K_Ca channel insensitivity to Ca²⁺ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells

Abstract

In smooth muscle cells, localized intracellular Ca²⁺ transients, termed “Ca²⁺ sparks,” activate several large-conductance Ca²⁺-activated K⁺ (K_Ca) channels, resulting in a transient K_Ca current. In some smooth muscle cell types, a significant proportion of Ca²⁺ sparks do not activate K_Ca channels. The goal of this study was to explore mechanisms that underlie fractional Ca²⁺ spark-K_Ca channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca²⁺ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of −40, 0, and +40 mV, mean transient K_Ca current frequency was ∼0.18, 0.43, and 0.26 Hz and K_Ca channel activity [number of K_Ca channels activated by Ca²⁺ sparks × open probability of K_Ca channels at peak of Ca²⁺ sparks (NP_o)] at the transient K_Ca current peak was ∼4, 12, and 24, respectively. Depolarization between −40 and +40 mV increased K_Ca channel sensitivity to Ca²⁺ sparks and elevated the percentage of Ca²⁺ sparks that activated a transient K_Ca current from 59 to 86%. In a Ca²⁺-free bath solution or in diltiazem, a voltage-dependent Ca²⁺ channel blocker, steady membrane depolarization between −40 and +40 mV increased transient K_Ca current frequency up to ∼1.6-fold. In contrast, caffeine (10 μM), an RyR channel activator, increased mean transient K_Ca current frequency but did not alter Ca²⁺ spark-K_Ca channel coupling. These data indicate that coupling is increased by mechanisms that elevate K_Ca channel sensitivity to Ca²⁺ sparks, but not by RyR channel activation. Overall, K_Ca channel insensitivity to Ca²⁺ sparks is a prominent factor underlying fractional Ca²⁺ spark uncoupling in newborn cerebral artery myocytes.

Arterial smooth muscle cell contractility is differentially regulated by local and global elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (16). Global nanomolar [Ca²⁺]_i elevations, caused by Ca²⁺ influx from the extracellular space and release from intracellular stores, stimulate contraction via the activation of Ca²⁺/calmodulin-dependent myosin light chain kinase (8, 16). In contrast, localized micromolar [Ca²⁺]_i transients, termed “Ca²⁺ sparks,” oppose contraction (16, 21).

Ca²⁺ sparks are induced by activation of several ryanodine-sensitive Ca²⁺ release (RyR) channels on the sarcoplasmic reticulum (SR) (5, 16). In smooth muscle cells, a Ca²⁺ spark can activate multiple large-conductance Ca²⁺-activated K⁺ (K_Ca) channels, resulting in a transient K_Ca current (3, 16, 21). In the arterial wall, transient K_Ca currents induce membrane hyperpolarization, which reduces voltage-dependent Ca²⁺ channel activity and, thus, global [Ca²⁺]_i and contractility (21). The differential regulation of arterial smooth muscle contractility by local and global Ca²⁺ signals exemplifies how a single signaling element can control opposing cellular functions in the same cell.

Several important features facilitate differential regulation of arterial smooth muscle contractility by local and global [Ca²⁺]_i elevations, including the spatial and temporal nature of the Ca²⁺ signals and the proximity and Ca²⁺ sensitivity of downstream target proteins (16). One important feature of Ca²⁺ sparks that allows specificity of signaling is that these events do not contribute significantly to global [Ca²⁺]_i because of their rapid and localized properties (16, 21). Another important aspect is that K_Ca channels are sensitive to micromolar [Ca²⁺]_i elevations, such as those generated by Ca²⁺ sparks (22, 33). As such, K_Ca channels are relatively insensitive to global nano-molar [Ca²⁺]_i elevations that signal contraction (22, 33).

In adult rat cerebral artery smooth muscle cells, the effective coupling of Ca²⁺ sparks to K_Ca channels is strong, and at a physiological membrane potential of −40 mV, essentially all Ca²⁺ sparks activate a transient K_Ca current (6, 23). However, in adult human and newborn porcine cerebral arterial, adult feline esophageal, and Bufo marinus stomach smooth muscle cells, the effective coupling of Ca²⁺ sparks to K_Ca channels is considerably weaker (14, 18, 27, 32). In these smooth cell types, a significant proportion of Ca²⁺ sparks do not activate a transient K_Ca current (∼20–40%), and the amplitude correlation between these events is less robust than in rat cerebral artery smooth muscle cells (14, 18, 27, 32). However, underlying causes of weak coupling and mechanisms that enhance coupling in these smooth muscle cell types are unclear.

