INTRODUCTION

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NUMEROUS STUDIES have documented a heterogeneity of responses to both physiological and pharmacological stimuli along the coronary vascular network (see Ref. 26). In a recent review, Jones et al. (21) proposed the concept of vascular microdomains as a means of distinguishing different portions of the coronary microcirculation by functional rather than anatomic attributes. Thus, within each microdomain, vascular tone appears governed by one predominant regulatory mechanism (21). Functional microdomains are often distinguished by vessel diameter. Heterogeneous vascular responses based on vessel diameter have been noted to increase myocardial work (24), -adrenergic stimulation (8), serotonin (27), adenosine (15, 23, 26), endothelium-dependent agonists (26), nitric oxide (23), flow-induced vasodilation (26), and myogenic tone (26). The cellular basis for this heterogeneity is poorly understood; however, a differential distribution of smooth muscle ion channels (13, 30, 36) and pumps (31) has been noted in vessels of different sizes within the same vascular tree, providing evidence that heterogeneity of ion channel distribution may contribute to the heterogeneity of vasoactive responses.

Numerous studies have demonstrated the importance of L-type Ca²⁺ channel activation in the maintenance of arterial tone (for review see Ref. 33). Vasoconstrictor agonists, including serotonin, endothelin, and norepinephrine, increase arterial tone directly and/or indirectly through activation of L-type Ca²⁺ channels (3, 17, 18, 35). Conversely, many vasodilators, such as adenosine (10), decrease arterial tone, in part, through activation of K⁺ channels, which hyperpolarize the cell membrane and inactivate voltage-gated Ca²⁺ channels (33). In addition, L-type Ca²⁺ channels contribute significantly to development of myogenic tone (21, 44). Numerous studies have shown that, within a vascular network, an inverse relationship exists between artery size and myogenic responsiveness (9, 11, 26). Within the coronary vasculature, Kuo et al. (26) demonstrated a heterogeneous development of myogenic tone in pressurized microvessels, i.e., myogenic tone was less in small arteries (140-280 µm) compared with large (80-130 µm), intermediate (50-70 µm), and small (25-45 µm) arterioles. Although incompletely understood, the underlying mechanisms producing myogenic tone in vascular smooth muscle appear to be initiated by the mechanical stimulus of stretch, by increasing either intravascular pressure or length (29). Mechanical stretch or increased intravascular pressure depolarizes smooth muscle, likely through activation of a nonselective stretch-activated cation channel (12, 29). As currently modeled (29, 34), this depolarization activates dihydropyridine-sensitive, voltage-gated (L-type) Ca²⁺ channels allowing Ca²⁺ influx, in addition to Ca²⁺ entry directly through stretch-activated channels, thus producing smooth muscle contraction.

The above studies demonstrate that both vasoconstrictor and vasodilator agonists can influence arterial tone via voltage-gated Ca²⁺ channel regulation. Recently, an increased Ca²⁺ channel density was found in small cerebral arteries compared with larger basilar arteries (30) and in resistance versus conduit arteries in the pulmonary vasculature (13), providing indirect evidence for a role of differential Ca²⁺ channel density in the heterogeneity of arterial tone regulation. However, despite the potentially crucial role of voltage-gated Ca²⁺ channels in coronary autoregulation, no studies to date have examined the distribution of Ca²⁺ current density within the coronary arterial vasculature. The purpose of the present study was to test the hypothesis that smooth muscle voltage-gated Ca²⁺ channel density would be heterogeneously distributed within the coronary arterial circulation. Furthermore, we hypothesized an inverse relationship between Ca²⁺ current density and arterial diameter similar to the reported inverse relationship between myogenic tone and coronary arterial diameter. Using whole cell voltage clamp, we compared Ca²⁺ current densities in coronary smooth muscle from conduit arteries (>1.0 mm), small arteries (200-250 µm), and large arterioles (75-125 µm). In support of our hypothesis, we found that smooth muscle L-type Ca²⁺ current density was inversely related to arterial diameter within the coronary circulation.

	MATERIALS AND METHODS

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Animals. Adult female miniature swine weighing 25-40 kg were obtained from the breeder (Charles River) and housed in pens at the College of Veterinary Medicine until use. Animal protocols were approved by the University of Missouri Animal Care and Use Committee in accordance with the "U. S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training."

