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(Circulation Research. 1995;77:121-130.)
© 1995 American Heart Association, Inc.

Articles

[Ca²⁺]_i Inhibition of K⁺ Channels in Canine Renal Artery

Novel Mechanism for Agonist-Induced Membrane Depolarization

Craig H. Gelband, Joseph R. Hume

From the Department of Physiology, University of Nevada School of Medicine, Reno.

Correspondence to Dr Joseph R. Hume, Department of Physiology, University of Nevada School of Medicine, Reno, NV 89557-0004.

	Abstract

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Abstract The patch-clamp technique was used to examine the inhibition of delayed rectifier K⁺ channels by agents that release intracellular Ca²⁺. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 µmol/L) present to inhibit delayed rectifier K⁺ current (I_K(dr)) and Ca²⁺-activated Cl^- current (I_Cl(Ca)), angiotensin II (Ang II) and caffeine increased Ca²⁺-activated K⁺ current (I_K(Ca)) between -25 and 80 mV (n=5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 µmol/L) present to inhibit I_K(Ca) and I_Cl(Ca), Ang II and caffeine only caused inhibition of I_K(dr). Block was achieved within 15 seconds of drug application and was reversible upon washout (n=5). The effects of Ang II on I_K(Ca) and I_K(dr) were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n=3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both I_K(Ca) and I_K(dr). In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 µmol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26±2.9 mV depolarization from -54±3.1 to -28±1.7 mV (n=6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca²⁺ indicator indo 1 (100 µmol/L), Ang II, caffeine, and 4-AP increased [Ca²⁺]_i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca²⁺]_i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca²⁺]_i (n=4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca²⁺-activated K⁺ channels, a 45±7-picosiemen 4-AP–sensitive K⁺ channel was identified that was sensitive to cytoplasmic Ca²⁺ (n=6). Increasing intracellular Ca²⁺ decreased channel opening probability [NxP(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca²⁺ from <5 to 200 and 600 nmol/L free Ca²⁺ decreased NxP(open) by 52±3% and 73±7%, respectively (n=6). The decrease in opening probability of the delayed rectifier K⁺ channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively. These results suggest that agonist-induced changes in intracellular Ca²⁺ can alter vascular smooth muscle membrane potential through regulation of delayed rectifier K⁺ channels.

Key Words: renal artery • [Ca²⁺]_i • K⁺ channels • membrane potential regulation

	Introduction

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A change in free [Ca²⁺]_i is an important factor in the regulation of vascular smooth muscle tone.¹ Excitation-contraction coupling of renal vascular smooth muscle is mainly regulated by circulating hormones, peptides, and neurotransmitters. In general, when a vasoconstrictor agonist binds to its appropriate receptor, a number of biochemical and electrical events take place in a very short period of time. First, the agonist, receptor, and GTP binding protein (G protein) form a tertiary complex. This complex activates phospholipase C (PLC), which breaks down the membrane phospholipid phosphatidylinositol diphosphate to inositol 1,4,5-tris-phosphate (IP₃). IP₃ initiates Ca²⁺ release from the sarcoplasmic reticulum (SR) via activation of a Ca²⁺-release channel. The net result is a rapid increase in free [Ca²⁺]_i. Ca²⁺ then binds to calmodulin and activates myosin light chain kinase, which in turn phosphorylates the smooth muscle myosin light chains, initiating cross-bridge cycling and contraction. Relaxation ensues when the agonist dissociates from its receptor, [Ca²⁺]_i is lowered through activation of sarcolemmal and SR Ca²⁺-ATPases, and cross-bridge cycling ceases.

While these biochemical events are taking place, vasoconstrictor agonists may also cause membrane depolarization and modulate ion channel activity. [Ca²⁺]_i is maintained at high levels because of a Ca²⁺ flux derived from the extracellular space through activation of voltage-dependent Ca²⁺ channels or receptor-operated cation channels. To date, three mechanisms have been described in which agonist binding would cause membrane depolarization. For example, during -adrenoceptor stimulation, there is activation of voltage-dependent Ca²⁺ channels² and Ca²⁺-activated Cl^- channels.³ Also ATP, an excitatory agonist coreleased with norepinephrine from sympathetic nerve terminals, activates a cation-selective receptor-operated channel in rabbit ear artery.⁴ Therefore, agonist activation of vascular smooth muscle can modulate a number of different membrane conductances that play various roles in excitation-contraction coupling.

Recently, we have demonstrated that changes in [Mg²⁺]_i⁵ and [Ca²⁺]_i⁶ depolarize vascular smooth muscle by inhibiting a 4-aminopyridine (4-AP)–sensitive delayed rectifier K⁺ current (I_K(dr)). Not only did [Mg²⁺]_i and [Ca²⁺]_i inhibit I_K(dr), but they depolarized renal arterial cells 25 mV from the resting membrane potential. Similarly, single delayed rectifier K⁺ channels were inhibited by millimolar internal Mg²⁺ in a concentration- and voltage-dependent manner. In the present study, our aim was to investigate whether divalent cation block of delayed rectifier K⁺ channels plays a physiological role in agonist-induced depolarization of canine renal artery.