The goal of this study was to investigate mechanisms that underlie fractional Ca²⁺ spark-K_Ca channel coupling in smooth muscle cells. Ca²⁺ spark-K_Ca channel coupling was studied in newborn porcine cerebral artery smooth muscle cells, which exhibit a weak coupling phenotype similar to that observed in other smooth muscle cell types, including human cerebral artery smooth muscle cells (27). We investigated whether an increase in K_Ca channel Ca²⁺ sensitivity or RyR channel activation enhances coupling. Data suggest that coupling is determined primarily by K_Ca channel sensitivity to Ca²⁺ sparks and indicate that RyR channel activation alone does not influence coupling.

MATERIALS AND METHODS

Tissue preparation

All procedures used were approved by the University of Tennessee Animal Care and Use Committee. Newborn pigs (1–3 days old, 1–2.5 kg body wt) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im). The brain was removed and maintained in ice-cold HEPES-buffered physiological saline solution (PSS) containing (in mM) 134 NaCl, 6 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH. Isolated arteries (50–200 μm) were dissected from the brain and cleaned to remove basolateral connective tissue. Individual smooth muscle cells were dissociated from cerebral arteries by a procedure described previously (13).

Confocal Ca²⁺ imaging

Arterial smooth muscle cells were placed in HEPES-buffered PSS containing 10 μM fluo 4-AM for 20 min at room temperature. The cells were then washed with HEPES-buffered PSS for 30 min to allow indicator deesterification. Fluo 4 was imaged using a laser scanning confocal microscope (Oz, Noran Instruments, Middleton, WI) and a ×60 water immersion objective (1.2 NA) attached to a microscope (model TE300, Nikon). Fluo 4 was illuminated at 488 nm with use of a krypton-argon laser, and emitted light >500 nm was captured. Images (56.3 × 52.8 μm) were recorded every 8.3 ms (i.e., 120 images per second). When a slit width of 100 μm was used, the z resolution (full width at half-maximal amplitude) of the imaging system was 7 μm, as determined by subresolution (100-nm-diameter) fluorescent beads. Electrophysiological and fluorescence measurements were synchronized using a light-emitting diode placed above the recording chamber that was triggered during acquisition. Each isolated smooth muscle cell was imaged for 10 s under each condition. Custom analysis software (kindly provided by Dr. M. T. Nelson, University of Vermont) was used to detect Ca²⁺ sparks in smooth muscle cells. For detection of Ca²⁺ sparks, an area 1.54 × 1.54 μm (7 × 7 pixels, i.e., 2.37 μm²) in each image (F) was divided by a baseline (F₀) that was determined by averaging 10 images without Ca²⁺ spark activity. The entire image area was analyzed to detect Ca²⁺ sparks. A Ca²⁺ spark was identified as a local increase in F/F₀ that was >1.2. Mean Ca²⁺ spark frequency and standard error of the mean under each condition were calculated by averaging individual cellular frequencies. Spatial spread of the Ca²⁺ spark was calculated at half-maximal amplitude. Changes in local or global [Ca²⁺]_i were calculated using the pseudoratio method (5)

$$\left[{\mathrm{Ca}}^{2+}\right]=\frac{K\mathrm{R}}{K∕{\left[{\mathrm{Ca}}^{2+}\right]}_{\text{rest}}+1-\mathrm{R}}$$

where K is the apparent affinity of fluo 4 for Ca²⁺ [770 nM (28)], R is the fractional fluorescence increase (F/F₀), and [Ca²⁺]_rest is [Ca²⁺]_i at F₀. Global Ca²⁺ fluorescence was calculated from the same images used for Ca²⁺ spark analysis and was the mean pixel value of 100 different images acquired over 10 s. Global [Ca²⁺]_i at 0 and +40 mV were calculated from the cellular change in F/F₀ from −40 mV (determined with fura 2; see Intracellular Ca²⁺ measurements using fura 2).

