Regulation of Spontaneous Transient Outward Potassium Currents in Human Coronary Arteries
Background Spontaneous transient outward potassium currents (STOCs) induce myogenic relaxation in small cerebral vessels. We found STOCs in human coronary artery vascular smooth muscle cells (VSMCs) and studied their regulation.
Methods and Results K+ currents were recorded in human coronary VSMCs by current- and voltage-clamp techniques. STOCs were recorded in the presence of 200 μmol/L Cd2+ and 10 μmol/L verapamil, which block voltage-dependent Ca2+ channels. STOCs were inhibited by iberiotoxin (100 nmol/L), a selective blocker of Ca2+-activated potassium channels (BKCa), and disappeared in a Ca2+-free bath. Iberiotoxin depolarized the VSMCs within 20 minutes from −44±7 to −18±5 mV (n=17). The Ca2+ ionophore A23187 increased intracellular Ca2+ and stimulated whole-cell BKCa current. Depletion of Ca2+ from the sarcoplasmic reticulum with caffeine (4 mmol/L) abolished STOCs for several minutes. Ryanodine (50 μmol/L) transiently stimulated STOCs but then completely inhibited STOCs within 10 minutes. The firing frequency of STOCs was directly correlated with intracellular Na+ concentrations from 0 to 24 mmol/L. Lowering intracellular Na+ to zero abolished STOCs. We next gave monensin (30 μmol/L) to increase intracellular Na+. This maneuver resulted in an increase in whole-cell current fluctuations and STOCs. Monensin-induced STOCs were abolished by either lowering extracellular Ca2+ to zero or chelating Ca2+ intracellularly with BAPTA-AM (30 μmol/L).
Conclusions STOCs resulted from BKCa activity and were dependent on extracellular Ca2+ but not significantly on voltage-dependent Ca2+ channels. STOCs were dependent on intracellular Na+ and intracellular calcium store refilling state. We suggest that Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchange determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.
Potassium channels are a diverse group that fulfill important cellular functions, including regulation of membrane excitability, resting membrane potential, action potential waveforms and frequency, excitation threshold, and vascular tone. We recently characterized five different kinds of vascular K+ currents in human coronary artery VSMCs.1 During our studies, we observed STOCs, which had not been described previously in human coronary arteries. STOCs influence myogenic arterial constriction and thus affect resistance and flow.2 Recently, Nelson et al3 were able to show in small cerebral arteries that STOCs are most likely responsible for a specific function, namely vasodilation. Their seminal work indicated that fast, fluctuant increases in local subsarcolemmal Ca2+ concentrations called “Ca2+ sparks” are important in STOC generation. Functional coupling of ryanodine-sensitive release units of the sarcoplasmic reticulum and the plasmalemmal BKCa channels is involved in the production of STOCs. We studied the regulation of STOCs in human coronary VSMCs and found that mainly Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchanger determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.