	Materials and Methods

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Electrophysiological Measurements
Single smooth muscle cells from canine renal artery were enzymatically dissociated by using previously described methods.^{5 7} Single cells were voltage-clamped, and membrane currents were measured by using the whole-cell or inside-out configuration of the patch-clamp technique.⁸ Patch pipettes for whole-cell and inside-out patch-clamp recordings were made from borosilicate glass capillaries and had resistances of 1 to 3 and 7 to 10 M when filled with the appropriate solutions. Voltage-clamp command potentials were applied to the cells, and membrane currents were recorded with an Axopatch-1D patch-clamp amplifier. Membrane current was monitored on a digital oscilloscope, digitized on-line (0.5 to 2.0 kHz), and stored on a computer. Single-channel currents were filtered at 2.0 kHz and digitized at 10.0 kHz. Resting membrane potentials were measured in the I=0 position of the patch-clamp amplifier. Values for channel opening probability [NxP(open), where N is the number of functional channels in a patch and P(open) is the opening probability] and mean open times were obtained from 3-minute steady state recordings of data. Data analysis was performed with PCLAMP 5.5.1 and 6.0 software (Axon Instruments). Single-channel openings were identified by an algorithm that uses both amplitude and slope information, measured with an interactive threshold detection program in the PCLAMP software. The threshold for detecting events was set at 50% of the expected single-channel amplitude. Mean open times, opening probability, and amplitude histograms were calculated from values obtained from this program. Specifically, NxP(open) was determined by using the following equation:

where t_s is the total time spent at each current level corresponding to s=1, 2, . . . n and T is the total time of the recording. Finally, all experiments were performed at room temperature.

Measurement of Intracellular Ca²⁺
The Ca²⁺ indicator indo 1 (pentapotassium salt, 100 µmol/L) was included in the patch pipette solution and dialyzed into the cell. This method of loading indo 1 prevents some of the difficulties that occur when cells are loaded with the ester form of the dye (ie, leakage from the cell, incomplete hydrolysis of the ester form, or sequestration into internal organelles). Background autofluorescence was measured before gaining access to the cell interior and subtracted from all fluorescence measurements. Loading of the dye was assessed by monitoring changes in fluorescence intensity. Sufficient loading had taken place 5 to 10 minutes after access to the cell interior. A 10-µm diameter region of the cell was irradiated with UV light at a wavelength of 340 nm with a mercury lamp. The light emitted from this region was collected from the optics of an epifluorescence microscope (Nikon) and measured at 400 and 500 nm by means of a microfluorometer and matched photomultiplier tubes.⁹ The voltages from the two photomultiplier tubes and an analog ratio of the two fluorescent signals (400/500 nm) was recorded on videotape. Changes in [Ca²⁺]_i were displayed as a change from control in the ratio of the 400- and 500-nm signals.

Solutions
The Krebs' solution contained (mmol/L) NaCl 120, NaHCO₃ 25, KCl 4.8, MgCl₂ 1.2, CaCl₂ 2.5, and D-glucose 10. For all whole-cell voltage-clamp experiments examining outward currents, the bath solution contained (mmol/L) NaCl 130, NaHCO₃ 10, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 1.5, D-glucose 5.5, and HEPES 10 (pH 7.4 with NaOH). The pipette solution contained (mmol/L) KCl 140, ATP (potassium salt) 5, and HEPES 10 (pH 7.2, with KOH). The calculated free [Ca²⁺] for this solution is <3 nmol/L, since the Ca²⁺ contamination of the salts was low (Sigma Chemical Co) and the water was double-distilled (Sigma). For inside-out single-channel recordings, the bath solution contained (mmol/L) KCl 140 and HEPES 10 (pH 7.2 with KOH). The free [Ca²⁺] in this solution (<5, 200, and 600 nmol/L) was made with CaCl₂ (0.3, 5.7, and 8 mmol/L) and EGTA (10 mmol/L) according to association constants from Fabiato and Fabiato.¹⁰ The pipette solution contained (mmol/L) NaCl 140, KCl 5.4, charybdotoxin (ChTX) 0.0002, D-glucose 5.5, and HEPES 10 (pH 7.4 with NaOH). In the experiments in which angiotensin II (Ang II) and caffeine were picospritzed (General Valve Corp) on cells, final concentrations of 100 nmol/L and 10 mmol/L, respectively, were made in external solution and placed in the picospritzer pipette. ChTX was obtained from Peninsula Laboratories, Inc, and the stock was 10^-4 mol/L in 150 mmol/L NaCl. Losartan was provided by Du Pont-Merck. All other chemicals were from Sigma.