Patch-clamp electrophysiology

Isolated cells were allowed to adhere to a glass coverslip in the bottom of a chamber for 10 min before experimentation. K⁺ currents were measured using the perforated-patch configuration of the patch-clamp technique with an Axo-patch 200B amplifier (Axon Instruments, Union City, CA). The bath solution was HEPES-buffered PSS. Where appropriate, Ca²⁺-free bath solution was prepared by substitution of equimolar CaCl₂ with NaCl and addition of 1 mM EGTA. The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES, and 0.05 EGTA, with pH adjusted to 7.2 with KOH. Membrane currents were filtered at 1 kHz and digitized at 4 kHz. In each patch under each condition, transient K_Ca current frequency and amplitude were calculated from ≥5 min of continuous gap-free data. At −40, 0, and +40 mV, in the presence of thapsigargin (500 nM), an SR Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks (16), a maximum of two, three, and six K_Ca channel openings, respectively, were observed (n = 5 cells). Therefore, at −40, 0, and +40 mV in control, a transient K_Ca current was defined as the simultaneous opening of three, four, or seven K_Ca channels, respectively. Single K_Ca channel current amplitude at each voltage was calculated using amplitude histograms.

Intracellular Ca²⁺ measurements using fura 2

Cerebral arteries were incubated in HEPES-buffered PSS containing 5 μM fura 2-AM and 0.05% Pluronic F-127 for 45 min at room temperature. After they were washed, the arteries were allowed 15 min for indicator deesterification. Fura 2 was alternately excited with 340- or 380-nm light with use of a xenon arc lamp and a personal computer-driven hyperswitch (Ionoptix, Milton, MA). Background corrected ratios were collected every 1 s at 510 nm with use of a photomultiplier tube (Ionoptix). For calibration of confocal Ca²⁺ imaging data, the extracellular K⁺ concentration was elevated from 6 to 30 mM by substitution of equimolar K⁺ for Na⁺; 30 mM K⁺ depolarizes arterial smooth muscle cells to ∼−40 mV (10), a voltage applied in transient K_Ca current measurements. [Ca²⁺]_i values were calculated from fura 2 fluorescence measurements using the following equation (9)

$$\left[{\mathrm{Ca}}^{2+}\right]=K_{\mathrm{d}}\frac{(\mathrm{R}-{\mathrm{R}}_{\min })}{({\mathrm{R}}_{\max }-\mathrm{R})}\frac{\left({\mathrm{S}}_{\mathrm{f}2}\right)}{\left({\mathrm{S}}_{\mathrm{b}2}\right)}$$

where R is the ratio of fluorescence at 340 nm to fluorescence at 380 nm, R_min and R_max are the minimum and maximum fluorescence ratios determined in Ca²⁺-free and saturating Ca²⁺ solutions, respectively, S_f2/S_b2 is the ratio of Ca²⁺-free to Ca²⁺-replete emissions at 380-nm excitation, and K_d is the dissociation constant for fura 2 [282 nM (19)]. For determination of R_min, R_max, S_f2, and S_b2 at the end of the experiments and in separate experiments, the Ca²⁺ permeability of smooth muscle cells was increased with 10 μM ionomycin and the cells were perfused with a high-Ca²⁺ (10 mM) or Ca²⁺-free (no added Ca²⁺, 5 mM EGTA) solution. Elevation of extracellular K⁺ from 6 to 30 mM or from approximately −60 to −40 mV increased arterial wall Ca²⁺ from 104 ± 17 to 244 ± 29 nM (n = 7 arteries, P < 0.05).

Statistical analysis

Values are means ± SE; n refers to the number of events analyzed, unless otherwise specified. Student's t-tests were used for comparison of paired or unpaired data and Student-Newman-Keuls test for comparison of multiple data sets. When data sets were not normally distributed, the Kruskal-Wallis test with Dunn's multiple comparisons test was used for statistical comparison. Linear regression was used to calculate statistical correlation between the amplitude of Ca²⁺ sparks and evoked transient K_Ca currents (Origin, OriginLab, Northampton, MA). Analysis of covariance of linear regression was used to compare amplitude correlation data sets (Graphpad Prism, San Diego, CA). P < 0.05 was considered significant.

Chemicals

Unless otherwise stated, all chemicals were obtained from Sigma Chemical (St. Louis, MO). Papain was purchased from Worthington Biochemical (Lakewood, NJ) and fluo 4-AM from Molecular Probes (Eugene, OR).