Human coronary arteries were obtained from hearts of patients with heart failure (dilated cardiomyopathy without significant arteriosclerosis) after orthotopic heart transplantation and from one nonfailing donor heart that could not be transplanted for technical reasons. The tissue was immediately placed in cold (8°C) Hanks' solution (in mmol/L: NaCl 119, KCl 4.7, KH2PO4 1.2, NaHCO3 25, MgSO4 1.2, glucose 11.1, EDTA 0.026, and CaCl2 2.5, plus 5% CO2 and 95% O2) during transportation to the laboratory for further dissection. Left coronary arteries, right coronary arteries, or branches from left, right, or circumflex coronary arteries (diameter, ≈1.5 mm) were dissected and cleaned of adhering tissue and fat in Hanks' solution. All coronary arteries were stored in Hanks' solution at 4°C before use and were studied within 48 hours. Smooth muscle cells were isolated as previously described for porcine and human coronary artery, with some modifications.1 4 The vessels were cut into small segments (≈4 to 8 mm long) and placed in a Ca2+-free Hanks' solution (in mmol/L: NaCl 137, KCl 5.4, KH2PO4 0.44, NaH2PO4 0.42, MgCl2 2, Ca2+ 0.05, glucose 11.1, and HEPES 10; pH 7.4 with NaOH) for 2 to 4 minutes at room temperature (20°C to 24°C). After a longitudinal section was cut, the segments were washed twice in this solution. The segments were then placed in the Ca2+-free solution containing 3 mg/mL collagenase (Sigma, type IA), 10 mg/mL BSA (Sigma), and 1 mg/mL elastase (Sigma, type IIA) and were incubated for 30 to 50 minutes with gentle agitation in the Ca2+-free Hanks' solution. The cells were stored in Hanks' solution (Ca2+, 0.12 mmol/L) containing 1 mg/mL BSA or in a solution containing (in mmol/L) NaCl 90, KH2PO4 1.2, MgCl2 5, glucose 5, taurine 20, and HEPES 5; pH 7.1 with NaOH at 4°C. Whole-cell K+ currents were measured according to the conventional patch-clamp method or the perforated-patch method with nystatin. The pipette solution contained (in mmol/L) potassium aspartate 80, KCl 50, NaCl 12, MgCl2 1, magnesium ATP 3, EGTA 10, potassium HEPES 5; pH 7.4. The extracellular solution contained (in mmol/L) NaCl 140, CaCl2 1.8, MgCl2 1, KCl 5.4, CdCl2 0.1 (or 0.2), glucose 10, and sodium HEPES 10; pH 7.4. Nystatin (Sigma) was dissolved in DMSO and diluted into the pipette solution to give a final concentration ranging from 50 to 100 μg/mL. A detailed description of the patch-clamp technique is given elsewhere.5 6
Iberiotoxin was obtained from RBI. EGTA, HEPES, A23187, ryanodine, and caffeine were purchased from Sigma-Aldrich. All salts were obtained from Merck. BAPTA-AM 30 mmol/L, a Ca2+ chelator that diffuses through membranes (Calbiochem), was used to chelate intracellular Ca2+.
All values are given as mean±SEM. We performed parametric (Student's t test) and nonparametric (Mann-Whitney test) and linear regression analysis as appropriate. A value of P<.05 was considered significant. The terms increase and decrease are used only when the results were statistically significant.
Electrophysiological Properties of STOCs
To investigate the ionic mechanism involved in the generation of STOCs, we performed perforated patch-clamp experiments with nystatin (n=124); ≈70% of all cells investigated possessed STOCs. The VSMCs exhibiting STOCs had resting membrane potential of −52±15 mV (n=21). Spontaneous transient negative hyperpolarization potentials (negative voltage spikes) were observed in our cells under current-clamp conditions. The decay phase of the negative voltage spikes was fitted by means of a multiexponential function. The duration of negative spikes varied from 50 to >100 ms, and their amplitude varied from 2 to 15 mV (Fig 1A⇓). Iberiotoxin (100 nmol/L) completely blocked generation of negative spikes and depolarized the VSMCs within 20 minutes from −44±7 to −18±5 mV (n=17).
Measurement of the amplitudes from individual STOCs revealed that each cell generated STOCs with several amplitudes and uniform duration of 121±35 ms (n=7) at all ranges of physiological potentials from −60 to +20 mV (Fig 1B⇑). STOC amplitude and frequency increased with increasing membrane potential (Fig 1B⇑). Fig 1B⇑, bottom, shows a composite picture of the discharges. A superimposition of STOCs, which occurred when the next discharge appeared during the decay phase of the previous discharge (latency period, <100 ms), increased the STOC duration up to 200 to 400 ms. The individual STOC duration was stable and was independent of the membrane potential. To show the integral characteristics, including discharge frequency and amplitude changes at different membrane potentials, we calculated the AUC of current traces recorded in steady-state potentials. AUC values were obtained from traces recorded at different holding potentials from five different cells. The values are plotted as a function of membrane potential (Fig 1C⇑). The reversal potential of STOCs obtained by linear fit was −65 mV.
