abstract To 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.

Developmental changes in long-opening behavior of L-type Ca2+ channels in embryonic chick heart cells.

In the early (3-day) stage of development, long-lasting openings of the L-type Ca2+ channels (mode 2) occur in embryonic chick heart cells. Since mode-2 behavior is infrequently observed in adult heart cells of other species, in the present study, developmental change in behavior of the Ca2+ channel was examined in young (3-day) and old (17-day) embryonic chick heart cells. In the whole-cell voltage clamp, the L-type Ca2+ current carried by Ca2+ ions was smaller in amplitude and had a faster inactivation in 17-day cells than in 3-day cells. The peak current density was 8.1 +/- 0.2 microA/cm2 (mean +/- SEM, n = 5) and 5.1 +/- 0.3 microA/cm2 (n = 5) in 3-day and 17-day cells, respectively. When the charge carrier was Ba2+, the L-type Ca2+ channel current density was also smaller in 17-day cells (22.7 +/- 1.8 microA/cm2) than in 3-day cells (28.3 +/- 2.1 microA/cm2). In single-channel recordings, the mode-2 behavior was infrequent in 17-day cells compared with 3-day cells. High-open probability sweeps (with an open probability of greater than 0.25), reflecting mode-2 behavior, accounted for 20.2% and 3.7% in 3-day and 17-day cells, respectively. The ensemble-averaged currents in 17-day cells was 37% of that current in 3-day cells. In addition, decay of the averaged current appeared to be faster in 17-day cells than in 3-day cells. All data from the single-channel analysis agreed with the data from the whole-cell voltage clamp.(ABSTRACT TRUNCATED AT 250 WORDS)