RESULTS

Membrane depolarization elevates transient K_Ca current frequency and activity in newborn cerebral artery smooth muscle cells

Steady membrane depolarization between −40 and 0 mV increased mean transient K_Ca current frequency from ∼0.18 to 0.43 Hz (Fig. 1, A and B). Further depolarization to +40 mV reduced transient K_Ca current frequency to ∼0.26 Hz (Fig. 1, A and B). In contrast, depolarization between −40 and +40 mV continually increased mean transient K_Ca current amplitude (Fig. 1, A and C). Transient K_Ca current amplitude (I) is dependent on the number of K_Ca channels activated by a Ca²⁺ spark (N), the open probability of K_Ca channels at the Ca²⁺ spark peak (P_o), and single K_Ca channel amplitude (i), giving iNP_o. Membrane depolarization increases the driving force for K⁺ and, thus, i. Therefore, transient K_Ca current amplitude data were normalized for voltage-dependent changes in driving force as follows: NP_o = I/i. In the same patches used for transient K_Ca current analysis, single K_Ca channel amplitudes at −40, 0, and +40 mV were 2.8 ± 0.1, 4.8 ± 0.1, and 9.0 ± 0.1 pA, respectively (n = 13). Over the voltage range of −40 to +40 mV, transient K_Ca channel activity (i.e., NP_o) increased from 4 to 23 (Fig. 1D). These data indicate that membrane depolarization elevates transient K_Ca current frequency and Ca²⁺ spark-induced K_Ca channel activity in newborn porcine cerebral artery smooth muscle cells.

Fig. 1.

Voltage dependence of transient Ca²⁺-activated K⁺ (KCa) current frequency, amplitude, and activity. A: original recordings obtained in the same newborn arterial smooth muscle cell illustrating effect of steady membrane potentials between −40 and +40 mV on transient K_Ca current frequency and amplitude. B: voltage dependence of transient K_Ca current frequency (n = 13 cells). C: depolarization-induced elevation in transient K_Ca current amplitude (n = 13 cells). D: depolarization-induced elevation in transient K_Ca current activity (n = 13 cells). NP_o, number of K_Ca channels activated by Ca²⁺ sparks × open probability of K_Ca channels at peak of Ca²⁺ sparks. *P < 0.05 vs. −40 mV.

Membrane depolarization activates Ca²⁺ sparks and augments Ca²⁺ spark-induced K_Ca channel activation

To examine the mechanisms by which membrane depolarization elevates transient K_Ca current frequency and activity in newborn arterial smooth muscle cells, simultaneous measurements of Ca²⁺ sparks and transient K_Ca currents were acquired using confocal Ca²⁺ imaging in combination with patch-clamp electrophysiology.

At −40 mV, ∼59% of Ca²⁺ sparks activated a transient K_Ca current (Fig. 2, Table 1). Steady membrane depolarization from −40 to 0 mV elevated global F/F₀ 1.33-fold, which translates to an increase in global [Ca²⁺]_i from 224 ± 29 nM (see materials and methods) to 363 nM. Depolarization from −40 to 0 mV elevated the amplitude of coupled and uncoupled Ca²⁺ sparks, with the mean amplitude of all Ca²⁺ sparks increasing from ∼874 to 1,424 nM. In contrast, mean Ca²⁺ spark spread was smaller and decay was faster at 0 mV that at −40 mV. Depolarization from −40 to 0 mV increased the percentage of Ca²⁺ sparks that activated a transient K_Ca current to ∼77%. Further depolarization from 0 to +40 mV reduced global [Ca²⁺]_i to 271 nM, which is expected because of a reduction in the driving force for Ca²⁺ influx, decreased mean Ca²⁺ spark amplitude to ∼1,121 nM and reduced coupled and uncoupled Ca²⁺ spark amplitude. However, depolarization to +40 mV increased the percentage of Ca²⁺ sparks that activated a transient K_Ca current to ∼86%. Taken together, membrane depolarization between −40 and +40 mV is estimated to increase K_Ca channel sensitivity to Ca²⁺ sparks from ∼0.015 to 0.026 NP_o/nM Ca²⁺ when the [Ca²⁺]_i detected by fluo 4 is taken as an indicator of Ca²⁺ spark amplitude.

Fig. 2.