Pharmacological Characterization of STOCs
Iberiotoxin (100 nmol/L) (Fig 2A⇓, top), a specific blocker of BKCa, abolished STOCs completely (n=9). STOCs were also blocked completely with the addition of BAPTA-AM 30 μmol/L (n=10) (Fig 2A⇓, middle). Removal of external Ca2+ depressed STOCs completely (n=12) within 1 minute (Fig 2A⇓, bottom), suggesting that Ca2+ influx from external sources is important for the generation of STOCs. STOCs were observed not only in steady-state holding potentials but also superimposed onto K+ currents elicited by ramps (from −100 to +100 mV) or by step voltage pulses in the perforated patch configuration (Fig 2B⇓, top left). The STOCs had a duration of 100 ms, and their amplitude increased when the membrane potential became increasingly positive. Threshold activation of macroscopic current fluctuations (STOCs superimposed onto currents elicited by voltage step pulses or ramps) was about −59±8 mV (n=17). Spontaneous fluctuations of whole-cell currents were abolished when the cells were placed in a Ca2+-free solution (n=14) (Fig 2B⇓, bottom left). The AUC, calculated for every trace of current induced by ramp pulses with a rate of 0.2 Hz, was plotted as a function of time (Fig 2B⇓, top right). AUC fluctuations disappeared completely in Ca2+-free solution (Fig 2B⇓, bottom right). BAPTA-AM and iberiotoxin also decreased the amplitude of whole-cell currents and blocked STOCs superimposed on potassium currents elicited by ramps or voltage pulses (data not shown). The calcium ionophore A23187 (30 μmol/L) appeared to induce superposition of STOCs several minutes after application (data not shown). Ca2+ influx with the calcium ionophore also increased the whole-cell current amplitude from 220±26 to 367±31 pA, with a shift in the threshold activation from −30±11 to −60±9 mV (n=12). STOCs were never observed after full activation of calcium-dependent potassium currents induced by Ca2+ influx through the ionophore, either during steady-state potentials or during whole-cell current configurations elicited by voltage pulses.
Influence of Ryanodine and Caffeine on STOCs
We next examined a potential role for internal Ca2+ stores in STOC generation in more detail. To prevent Ca2+ influx through voltage-dependent Ca2+ channels7 and to discriminate a pure effect of Ca2+ release from internal stores, all experiments were performed with 200 μmol/L Cd2+ and 10 μmol/L verapamil in the bath solution. Cd2+ ions reduced the frequency of STOCs in human VSMCs by 18% (n=6) but had no effect on duration and amplitude of STOCs (Fig 3A⇓). The durations were 99±35 ms and 104±30 ms before and after Cd2+, respectively (n=6). Subsequent application of verapamil (10 μmol/L), an organic voltage-dependent L-type Ca2+ channel blocker, to the bath solution had no effect or transiently decreased STOC amplitudes (compare Fig 3A⇓ versus 3B). The frequency and duration of STOCs, however, were not further reduced by verapamil (duration of STOCs in the presence of Cd2+ plus verapamil, 116±40 ms; n=6). Ryanodine (50 μmol/L) produced a transient stimulation (n=6) of STOC firing frequency (Fig 3A⇓) and transiently increased the whole-cell potassium current. STOCs were completely blocked by 50 μmol/L ryanodine within 10 minutes. A ryanodine washout for >20 minutes never produced recovery of STOC firing frequency. To further test the notion of Ca2+ release from internal stores, we added caffeine (4 mmol/L) in the presence of Cd2+ and verapamil. The first application of caffeine (n=9) transiently increased steady-state current (hump), followed by a temporary inhibition of STOCs (Fig 3B⇓). STOCs recovered to control values within 7 to 12 minutes after the removal of caffeine from the bath. After the internal Ca2+ stores were replenished and STOCs had recovered, caffeine again had a transient stimulatory effect on steady-state current (hump) (Fig 3B⇓). Caffeine 4 mmol/L was not able to induce a transient stimulatory effect on steady-state current (hump) during caffeine-induced temporary inhibition of STOCs (not shown).