Statistics
Results are expressed as mean±SEM. Statistical significance was evaluated by using Student's t test for unpaired observations. Differences were considered significant at P<.05; n corresponds to the number of cells examined. Membrane currents were measured from the zero current level. In the figures, the zero current level is indicated with a line.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Intracellular Ca²⁺ Release on K⁺ Current
Voltage-ramp protocols were used to investigate the effects of intracellular Ca²⁺ on K⁺ current. Depolarizing voltage ramps have been a paradigm useful in the investigation of the pharmacological and biophysical characteristics of K⁺ current in canine renal artery.^{5 7} Fig 1 illustrates an experiment at room temperature in which currents elicited by 1-second voltage steps are compared with currents elicited by 1-second voltage ramps from -80 to +80 mV in a canine renal arterial cell. The magnitudes of voltage-dependent outward currents were essentially identical when elicited by either voltage-step or ramp protocols. With slower voltage ramps, there is some voltage-dependent inactivation of K⁺ current; however, in the present study, in which 1-second voltage ramps were used, little inactivation is produced. As previously shown,^{5 7} I_K(dr) in these cells activates between -40 and -30 mV, is relatively small in amplitude, and displays voltage- and time-dependent activation.^{5 7} I_K(dr) is blocked by 4-AP (K_i, 700 µmol/L) and insensitive to tetraethylammonium (TEA; K_i, 1 mmol/L) and ChTX.^{5 7} The Ca²⁺-activated K⁺ current (I_K(Ca)) activates at more positive potentials, is noisy and large in amplitude, exhibits voltage and time dependence, and is ChTX and TEA sensitive (K_i, 250 µmol/L) and 4-AP insensitive.^{5 7}

View larger version (10K): [in this window] [in a new window]   Figure 1. Current recordings showing voltage and time dependence of a voltage step and ramp paradigm. A, K+ current elicited by voltage steps from a holding potential of -80 mV to the test potentials stated at the right of each current tracing. K+ current shows very little inactivation during 1-second voltage steps. B, K+ current elicited during a 1-second voltage ramp. Note that the current elicited during the ramp is not significantly different in magnitude from that elicited during the voltage step paradigm.

Fig 2 demonstrates the effects of a brief (1-second) application of Ang II (100 nmol/L) and caffeine (10 mmol/L) on whole-cell K⁺ current during a voltage-ramp depolarization. Currents were recorded 3 seconds after rapid application of the agonist with a picospritzer. Both agents caused two visible effects: a reduction in I_K(dr) (evident at negative potentials) and increase in I_K(Ca) (evident at positive potentials). The effects at first seem very small, but the two effects may oppose one another, which would tend to negate any significant change in net K⁺ current. It is also noteworthy that the scale bar for the current is in nanoamps, reflecting significant changes in K⁺ current. Since it is very difficult to analyze the effects of pharmacological agents on multiple components of currents, I_K(Ca) and I_K(dr) were pharmacologically isolated, and the actions of Ang II and caffeine were investigated on each current individually.

View larger version (14K): [in this window] [in a new window]   Figure 2. Current recordings showing that angiotensin II (Ang II, 100 nmol/L; A) and caffeine (10 mmol/L, B) cause a biphasic effect on whole-cell K+ current. When rapidly applied to cells, both Ang II and caffeine caused a reduction in delayed rectifier K+ current and an increase in Ca2+-activated K+ current. Similar results were obtained in six cells. The rate of depolarization was 0.16 mV/ms.

To isolate and assess the effects of increasing [Ca²⁺]_i on I_K(Ca), I_K(dr), and membrane potential, contributions of a combination of conductances must be pharmacologically inhibited. 4-AP, ChTX, and niflumic acid are relatively specific inhibitors of I_K(dr), I_K(Ca), and Ca²⁺-activated Cl^- current (I_Cl(Ca)) in vascular smooth muscle, respectively.^{5 7 11 12} 4-AP has no significant effect on other K⁺ currents in vascular smooth muscle, including I_K(Ca)^{5 7 13 14} and the inward rectifying K⁺ current.¹⁵ Finally, ATP-sensitive K⁺ channels do not pose a potential complicating problem in these experiments, since 5 mmol/L ATP was included in the internal solution, which is expected to block most of this current in vascular smooth muscle.¹⁶

Therefore, to isolate I_K(Ca), experiments (Fig 3) were performed in the presence of 4-AP (5 mmol/L) and niflumic acid (100 µmol/L) to inhibit I_K(dr) and I_Cl(Ca), respectively. The large-conductance Ca²⁺-activated K⁺ channel acts as an internal control in these experiments to sense changes in [Ca²⁺]_i. As others have shown, the expected result of an increase in [Ca²⁺]_i would be an increase in I_K(Ca) at more negative membrane potentials and a shift of its activation curve in the hyperpolarizing direction.¹⁷ Upon rapid application of Ang II (100 nmol/L) or caffeine (10 mmol/L), I_K(Ca) increased between -25 and +80 mV (n=5, Fig 3). These data suggest that changes in [Ca²⁺]_i can be "sensed" by ion channels in the plasma membrane, which may thereby regulate resting membrane potential.