Membrane depolarization elevates KCa channel sensitivity to Ca²⁺ sparks. A: simultaneous recordings of whole cell current (top traces) and Ca²⁺ sparks (bottom traces) at −40, 0, and +40 mV. Fluo 4 fluorescence changes (F/F₀) were measured in 2 different 1.54 × 1.54 μm (i.e., 2.37 μm²) areas of the cell in which Ca²⁺ sparks occurred. At −40 mV, 1 Ca²⁺ spark occurred at 1 location; at 0 mV, 4 Ca²⁺ sparks were observed at 2 locations; at +40 mV, 3 Ca²⁺ sparks occurred at 1 location. *, Ca²⁺ spark at its peak in the pseudocolored inset image. B: voltage dependence of relation between peak Ca²⁺ spark amplitude and activity (NP_o) of evoked transient KCa currents (n = 14, 13, and 10 cells for −40, 0, and +40 mV, respectively). Linear regression with 95% confidence bands is illustrated with slopes of 0.005, 0.010, and 0.016 for −40, 0, and +40 mV, respectively. Membrane depolarization elevated linear correlation coefficient as follows: 0.04 for −40 mV, 0.28 for 0 mV, and 0.51 for +40 mV. Amplitudes of Ca²⁺ sparks and evoked transient KCa currents were significantly correlated at each voltage (P < 0.05 for each).

Table 1.

Regulation of Ca²⁺ spark and transient K_Ca current properties by membrane potential

	−40 mV	0 mV	+40 mV
Ca2+ sparks
Amplitude, All nM	874 ± 89 (65)	1,424 ± 100*(74)	1,121 ± 147 (62)
Coupled	927 ± 123 (45)	1,377 ± 114*(58)	1,187 ± 180 (50)
Uncoupled	755 ± 77 (20)	1283 ± 97*(16)	849 ± 99 (12)
Spread, μm2	3.56 ± 0.35 (17)	3.08 ± 0.39*(17)	3.58 ± 0.30 (17)
Decay (t1/2), ms	61.3 ± 5.3 (17)	52.6 ± 3.0*(17)	59.2 ± 3.6 (17)
Coupling, %	58.6 ± 6.4	76.5 ± 4.2*	86.2 ± 5.1*
Global Ca2+, nM	224 ± 29	363	271
KCa transients
Amplitude, pA	34.7 ± 5.9 (39)	88.4 ± 11.6*(52)	251.5 ± 24.3*(41)
NPo	13.9 ± 2.4 (39)	22.1 ± 2.9*(52)	31.4 ± 3.0*(41)

Values are means±SE of number of events in parentheses. t_1/2, half time; K_Ca, Ca²⁺-activated K⁺; NP_o, number of K_Ca channels activated by Ca²⁺ spark × open probability of K_Ca channels at peak of Ca²⁺ spark.

^*P < 0.05 vs. −40 mV.

Diltiazem or removal of extracellular Ca²⁺ blocks depolarization-induced elevations in transient K_Ca current frequency, but not activity

To investigate the contribution of Ca²⁺ influx to the depolarization-induced increase in Ca²⁺ spark-K_Ca channel coupling, voltage-dependent transient K_Ca current regulation was measured in a Ca²⁺-free bath solution or in the presence of diltiazem (50 μM), a voltage-dependent Ca²⁺ channel blocker.

At −40 mV, removal of extracellular Ca²⁺ reduced transient K_Ca current frequency to 0.41 ± 0.10 of control (P < 0.05) but did not change transient K_Ca current amplitude (0.99 ± 0.05 of control, P > 0.05, n = 5 cells). At −40, 0, and +40 mV, 50 μM diltiazem reduced transient K_Ca current frequency to 0.43 ± 0.06, 0.24 ± 0.03, and 0.46 ± 0.03 of control, respectively (P < 0.05 for each), but did not alter transient K_Ca current amplitude (1.00 ± 0.10, 1.17 ± 0.10, and 1.07 ± 0.05 of control, respectively, P > 0.05 for each, n = 6 cells). More importantly, in the absence of extracellular Ca²⁺ or in the continued presence of diltiazem, steady membrane depolarization between −40 and +40 mV increased mean transient K_Ca current frequency up to 1.6-fold (Fig. 3A). Over the same voltage range, transient K_Ca current activity (NP_o) increased up to approximately fourfold (Fig. 3B). These data indicate that steady membrane depolarization elevates transient K_Ca current frequency and activity in the absence of extracellular Ca²⁺ entry or voltage-dependent Ca²⁺ channel activation.

Fig. 3.

In the absence of Ca²⁺ influx, membrane depolarization elevates transient KCa current frequency and amplitude. In a Ca²⁺-free bath solution (n = 5 cells) or in the continued presence of 50 μM diltiazem (n = 6 cells), steady membrane depolarization elevates transient KCa current frequency (A) and activity (B). Dotted lines indicate control level. *P < 0.05 vs. −40 mV. #P < 0.05 vs. 0 mV.