Intracellular Na+ and Intracellular and Extracellular Ca2+ and STOCs
To test for a possible role of the Na+/Ca2+ exchanger in the generation of STOCs, we used the sodium ionophore monensin to increase intracellular Na+ concentration.8 Monensin 30 mmol/L (n=9) increased STOC frequency and the duration of serial STOC discharges (Fig 4A⇓, top and middle). The increased intracellular Na+ concentration did not affect the duration of STOC decay. Monensin also increased the amplitude of whole-cell outward currents evoked by voltage pulses from 245±26 to 394±47 pA (n=10) at +40 mV. When extracellular Ca2+ was reduced to zero (Fig 4A⇓, bottom), monensin no longer had a stimulatory effect on STOCs. The same effect on the monensin-induced increase in STOCs was produced by BAPTA-AM (data not shown). The relationship between STOC AUC and the membrane potential with monensin was compared with the same relationship under control conditions (Fig 4B⇓). A significantly steeper relationship with monensin compared with controls was identified.
We next tested the dependency of STOC generation on the intracellular Na+ concentration. STOCs were not observed when intracellular Na+ was adjusted to zero (n=14) (Fig 4C⇑). STOC firing frequency increased significantly when the pipette solution contained 12 mmol/L Na+ (n=10). A further increase was observed when the pipette solution contained 24 mmol/L Na+ (n=14). An increase in the intracellular Na+ concentration with monensin induced whole-cell outward current increases from 218±42 to 367±24 pA, even when the initial intracellular Na+ concentration was zero (n=9) (Fig 4D⇑). During the first several minutes after application of monensin to the bath solution, the current/voltage relationship elicited by ramp voltage pulses exhibited spontaneous changes in amplitude and a shift of current activation threshold to negative potentials from −32±7 to −61±9 mV. Some cells with 0 mmol/L intracellular Na+ concentration (4 of 18 cells investigated) exhibited STOCs because of increases in the intracellular Na+ concentration elicited by monensin. The stimulatory effect of monensin on spontaneous current fluctuations in the cells containing 0 mmol/L Na+ was blocked by BAPTA-AM (n=6) and by perfusion of the cells with a Ca2+-free bath solution (n=7).
The important observations of this study are that STOCs are present in human coronary VSMCs and that mainly Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchanger determines calcium store refilling, which in turn regulates STOC generation. We identified a novel pathway for regulation of STOCs, and therefore arterial tone, that differs from those reported in several other, nonhuman, smooth muscle preparations.3 9 10 11 12 13 We showed that STOCs depend on intracellular Ca2+ and obtained strong evidence that the BKCa channel is responsible for STOC generation. However, blockade of voltage-dependent Ca2+ channels by Cd2+ and verapamil had only a minor inhibitory effect on STOCs. Thus, STOCs were related to BKCa channels but were not dependent on Ca2+ entry through voltage-dependent Ca2+ channels. We next used ryanodine and caffeine to further elucidate the role of internal Ca2+ stores.3 9 10 11 12 We observed that ryanodine initially increased STOCs but eventually resulted in a decrease in STOC firing frequency and block. Caffeine led to a temporary blockade of STOCs. After recovery of STOCs, caffeine again transiently increased the steady-state current (hump), indicating that the internal Ca2+ stores had been replenished and STOCs were merely dependent on the refilling state of the internal Ca2+ stores.
Since STOCs were not dependent on voltage-dependent Ca2+ channels but rather were solely dependent on internal Ca2+ stores, we reasoned that an additional transporter for Ca2+ ions must be responsible for modulating membrane conductance and for the generation of STOCs.14 The Na+/Ca2+ exchanger, which is present in cardiac, neuronal, retinal, and smooth muscle cells,8 15 16 17 would explain Ca2+ entry into our cells. This exchanger is known to be electrogenic and is a major route for transmembrane Ca2+ fluxes. To test for a possible role of the Na+/Ca2+ exchanger in the generation of STOCs, we first lowered intracellular Na+ to zero, which abolished STOCs. We next identified a direct correlation between STOC firing and intracellular Na+ concentration. We then used the sodium ionophore monensin to increase intracellular Na+ concentration.8 A fundamental feature of the Na+/Ca2+ exchanger is that an elevated [Na+]i concentration promotes the reverse mode of the Na+/Ca2+ exchanger. Thus, when intracellular Na+ is exchanged for extracellular Ca2+, a change in STOC firing frequency would be expected. The modulation of the STOC firing frequency by monensin provides strong evidence that intracellular Ca2+ values are influenced by an increase in the intracellular Na+ concentration.