Preparation of coronary arteries. Pigs were anesthetized with ketamine (30 mg/kg) and pentobarbital sodium (35 mg/kg), and heparin was administered. The hearts were removed and placed in iced (4°C) Krebs bicarbonate solution during vessel isolation. Conduit (>1.0 mm ID) segments of right coronary artery were trimmed of fat and connective tissue in sterile modified Eagle's minimal essential storage medium containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; EH) plus 2% horse serum on ice. Small arteries (200-250 µm ID) and large arterioles (75-125 µm ID) were dissected free from the subepicardial wall. Small arteries and large arterioles between branch points were selected. All arteries were stored under sterile conditions in EH at 4°C until use (0-1 day).

Cell dispersion. All experiments were performed on freshly dispersed cells using methods modified from those described previously (38-40, 42, 43). Small arteries and large arterioles were incubated in 200 µl of enzyme solution consisting of low-Ca²⁺ (0.5 mM) physiological solution plus 294 U/ml collagenase (CLS II, Worthington), 6.5 U/ml elastase (Worthington), 2 mg/ml bovine serum albumin (fraction V, Sigma), 1 mg/ml soybean trypsin inhibitor (type I-S, Sigma), and 0.4 mg/ml deoxyribonuclease I (type IV, Sigma). Cells were enzymatically dispersed by incubation for 45-60 min in a shaking water bath at 37°C. Immediately after incubation, the enzyme solution was replaced with enzyme-free low-Ca²⁺ solution and isolated single cells were obtained with gentle trituration by micropipette. For conduit coronary arteries, single cells were dispersed according to previously published techniques (38-40, 42, 43). Briefly, coronary arteries were opened longitudinally and pinned, lumen side up, in ~2 ml of low-Ca²⁺ enzyme solution. Cells were enzymatically dispersed for 45-60 min in a shaking water bath at 37°C. Enzyme solution was replaced with enzyme-free low-Ca²⁺ solution, and isolated single cells were obtained by repeatedly directing a stream of low-Ca²⁺ solution over the artery by Pasteur pipette. All solutions used for conduit and resistance vessels were identical. Cell suspensions were stored in low-Ca²⁺ (0.5 mM) buffer at 4°C until use (0-6 h).

Whole cell voltage clamp. Whole cell currents were determined using a standard whole cell voltage-clamp technique (37) as used routinely (39-42). Cells were initially superfused with physiological saline solution (PSS) containing (in mM) 2 CaCl₂, 138 NaCl, 1 MgCl₂, 5 KCl, 10 HEPES, and 10 glucose, pH 7.4, during gigaseal formation. After whole cell configuration, the superfusate was switched to PSS with tetraethylammonium chloride (TEACl) substituted for NaCl and either 2 mM Ca²⁺ or 10 mM Ba²⁺ as the charge carrier. The pipette solution contained (in mM), 120 CsCl, 10 TEACl, 1 MgCl₂, 20 HEPES, 2 MgATP, 0.5 tris(hydroxymethyl)aminomethane · GTP, 5 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.2 fura 2 (pentapotassium salt, pH 7.1). Ionic currents were amplified by a Warner PC-501 patch-clamp amplifier with a 10-G headstage. Whole cell currents were filtered through an eight-pole low-pass filter with a cutoff frequency of 400 Hz, digitized at 600-µs intervals, and stored and analyzed on computer with customized AxoBASIC 1.0 software (Axon Instruments). Current densities (pA/pF) were obtained for each cell by normalization of whole cell current to cell capacitance. Capacity currents were measured for each cell during 10-ms pulses from a holding potential of 80 mV to a test potential of 70 mV. Capacity currents were filtered at a low-pass cut-off frequency of 8.4 kHz and digitized at 25-µs intervals. Leak subtraction was not performed. Data acquisition and analysis were accomplished using a Labmaster analog-to-digital converter and microcomputer equipped with AxoBASIC 1.0 data acquisition software (Axon Instruments). All experiments were conducted at room temperature (22-25°C). Cells were continually superfused under gravity flow. Stock solutions of nifedipine were dissolved in 100% ethanol and diluted 1,000-fold for final solutions.

Statistics. Data are expressed as means ± SE, with each cell counting as one observation (n). Within each experiment, cells were obtained from more than one animal. Analysis of variance was used to compare current-voltage (I-V) relationships in cells from conduit arteries, small arteries, and large arterioles, with unpaired t-test used for post hoc analysis. A P value <0.05 was set as the criterion for significance in all comparisons.