Caffeine activates Ca²⁺ sparks and transient K_Ca currents

To determine whether RyR channel activation alters Ca²⁺ spark-K_Ca channel coupling in arterial smooth muscle cells, we studied transient K_Ca current regulation by caffeine, an RyR channel activator.

At −40, 0, and +40 mV, 10 μM caffeine increased transient K_Ca current frequency ∼1.5-, 1.6-, and 1.5-fold, respectively (Fig. 4A). In contrast, over the same voltage range, caffeine did not alter transient K_Ca channel activity (NP_o; Fig. 4B). To investigate the effects of caffeine on Ca²⁺ spark properties and Ca²⁺ spark-K_Ca channel coupling, we used simultaneous patch-clamp electrophysiology and confocal Ca²⁺ imaging. Experiments were performed at 0 mV, because caffeine was most effective at activating transient K_Ca currents at this voltage. Caffeine increased mean global Ca²⁺ from ∼363 to 419 nM but reduced mean peak Ca²⁺ spark amplitude from ∼1,956 to ∼1,375 nM (Table 2). Caffeine also increased mean Ca²⁺ spark spatial spread from ∼2.9 to 3.6 μm². Caffeine did not alter Ca²⁺ spark decay, the percentage of Ca²⁺ sparks that activated a transient K_Ca current, the amplitude relation between sparks and transient K_Ca currents, or transient K_Ca channel activity (NP_o; Table 2, Fig. 5). These data indicate that RyR channel activation decreases Ca²⁺ spark amplitude (i.e., the local subsarcolemmal [Ca²⁺]_i activating K_Ca channels) and elevates spatial spread of Ca²⁺ sparks, which would increase the number of K_Ca channels impacted by the spark. The combination of these changes in Ca²⁺ spark properties results in no net change in Ca²⁺ spark-K_Ca channel coupling.

Fig. 4.

Caffeine elevates transient KCa current frequency, but not activity. A: 10 μM caffeine increased transient KCa current frequency at steady membrane potentials of −40, 0, and +40 mV (n = 6 cells). B: caffeine did not alter transient KCa current activity at −40, 0, and +40 mV (n = 6 cells). *P < 0.05 vs. control at the same voltage.

Table 2.

Regulation of Ca²⁺ spark and transient K_Ca current properties by caffeine

	Control	Caffeine (10 μM)
Ca2+ sparks
Amplitude: all, nM	1,915 ± 213 (33)	1,375 ± 81*(42)
Spread, μm2	2.92 ± 0.29 (20)	3.61 ± 0.31*(20)
Decay (t½), ms	54.5 ± 3.9 (16)	50.7 ± 5.7 (16)
Coupling, %	75.4 ± 4.9	75.8 ± 5.1
Global Ca2+, nM	363	419 ± 9*
KCa transient activity (NPo)	15.5 ± 1.4 (25)	15.7 ± 1.0 (32)

Values are means ± SE of number of events in parentheses.

^*P < 0.05 vs. control.

Fig. 5.

Caffeine does not alter Ca²⁺ spark-KCa channel coupling. Caffeine did not alter relation between peak Ca²⁺ spark amplitude and transient KCa current activity (NP_o). Control and caffeine data were obtained from the same 7 cells. Linear regression with 95% confidence bands are illustrated with slopes of 0.009 and 0.011 for control and caffeine, respectively. Correlation coefficients were 0.11 and 0.53 for control and caffeine data, respectively. Amplitudes of Ca²⁺ sparks and evoked transient KCa currents were significantly correlated for control and caffeine (P < 0.0001 for each).

DISCUSSION

The regulation of Ca²⁺ spark-K_Ca channel coupling by mechanisms that activate K_Ca and RyR channels was studied in newborn cerebral artery smooth muscle cells, in which a significant proportion of Ca²⁺ sparks do not activate a transient K_Ca current. Membrane depolarization between −40 and +40 mV increased 1) transient K_Ca current frequency and activity (NP_o), 2) the percentage of Ca²⁺ sparks that activated a transient K_Ca current from 59 to 86%, and 3) the sensitivity of K_Ca channels to Ca²⁺ sparks. Ca²⁺ influx or voltage-dependent Ca²⁺ channel activation was not obligatory for membrane depolarization to elevate transient K_Ca current frequency and activity. In contrast, RyR channel activation elevated transient K_Ca current frequency solely by causing an increase in Ca²⁺ spark frequency. RyR channel activation did not change Ca²⁺ spark-K_Ca channel coupling or transient K_Ca current activity. These data indicate that K_Ca channel Ca²⁺ sensitivity, rather than RyR channel activity, is a principal factor that underlies fractional Ca²⁺ spark coupling in newborn cerebral artery smooth muscle cells.