View larger version (17K): [in this window] [in a new window]   Figure 3. Current recordings showing that angiotensin II (Ang II, 100 nmol/L; A) and caffeine (10 mmol/L, B) increase Ca2+-activated K+ current (IK(Ca)). Increasing intracellular Ca2+ activated IK(Ca) in a time-dependent manner. 4-Aminopyridine (10 mmol/L) and niflumic acid (100 µmol/L) were present to inhibit delayed rectifier K+ current and Ca2+-activated Cl- current, respectively. Similar results were obtained in five cells. The rate of voltage depolarization was 0.16 mV/ms.

To investigate agonist inhibition of I_K(dr) (Fig 4), experiments were performed in the presence of ChTX (100 nmol/L) and niflumic acid (100 µmol/L). We have previously shown that ChTX selectively inhibits I_K(Ca) in canine renal arterial cells.⁷ Rapid application of Ang II (100 nmol/L) or caffeine (10 mmol/L) under these conditions caused inhibition of I_K(dr) (Fig 4). The time course for the inhibition of I_K(dr) is illustrated in Fig 5. At a test potential of +60 mV, maximum inhibition with either agent was reached in 15 seconds (Fig 5), and the drug-induced inhibition of I_K(dr) was reversible after 20 seconds of washout. In a number of experiments, Ang II (n=5) and caffeine (n=5) caused a 92±4% and 93±8% decrease of I_K(dr) at 0 mV and an 81±3% and 83±4% decrease in I_K(dr) at +60 mV.

View larger version (20K): [in this window] [in a new window]   Figure 4. Current recordings showing that angiotensin II (Ang II, 100 nmol/L; A) and caffeine (10 mmol/L, B) inhibit delayed rectifier K+ current (IK(dr)). Increasing intracellular Ca2+ inhibited IK(dr). Charybdotoxin (100 nmol/L) and niflumic acid (100 µmol/L) were present to inhibit Ca2+-activated K+ current and Ca2+-activated Cl- current, respectively. Similar results were obtained in five cells. The rate of voltage depolarization was 0.16 mV/ms.

View larger version (13K): [in this window] [in a new window]   Figure 5. Time courses of inhibition of delayed rectifier K+ current (IK(dr)) by angiotensin II (A) and caffeine (B). Angiotensin II (100 nmol/L) and caffeine (10 mmol/L) caused a significant decrease in IK(dr) within 15 seconds. After washout, IK(dr) returned to control levels in 30 seconds. Similar results were obtained in five cells. Holding potential was -80 mV; test potential, 60 mV.

Finally, if Ang II or caffeine were exerting their effects through receptor binding or by increasing [Ca²⁺]_i and not by a direct effect, then an agent that blocks the appropriate Ang II (AT₁) receptor in vascular smooth muscle¹⁸ or chelates Ca²⁺ should inhibit the effects of either agent. Fig 6 illustrates an experiment in which the specific AT₁ receptor antagonist, losartan, inhibited the Ang II–induced effects on I_K(Ca) and I _K(dr). In the control condition, when I_K(Ca) was isolated with 4-AP (10 mmol/L) and niflumic acid (100 µmol/L), Ang II (100 nmol/L) caused a rapid increase in I_K(Ca) (Fig 6A, left). After pretreatment with losartan (1 µmol/L, 10 minutes), the Ang II–stimulated increase in Ca²⁺-activated K⁺ current was prevented (Fig 6A, right; n=3). Similarly, when I_K(dr) was isolated with ChTX (100 nmol/L) and niflumic acid (100 µmol/L), 10 minutes of pretreatment with losartan (1 µmol/L) prevented (Fig 6B, left) the Ang II–induced decrease in I_K(dr) (Fig 6B, right; n=3). These results suggest that the Ang II–induced changes in K⁺ current are initiated by receptor binding.

View larger version (28K): [in this window] [in a new window]   Figure 6. Current recordings showing that losartan prevents the angiotensin II (Ang II)–induced changes in Ca2+-activated K+ current (IK(Ca)) and delayed rectifier K+ current (IK(dr)). A, Ang II (100 nmol/L) caused a significant increase in IK(Ca) after 12 seconds (left). A second application of Ang II in the presence of the specific Ang II receptor antagonist, losartan (1 µmol/L), prevented any increase in IK(Ca) (right, n=3). B, Ang II (100 nmol/L) caused a significant decrease in IK(dr) after 12 seconds (left). A second application of Ang II in the presence of the losartan (1 µmol/L) prevented any decrease in IK(dr) (right, n=3). The rate of voltage depolarization was 0.16 mV/ms.

When cells were loaded with the Ca²⁺-chelating agent BAPTA (10 mmol/L) through the patch pipette, no effect of either agent was observed (Fig 7, n=6). These data demonstrate that the effects of Ang II and caffeine are not reflective of a direct action by the agent but one that involves increases in [Ca²⁺]_i. In another set of experiments, caffeine pretreatment (5 minutes) failed to prevent the effects of Ang II on I_K(Ca) and I_K(dr) (n=3, data not shown). This further suggests that in the case of Ang II, both stimulation of I_K(Ca) and inhibition of I_K(dr) were likely mediated by Ca²⁺ release from a distinct IP₃-sensitive store, since the effects were not affected by caffeine pretreatment.