	RESULTS

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Cell volume and arterial diameter. Table 1 compares surface area and series resistance in smooth muscle cells isolated from conduit arteries, small arteries, and large arterioles. Overall, mean cell surface area, measured as whole cell membrane capacitance, decreased as arterial diameter decreased. Series resistance during voltage clamp was similar between all groups. To account for differences in cell membrane surface area, Ca²⁺ current data were normalized to capacitance for comparisons between groups.

Heterogeneous distribution of Ca²⁺ current density. Representative families of current traces for voltage-gated Ca²⁺ current in smooth muscle cells from conduit arteries, small arteries, and large arterioles are shown in Fig. 1. Figure 2 compares the I-V relationship for voltage-gated Ca²⁺ current in smooth muscle cells from conduit arteries, small arteries, and large arterioles with either 2 mM external Ca²⁺ or 10 mM Ba²⁺ as the charge carrier. Voltage-gated Ca²⁺ current density was inversely related to arterial diameter, i.e., large arterioles > small arteries > conduit arteries. With Ca²⁺ as the charge carrier (Fig. 2A), peak inward current at 0 mV was increased approximately sixfold in large arterioles and approximately threefold in small arteries compared with cells from conduit arteries. The arterial size dependence of whole cell Ca²⁺ current became apparent at 40 mV in physiological Ca²⁺ concentrations (Fig. 2A), with inward current being significantly greater in small arteries compared with conduit arteries at this membrane potential. Although Ca²⁺ current in large arterioles was not significantly different at 40 mV, this appeared to be the result of an unexplained residual outward current at this potential in this group. At membrane potentials of 30 mV to +20 mV, the inverse relationship of Ca²⁺ current density and arterial size was significant for all comparisons, i.e., large arterioles > small arteries > conduit arteries. With Ba²⁺ as the charge carrier (Fig. 2B), an identical inverse relationship of arterial diameter and inward current was observed at membrane potentials of 20 to +30 mV, consistent with a positive shift of the voltage-gated Ca²⁺ current using Ba²⁺ as the external charge carrier. In all arterial sizes, peak inward current occurred at 0 mV with Ca²⁺ and +10 mV with Ba²⁺ as the external charge carrier, indicating an ~10-mV positive shift in the I-V relationship. This positive shift produced by Ba²⁺ is characteristic of L-type Ca²⁺ channels (1), supporting previous studies showing a predominance of L-type Ca²⁺ current in vascular smooth muscle (2, 5, 19, 28, 30).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Voltage-gated Ca2+ channel (VGCC) current in coronary arterial smooth muscle. Representative current families for inward VGCC current in cells from conduit (A) and small (B) arteries and large arterioles (C). Currents were elicited by step depolarizations to 10, 0, and +10 mV from a holding potential of 80 mV using 10 mM Ba2+ as external charge carrier. Membrane capacitance in these cells was 26, 20, and 16 pF for conduit artery, small artery, and arteriole, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Heterogeneity of VGCC density. Current-voltage (I-V) relationships for whole cell VGCC current in smooth muscle cells from conduit arteries, small arteries, and large arterioles are shown with 2 mM Ca2+ (ICa, A) or 10 mM Ba2+ (IBa, B) as external charge carrier. Currents are plotted as peak inward current measured during a 330-ms step depolarization to membrane potential (Vm) indicated from holding potential of 80 mV. Currents are normalized to cell membrane capacitance (pA/pF). VGCC current density was inversely related to arterial diameter, i.e., large arterioles > small arteries > large arterioles. In presence of Ba2+, VGCC current showed a 10-mV positive shift in I-V relationship compared with VGCC current in presence of Ca2+, which was similar in all arterial groups. Data are means ± SE; n values are as reported in Table 1 (B) or 34, 29, and 15 cells for conduit artery, small artery, and large arteriole, respectively, from same animals (A). * P < 0.05, arteriole > small artery > conduit; # P < 0.05, arteriole > conduit;  P < 0.05, small artery > conduit; + P < 0.05 vs. small artery and conduit.