Membrane depolarization between −40 and 0 mV increased global [Ca²⁺]_i, Ca²⁺ spark amplitude, K_Ca channel sensitivity to Ca²⁺ sparks, and the percentage of Ca²⁺ sparks that activated a transient K_Ca current. Further depolarization to +40 mV decreased Ca²⁺ spark amplitude and reduced global [Ca²⁺]_i, which was expected because of a reduction in driving force for Ca²⁺ influx. However, depolarization from 0 to +40 mV further increased the percentage of Ca²⁺ sparks that activated a transient K_Ca current and elevated K_Ca channel sensitivity to Ca²⁺ sparks. These data suggest that, in newborn arterial smooth muscle cells, effective coupling and percent coupling of Ca²⁺ sparks to K_Ca channels are modulated primarily by K_Ca channel sensitivity to Ca²⁺ sparks, rather than by RyR channel activity. An explanation for these findings is that membrane depolarization increases K_Ca channel apparent Ca²⁺ sensitivity, which would increase the impact of sparks on K_Ca channel P_o (4, 12, 20). The depolarization-induced elevation in transient K_Ca current frequency most likely occurs through an increase in the percentage of Ca²⁺ sparks that activate K_Ca channels. In support of this conclusion, in the presence of diltiazem or in the absence of extracellular Ca²⁺, both of which would block depolarization-induced Ca²⁺ spark activation (13, 17), depolarization elevated transient K_Ca current frequency and activity. In murine colonic myocytes, a reduction in extracellular Ca²⁺ reduced local intracellular Ca²⁺ transients but elevated transient K_Ca current frequency and amplitude by removing protein kinase C-mediated K_Ca channel inhibition (2). In contrast, in the present study, removal of extracellular Ca²⁺ or diltiazem reduced transient K_Ca current frequency but did not alter amplitude. These data suggest Ca²⁺ sparks are activated by Ca²⁺ influx through voltage-dependent Ca²⁺ channels, as previously reported (13, 17), and illustrate differences in the mechanisms by which Ca²⁺ spark-K_Ca channel coupling is modulated by Ca²⁺ influx pathways in colonic and arterial smooth muscle cells.

K_Ca channel “Ca²⁺ sensitivity” has previously been used to describe 1) the Ca²⁺ concentration that induces half-maximal activation at a given voltage, 2) the slope of the Ca²⁺-activity relation at a defined voltage, and 3) a shift in half-maximal potential for a given Ca²⁺ concentration change (4). Depolarization shifts the Ca²⁺ concentration-K_Ca channel activity relation leftward (4) and increases the percentage of Ca²⁺ sparks that activate K_Ca channels. The present data dispute the possibility that uncoupling occurs because K_Ca channels within the vicinity of Ca²⁺ spark sites are absent or incapable of activation. The K_d for Ca²⁺ of newborn porcine arteriole smooth muscle cell K_Ca channels is 31 μM at 0 mV, which is high compared with that of K_Ca channels in other smooth muscle cell preparations, including human coronary artery and rat cerebral artery (22, 26, 30). Conceivably, uncoupling may occur because K_Ca channel Ca²⁺ sensitivity is lower in uncoupled than in strongly coupled cell types. Other likely explanations are that uncoupled Ca²⁺ sparks are of lower amplitude (present study and Ref. 14) and/or the distance between uncoupled spark release sites and the sarcolemma is greater, both of which would result in lower spark-induced subsarcolemmal Ca²⁺ elevations. In B. marinus stomach smooth muscle cells, some Ca²⁺ spark sites generate sparks that reliably activate transient K_Ca currents, whereas other locations consistently generate uncoupled sparks (31). In the amphibian preparation, sites that generate uncoupled Ca²⁺ sparks may be located near sarcolemma that is devoid of K_Ca channels or populated by inactivatable K_Ca channels (31). However, in newborn cerebral artery smooth muscle cells, Ca²⁺ spark-K_Ca channel coupling is increased by membrane depolarization and carbon monoxide, which elevate K_Ca channel apparent Ca²⁺ sensitivity (14, 15, 30). Similarly, in guinea pig bladder smooth muscle cells, membrane depolarization between −50 and −20 mV elevated Ca²⁺ spark coupling (11). Thus K_Ca channel localization near Ca²⁺ spark sites and regulation by Ca²⁺ sparks appear to differ in mammalian and amphibian smooth muscle cells.