View larger version (14K): [in this window] [in a new window]   Figure 7. Current recordings showing that buffering [Ca2+]i prevents the angiotensin II (Ang II)–induced (A) or caffeine-induced (B) changes in outward currents. When BAPTA (10 mmol/L) was present in the dialyzing patch pipette, neither Ang II (100 nmol/L) nor caffeine (10 mmol/L) caused an effect on outward currents (Ca2+-activated K+ current or delayed rectifier K+ current). Similar results were obtained in six cells. The rate of voltage depolarization was 0.04 mV/ms.

Ang II and Caffeine Depolarize Renal Arterial Smooth Muscle Cells
We have previously shown that 4-AP and an increase in [Mg²⁺]_i can cause depolarization of canine renal arterial smooth muscle cells.⁵ Fig 8 examines the effect of caffeine on membrane potential of an isolated renal arterial cell. In current clamp, the mean resting membrane potential of these cells was -54±3.1 mV (n=6). Application of ChTX (100 nmol/L) and niflumic acid (100 µmol/L) caused little change in resting membrane potential (-52±3.5 mV, Fig 8A). However, subsequent application of caffeine (10 mmol/L) caused a 26±2.9-mV depolarization from -54±3.1 to -28±1.7 mV (P<.01, n=6). 4-AP (10 mmol/L), an inhibitor of I_K(dr), depolarized the tissue itself (27±2.1 mV, n=6) and blocked the caffeine-induced depolarization (Fig 8B). Similar results were obtained when Ang II was used to release intracellular Ca²⁺ (n=4, data not shown). These results suggest that when Ca²⁺ is released from intracellular stores, a significant inhibition of I_K(dr) is observed, which can regulate membrane potential of isolated renal arterial cells.

View larger version (14K): [in this window] [in a new window]   Figure 8. Recordings showing that caffeine depolarizes renal arterial cells by inhibition of delayed rectifier K+ current. Charybdotoxin (ChTX) and niflumic acid were used to inhibit Ca2+-activated K+ channels and Ca2+-activated Cl- channels. A, Application of ChTX and niflumic acid had no effect on resting membrane potential. Application of caffeine caused a transient depolarization. Similar results were obtained in six cells. B, Pretreatment of the cell with 4-aminopyridine (4-AP) depolarized the resting membrane potential and inhibited the caffeine-induced depolarization. Similar results were obtained in six cells.

To assay directly the effects of Ang II, caffeine, and 4-AP on intracellular Ca²⁺ release and changes in membrane potential, cells were current-clamped, and the membrane-impermeant form of the Ca²⁺ indicator indo 1 (100 µmol/L) was loaded into cells through the patch pipette. Fig 9 shows results from one such experiment. Application of ChTX (100 nmol/L) and niflumic acid (100 µmol/L) alone caused no change in resting fluorescence 400/500 nm ratio (control, 0.95±0.02; after ChTX and niflumic acid, 0.95±0.03; n=4) or membrane potential (control, -52±3.1 mV; after ChTX and niflumic acid, -50±2.7 mV; n=4). This suggests that under basal conditions, neither the large-conductance Ca²⁺-activated K⁺ channel nor a Ca²⁺-activated Cl^- conductance is playing a major role in the regulation of resting membrane potential or that the effects of both conductances oppose each other. Subsequent application of Ang II or caffeine caused a rapid rise in [Ca²⁺]_i, followed by depolarization of the membrane potential to -25±2.5 mV (Ang II, n=4) or -28±3.2 mV (caffeine, n=4). In contrast, exposure to 4-AP first caused membrane depolarization to -27±4.1 mV (n=4), which was followed by a rise in [Ca²⁺]_i, consistent with a dependence of extracellular Ca²⁺ entry. The fact that Ang II and caffeine induced an increase in [Ca²⁺]_i that preceded membrane depolarization suggests that these agents are acting through a similar mechanism: an initial increase in [Ca²⁺]_i that causes inhibition of I_K(dr).

View larger version (20K): [in this window] [in a new window]   Figure 9. Current recordings showing simultaneous measurement of membrane potential and [Ca2+]i. Angiotensin II (Ang II, 100 nmol/L), caffeine (Caff, 10 mmol/L), and 4-aminopyridine (4-AP, 10 mmol/L) depolarized renal arterial cells (top) and increased [Ca2+]i (bottom). Charybdotoxin (100 nmol/L) and niflumic acid (100 µmol/L) alone had no effect on either membrane potential or [Ca2+]i. Similar results were obtained in four cells.