An increase in intracellular Na+ with monensin induced whole-cell outward current increases and oscillations, even when the initial intracellular Na+ concentration was zero. The stimulatory effect of monensin was blocked by perfusion of the cells with a Ca2+-free bath solution and by BAPTA-AM. Our results demonstrate the existence of a functional Na+/Ca2+ exchanger operating under physiological conditions in VSMCs from human coronary arteries. Na+/Ca2+ exchange is able to replenish Ca2+-depleted sarcoplasmic reticulum and could regulate STOC generation via this pathway. Our data can be explained by a model in which BKCa channels and sarcoplasmic reticulum are linked to a Na+/Ca2+ exchange–dependent calcium compartment.18 19 We suggest that mainly Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchange determines calcium store refilling, which in turn regulates spontaneous STOC generation in human coronary VSMCs. We believe that STOCs are likely to be important in protecting human coronary arteries from vasoconstrictor influences. Elucidation of their regulation may provide new insight into the control of coronary tone and may also have future therapeutic implications.
Selected Abbreviations and Acronyms
|AUC||=||area under the curve|
|BKCa||=||Ca2+-activated potassium channel|
|STOC||=||spontaneous transient outward K+ current|
|VSMC||=||vascular smooth muscle cell|
This work was supported by the Deutsche Forschungsgemeinschaft (Ha 1388/2-3) and by a grant-in-aid from the Bundesministerium fu¨r Bildung und Forschung. Prof R. Hetzer of the Deutsches Herzzentrum, Berlin, kindly supplied us with tissue from human hearts during orthotopic heart transplants.
- Received June 24, 1996.
- Revision received September 3, 1996.
- Accepted September 6, 1996.
- Copyright © 1997 by American Heart Association
Gollasch M, Ried C, Bychkov R, Luft FC, Haller H. Potassium currents in single smooth muscle cells of human coronary arteries. Circ Res. 1996;78:676-689.
Fay FS. Calcium sparks in vascular smooth muscle: relaxation regulation. Science. 1995;270:588-589.
Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science. 1995;270:633-637.
Gollasch M, Bychkov R, Ried C, Behrend F, Luft FC, Haller H. Pinacidil relaxes pig and human coronary arteries by activating ATP-dependent potassium channels in smooth muscle cells. J Pharmacol Exp Ther. 1995;275:681-692.
Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988;92:145-159.
Gollasch M, Haller H, Schultz G, Hescheler J. Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways. Proc Natl Acad Sci U S A. 1991;88:10262-10266.
Barcenas-Ruiz L, Beuckelmann DJ, Wier WG. Sodium-calcium exchanger in heart: membrane currents and changes in [Ca2+]i. Science. 1987;238:1720-1722.
Desilets M, Driska SP, Baumgarten CM. Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Circ Res. 1989;65:708-722.
Ganitkevich V, Isenberg G. Isolated guinea-pig smooth muscle cells: acetylcholine induces hyperpolarization due to sarcoplasmic reticulum calcium release activating potassium channels. Circ Res. 1990;67:525-529.
Blaustein MP. Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and hypothesis. Am J Physiol. 1977;232:C165-C173.
Langer GA, Wang SY, Rich TL. Localization of Na/Ca exchanger-dependent Ca compartment in cultured neonatal rat heart cells. Am J Physiol. 1995;268:C119-C126.
Wendt-Gallitelli MF, Schwarz H, Isenberg G, Moore E, Fay F. Colocalization of Ca2+ transporting proteins and functional Ca2+ microdomains in smooth muscle. J Muscle Res Cell Motility. 1995;4:450. Abstract.