Regardless of voltage, caffeine, which elevates RyR channel Ca²⁺ sensitivity (24), induced a similar relative increase in transient K_Ca current frequency but did not change transient K_Ca channel activity. These data suggest that caffeine activates transient K_Ca currents by elevating Ca²⁺ spark frequency. Caffeine also elevated global [Ca²⁺]_i and reduced Ca²⁺ spark amplitude, presumably by causing SR Ca²⁺ leak and a reduction in SR Ca²⁺ load, respectively (6). Caffeine also increased Ca²⁺ spark spread, presumably by elevating the number of RyR channels that contribute to sparks through localized Ca²⁺-induced Ca²⁺ release. In pulmonary artery smooth muscle cells, 500 μM caffeine did not change Ca²⁺ spark amplitude (calculated as F/F₀) but elevated Ca²⁺ spark frequency, duration, and spread (25). In B. marinus stomach smooth muscle cells, caffeine increased the number of spark sites from ∼42 to 400 (31). Conceivably, caffeine may have also generated Ca²⁺ sparks at additional sites in newborn cerebral artery smooth muscle cells. However, the low Ca²⁺ spark frequency in newborn arterial smooth muscle cells and the 10-s time limit required for imaging to avoid laser-induced cell damage precluded systematic examination of this possibility. Nevertheless, the net effect of Ca²⁺ spark spatial and temporal changes was no net change in the mean percentage or effective Ca²⁺ spark-K_Ca channel coupling. Thus, in newborn porcine cerebral artery smooth muscle cells, RyR channel activation elevates transient K_Ca current frequency by elevating Ca²⁺ spark frequency, and not by altering Ca²⁺ spark-K_Ca channel coupling.

Caffeine, at low micromolar concentrations, induces a K_Ca channel-sensitive vasodilation in pressurized newborn cerebral arteries (1). Carbon monoxide increases K_Ca channel apparent Ca²⁺ sensitivity and Ca²⁺ spark-K_Ca channel coupling in smooth muscle cells and dilates newborn porcine cerebral arteries (14, 15, 30). These findings show that an elevation in Ca²⁺ spark frequency alone or an increase in Ca²⁺ spark-K_Ca channel coupling induces vasodilation through K_Ca channel activation. In the present study, membrane depolarization within the physiological range [i.e., ca. −60 to −20 mV (19)] would increase Ca²⁺ spark-Ca²⁺ channel coupling by only ∼10–15%. The increase in coupling alone would be predicted to have only a small effect on membrane potential. However, the combination of an increase in coupling and depolarization-induced transient K_Ca current frequency and amplitude elevation would increase K⁺ current through K_Ca channels, produce membrane hyperpolarization, and oppose pressure-induced constriction (16). Within the physiological range of voltages, Ca²⁺ spark-K_Ca channel coupling in newborn myocytes does not reach 100%, allowing additional mechanisms that enhance K_Ca channel Ca²⁺ sensitivity to augment coupling and further enhance K_Ca channel activity [e.g., carbon monoxide (14)]. As such, signaling elements that increase K_Ca channel Ca²⁺ sensitivity will be more effective vasodilators in myocytes that exhibit fractional coupling than in cells with 100% coupling. Furthermore, messengers that elevate Ca²⁺ spark frequency and coupling to K_Ca channels, including reactive oxygen species (7, 29) and carbon monoxide (14, 15), should produce the most significant K_Ca channel-dependent vasodilation.

In summary, the present data indicate that, in newborn porcine cerebral artery smooth muscle cells, fractional Ca²⁺ spark coupling occurs through K_Ca channel insensitivity to Ca²⁺ sparks. Uncoupled Ca²⁺ sparks can be coupled by mechanisms that elevate K_Ca channel Ca²⁺ sensitivity. In contrast, RyR channel activation alone reduces Ca²⁺ spark amplitude and increases Ca²⁺ spark spread, resulting in no net change in Ca²⁺ spark coupling.