Cytoplasmic Ca²⁺ Inhibits Delayed Rectifier K⁺ Channels
We next examined the direct effect of cytoplasmic Ca²⁺ on delayed rectifier K⁺ channels in inside-out patches of membrane. We have previously shown that in a physiological K⁺ gradient, the conductance of 4-AP–sensitive delayed rectifier K⁺ channels in renal arterial cells is 57±6 picosiemens (pS), whereas the large-conductance Ca²⁺-activated K⁺ channels have a conductance of 130±17 pS.^{5 7} In the present experiments, the pipette contained ChTX (200 nmol/L) to reduce the activity of large-conductance Ca²⁺-activated K⁺ channels. Fig 10 shows single I_K(dr)s recorded in an inside-out patch at a holding potential of 0 mV. In this membrane patch, at least two channels were present. In nominally Ca²⁺-free solution (<5 nmol/L, left tracings), NxP(open) was 0.43. When Ca²⁺ at 200 and 600 nmol/L was washed into the bath, channel opening probability significantly decreased by 51.2% and 72.1% to 0.21 (middle tracings) and 0.12 (right tracings), respectively. There was no significant change in single-channel current amplitude. Mean single-channel current amplitudes at a holding potential of 0 mV were 2.3±0.15, 2.15±0.10, and 2.10±0.13 pA in nominally Ca²⁺-free solution and solutions containing 200 and 600 nmol/L Ca²⁺, respectively (n=4).

View larger version (28K): [in this window] [in a new window]   Figure 10. Ca2+ inhibits delayed rectifier K+ channels in inside-out patches of renal arterial smooth muscle cells. Charybdotoxin (200 nmol/L) was in the patch pipette. Holding potential was 0 mV. All single-channel openings are upward deflections of current. Top, In nominally free Ca2+ (<5 nmol/L), openings of delayed rectifier K+ channels were recorded (left); NxP(open), where N is the number of functional channels in a patch and P(open) is the opening probability, was 0.43. Upon the application of 200 nmol/L Ca2+ to the bath, the delayed rectifier K+ channels were inhibited; NxP(open) was 0.21 (middle). When 600 nmol/L Ca2+ was applied to the patch, NxP(open) decreased to 0.12 (right). Data were collected from 3-minute recordings of steady state data. Bottom, Amplitude histograms show that single-channel amplitude did not change significantly. Similar effects were obtained in four cells.

Fig 11 shows the current-voltage relation for the Ca²⁺-sensitive delayed rectifier K⁺ channel and summarizes the effects of Ca²⁺ on channel opening probability (Fig 11B) and mean open time (Fig 11C). The single-channel slope conductance of the delayed rectifier K⁺ channels in Fig 10 was 45 pS, and the mean slope conductance in a number of experiments was 49±6 pS (n=6). At potentials of -20, 0, +20, and +40 mV, 200 and 600 nmol/L Ca²⁺ significantly decreased channel opening probability in a concentration-dependent manner (Fig 11B; *P<.05, **P<.01; n=6). Delayed rectifier K⁺ channels in canine renal artery frequently show a combination of long and short mean open times.⁵ The majority of single-channel currents recorded from patches in the present study had long mean open times. The Ca²⁺-dependent block was manifested through a decrease in mean open time of delayed rectifier K⁺ channels. At potentials of -20, 0, +20, and +40 mV, 200 and 600 nmol/L Ca²⁺ significantly decreased mean open time (Fig 11C; *P<.01, **P<.05; n=6). The block by cytoplasmic Ca²⁺ was voltage dependent, since at more depolarized potentials the degree of block by Ca²⁺ was greater (Fig 11C). For example, at -20 mV, 600 nmol/L Ca²⁺ decreased mean open time by 56.3±3.1%, whereas at +40 mV, the decrease in mean open time was 74.1±4.2%. These data are consistent with a voltage-dependent blocking mechanism in which Ca²⁺ is driven into the open channel pore by membrane depolarization. This type of divalent cation inhibition of delayed rectifier K⁺ channels is similar to that previously shown with internal Mg²⁺.⁵

View larger version (21K): [in this window] [in a new window]   Figure 11. Cytoplasmic Ca2+ decreases opening probability and mean open time of delayed rectifier K+ channels. A, Graph showing a representative single-channel current-voltage relation for the delayed rectifier K+ channels of Fig 10. The slope conductance () was 45 picosiemens. The solid line is a fit to the Goldman-Hodgkin-Katz current equation. B, Graph showing that when [Ca2+] was changed from <5 to 200 or 600 nmol/L Ca2+, opening probability significantly decreased in a dose-dependent manner (*P<.05, **P<.01; n=4). NxP(open) indicates the number of functional channels in a patch (N) times the opening probability [P(open)]. When error bars are not present, they are the same size as the symbol. C, Bar graph showing that an increase in the internal [Ca2+] from <5 to 200 and 600 nmol/L significantly decreased the mean open time of delayed rectifier K+ channels in a voltage-dependent manner (*P<.05, **P<.01; n=4).

The decrease in mean open time can be attributed to changing the open and closed time constants of the delayed rectifier K⁺ channel. Fig 12 shows the open and closed time distributions in the presence and absence of Ca²⁺ at a holding potential of -20 mV. In the presence of 600 nmol/L cytoplasmic Ca²⁺, there was a significant shift in the open time distribution to shorter events (Fig 12A and 12C), and there was a significant shift in the closed time distribution to longer events (Fig 12B and 12D). The histograms were best fit by one exponential. The open time constant significantly decreased from 7.9±1.8 to 2.8±1.4 milliseconds (P<.01, n=6) in the presence of 600 nmol/L Ca²⁺, whereas the closed time constant significantly increased from 446±13 to 841±23 milliseconds (P<.005, n=6). Thus, the decrease in mean open time reflects changes in both open and closed time distributions of the delayed rectifier K⁺ channel. It is interesting to note that the ability of [Ca²⁺]_i (600 nmol/L, Figs 10 through 12) to modulate delayed rectifier K⁺ channel kinetics is more potent than the effects previously observed with Mg²⁺ (10 mmol/L).⁵

View larger version (29K): [in this window] [in a new window]   Figure 12. Effect of Ca2+ on open and closed time distributions. The histograms were best fit by one exponential. The data are from the experiment shown in Fig 9, except the holding potential was -20 mV. A and C, When Ca2+ was increased from control to 600 nmol/L, the open time constant () decreased from 8.3 to 2.2 milliseconds. B and D, In the presence of 600 nmol/L Ca2+, the closed time constant () increased from 454 to 856 milliseconds.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data describe novel effects of increasing [Ca²⁺]_i by vasoconstrictor agents on membrane potential and outward current in canine renal arterial cells. First, a 4-AP–sensitive delayed rectifier K⁺ current, I_K(dr), was blocked by physiological concentrations of intracellular Ca²⁺ when released from intracellular stores. Similar results were previously obtained after histamine (H₁) receptor stimulation of isolated coronary artery cells.¹⁹ Second, 4-AP, caffeine, and Ang II significantly increased [Ca²⁺]_i and depolarized renal arterial smooth muscle cells through inhibition of I_K(dr). Ang II and caffeine indirectly inhibited I_K(dr) by releasing intracellular Ca²⁺, whereas 4-AP directly inhibited I_K(dr), depolarizing the cell, thus leading to an increase in [Ca²⁺]_i. Both of these mechanisms of membrane depolarization would cause the opening of voltage-dependent Ca²⁺ channels and sustained Ca²⁺ influx. Since the activation range of I_K(dr) is close to the average resting membrane potential of vascular smooth muscle cells, these results suggest that changes in intracellular Ca²⁺ may play a role in regulating membrane potential. Third, single delayed rectifier K⁺ channels were inhibited by cytoplasmic Ca²⁺. The mechanism of block by Ca²⁺ is similar to that of Mg²⁺ in that Ca²⁺ caused a decrease in opening probability via a decrease in mean open time.⁵ These results suggest the existence of a novel feedback pathway in smooth muscle by which intracellular Ca²⁺ may regulate sarcolemmal ionic permeability. Since agonist activation of smooth muscle is known to cause an increase in intracellular Ca²⁺, a significant inhibition of I_K(dr) is expected, which would cause membrane depolarization and activation of voltage-dependent Ca²⁺ channels, thus regulating smooth muscle tone (Fig 13). One caveat is that this positive-feedback loop can be antagonized by Ca²⁺ release activating I_K(Ca).

View larger version (33K): [in this window] [in a new window]   Figure 13. A schematic model for Ca2+ inhibition of delayed rectifier K+ channels (DRs) and agonist-induced depolarization of smooth muscle. Agonists binding to their appropriate receptors will cause an increase in [Ca2+]i and inhibition of DRs. This decrease in K+ current will cause membrane depolarization and the opening of voltage-dependent Ca2+ channels. SL indicates sarcolemma; SR, sarcoplasmic reticulum; A, agonist; R, receptor; Gp, GTP binding protein; PLC, phospholipase C; IP3, inositol 1,4,5-tris-phosphate; DAG, diacylglycerol; IP3R, IP3 receptor; VDCC, voltage-dependent Ca2+ channel; and Vm, membrane potential voltage sensor.

Since the sources of Ca²⁺ that are used in contraction of the canine renal artery have not been investigated in any detail, a number of agents that increase Ca²⁺ entry and release intracellular Ca²⁺ have been recently investigated in tissue rings of the canine renal artery.^{6 20} KCl (80 mmol/L), Ang II (100 nmol/L), caffeine (10 mmol/L), and 4-AP (1 mmol/L) caused contraction in Ca²⁺-containing solution. However, in Ca²⁺-free (2 mmol/L EGTA) solution, only Ang II and caffeine caused a contraction, while KCl and 4-AP had no effect. Ryanodine (10 mmol/L) treatment caused contraction on its own and further inhibited the caffeine contractions. Ryanodine had no effect on the KCl- or phenylephrine-induced contraction. These results suggest (1) that KCl and 4-AP require extracellular Ca²⁺ as a source for contraction, whereas Ang II and caffeine use intracellular Ca²⁺ for contraction, and (2) that the ryanodine- and caffeine-sensitive Ca²⁺ store in canine renal artery is a separate smaller store distinct from the larger IP₃-sensitive Ca²⁺ store. These data are consistent with the previous electrophysiological results showing that Ang II stimulation of I_K(Ca) and inhibition of I_K(dr) were mediated by Ca²⁺ release from a caffeine-insensitive store.

Inhibition of K⁺ channels is not the only mechanism by which an agonist can cause changes in membrane potential. During agonist-induced contraction of vascular smooth muscle, other electrical events are taking place that would cause depolarization of the muscle cells and regulate membrane potential. Upon -adrenoceptor stimulation, there is an opening of voltage-dependent Ca²⁺ channels² and Ca²⁺-activated Cl^- channels.³ ATP, another excitatory agonist coreleased with norepinephrine from sympathetic nerve terminals, activates a cation-selective receptor-operated channel in rabbit ear artery.⁴ These pathways and the agonist inhibition of K⁺ channels may combine to play a significant role in excitation-contraction coupling of vascular smooth muscle.

Recently, a number of K⁺ channels have been suggested to regulate membrane potential of smooth muscle cells. We⁵ and others²¹ have demonstrated that delayed rectifier K⁺ channels are important regulators of smooth muscle resting membrane potential. However, other K⁺ channels have also been suggested to play a role in the regulation of smooth muscle tone. Large-conductance Ca²⁺-activated K⁺ channels may represent a negative-feedback mechanism for cerebral arteries when pressurized.²² ATP-sensitive²³ and inwardly rectifying K⁺ channels¹⁵ have also been implicated in regulating tone in the small vessels of cerebral, pulmonary, and coronary circulation. Therefore, a combination of K⁺ channels or variation in K⁺ channel distribution may explain why different K⁺ channels are observed to regulate membrane potential in various vascular beds.

With respect to the inhibition of delayed rectifier K⁺ channels by intracellular cations, a number of investigators have shown similar effects in a number of different cell types, including neuronal, muscular, and secretory cells.²⁴ For example, internal Ba²⁺, Na⁺, Cs⁺, and Li⁺ inhibit I_K(dr) in squid giant axon and the node of Ranvier.^{25 26 27 28} Recently, two other groups have shown direct inhibition of delayed rectifier K⁺ channels by divalent cations. First, Lopatin and Nichols²⁹ have shown that internal Mg²⁺ and Na⁺ inhibit a cloned delayed rectifier K⁺ channel (DRK1). Finally, in parathyroid cells, Komwatana et al³⁰ have demonstrated that intracellular Ca²⁺ inactivates an outwardly rectifying K⁺ current. The current was decreased within a physiological range of Ca²⁺ concentrations (pCa 8 to 5). The blocking mechanism of internal divalent and monovalent cations is similar and involves a voltage-dependent block of the open channel. Therefore, block by divalent cations should produce a more significant block at more depolarized membrane potentials. This result is similar to that obtained in renal arterial cells for both Ca²⁺ and Mg²⁺.⁵

It should be noted that the macroscopic I_K(dr) in some smooth muscle preparations may be due to a mixture of underlying single K⁺ channels. Recently, in canine colonic smooth muscle cells, it was demonstrated that, by using both biophysical and pharmacological manipulations, multiple components of I_K(dr) exist.³¹ There have also been a variety of single-channel conductances that have been reported for delayed rectifier K⁺ channels in various smooth muscle cell preparations. These range from 7 to 55 pS.^{5 7 32 33 34} Therefore, the 4-AP–sensitive 45-pS K⁺ channel observed in the present experiments in canine renal artery may contribute to the membrane depolarization observed when intracellular Ca²⁺ is released by vasoconstrictor agonists, but our experiments do not rule out the potential involvement of other yet-to-be-identified K⁺ channels.

Based on these results with agents that release intracellular Ca²⁺, our data suggest that agonist modulation of I_K(dr) can influence the contractile state of vascular smooth muscle. This could have strong implications in a number of physiological and pathophysiological conditions involving different vascular beds. Besides being important in agonist-induced contraction of vascular smooth muscle, we have recently shown that hypoxic pulmonary vasoconstriction may also involve an initial increase in [Ca²⁺]_i, leading to inhibition of delayed rectifier K⁺ channels and membrane depolarization.³⁵ Pathophysiologically, if intracellular [Ca²⁺] is elevated above basal levels, ie, during renal vasospasm or hypertension, a significant decrease in I_K(dr) would occur, further exacerbating the contractile state of the muscle. Therefore, changes in I_K(dr) during a number of physiological and pathophysiological conditions may play a significant role in the regulation of smooth muscle membrane potential and tone.

	Acknowledgments

 
This study was supported by National Institutes of Health (NIH) grant HL-49254 (Dr Hume); Dr Gelband was supported by an NIH fellowship (HL-08531). Canine renal artery was graciously made available from a program project grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-41315). Expert technical assistance was provided by Michael Sokoloff and Sarena Keane. We thank Dr James Kenyon for comments on the manuscript.

Received August 15, 1994; accepted March 20, 1995.

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