Dihydropyridine sensitivity of Ca²⁺ current. L-type Ca²⁺ channels are highly sensitive to block by dihydropyridines such as nifedipine (2). Figure 3 shows the effect of nifedipine on voltage-gated Ca²⁺ current in conduit arteries, small arteries, and large arterioles. In smooth muscle cells from all arterial diameters, nifedipine completely blocked inward current. Peak inward current at +10 mV was 1.70 ± 0.32 vs. 0.01 ± 0.04 pA/pF in conduit arteries (n = 4), 3.38 ± 0.39 vs. 0.25 ± 0.18 pA/pF in small arteries (n = 4), and 6.30 ± 1.11 vs. 0.35 ± 0.08 pA/pF in large arterioles (n = 4), before and after nifedipine, respectively. Conversely, the dihydropyridine-sensitive Ca²⁺ channel activator BAY K 8644 (200 nM) produced a similar ~350% increase in peak inward current in cells from each arterial size, i.e., 344 ± 22, 359 ± 28, and 347 ± 19% for conduit artery (n = 7), small artery (n = 8), and large arteriole (n = 16), respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   VGCC current inhibition by nifedipine. Typical experimental tracings showing block of inward Ba2+ current by the dihydropyridine nifedipine (3 µm). Step depolarizations to +10 mV from holding potential of 80 mV before (control) and after (+nifedipine) nifedipine are shown for cells from conduit arteries (A), small arteries (B), and large arterioles (C). In presence of nifedipine, peak inward current at +10 mV was reduced 114, 100, and 95%, for conduit arteries, small arteries, and large arterioles (see text for group data), respectively, demonstrating predominance of dihydropyridine-sensitive Ca2+ channel current (L-type) in coronary smooth muscle in each arterial group. Similar inhibition was seen at all membrane potentials.

Voltage dependence of Ca²⁺ current activation and inactivation. The voltage dependence of activation and steady-state inactivation for whole cell Ca²⁺ current is shown in Fig. 4. Voltage-gated inactivation is shown by the decrease in normalized Ca²⁺ current with increasing steady-state membrane potential. The membrane potential at which Ca²⁺ current decreased to one-half was approximately 34 mV and was similar in smooth muscle cells from each arterial group. This value is similar to that reported in rabbit coronary smooth muscle (28). In contrast, the membrane potential producing half-maximal activation occurred at a more negative potential in cells from large arterioles compared with small and conduit arteries (13.23 ± 0.88, 6.22 ± 1.35, and 8.62 ± 0.81 mV, respectively; P < 0.05). Thus, in addition to an increased whole cell Ca²⁺ current density (Fig. 2), Ca²⁺ current in smooth muscle cells of large arterioles activates at a more negative membrane potential compared with cells from conduit and small arteries. To further test the possibility that differences in Ca²⁺ channel subtypes underlie the differences observed between arterial diameters, we examined the effect of steady-state depolarization on the I-V relationship. Two types of voltage-gated Ca²⁺ channels found in smooth muscle can be separated by holding potential (2, 19, 41). Compared with L-type channels, T-type channels activate and peak at more negative membrane potentials and are inactivated by steady holding potentials less than 40 mV (2, 41). Thus the presence of two types of voltage-gated Ca²⁺ channels can be discerned by a positive shift in the I-V relationship when the holding potential is increased (41). Figure 5 shows the effect of a steady-state decrease in holding potential on the I-V relationship in conduit arteries, small arteries, and large arterioles. Prolonged depolarization significantly reduced inward current in all groups, consistent with voltage-gated inactivation; however, there was no shift in the I-V relationship compared with that obtained with a more negative holding potential (80 mV; Fig. 2B), confirming that the inward Ca²⁺ current in all arteries is predominantly L-type Ca²⁺ current.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   VGCC current activation and inactivation. Normalized I-V relationships for whole cell Ca2+ current in smooth muscle cells from conduit arteries, small arteries, and large arterioles are shown using 10 mM Ba2+ as external charge carrier. Voltage-gated inactivation was determined by measuring peak inward current during a 330-ms depolarization to +10 mV after a 4-s prepulse to varying Vm indicated. Voltage-gated activation was determined by normalizing data from Fig. 1B to peak inward current for each cell. Curves were fit by a conventional Boltzmann distribution equation, I/Imax = 1/[1 + exp(V  V0.5)/k], where I represents current, Imax is maximal current, V0.5 is Vm producing half-maximal inactivation or activation, and k is slope factor. Inactivation curves were similar in all arterial groups. However, V0.5 for activation in cells from large arterioles showed a significant negative shift compared with cells from small or conduit arteries [13.23 ± 0.88, 6.22 ± 1.35, and 8.62 ± 0.81 mV for large arterioles (n = 14), small arteries (n = 10), and conduit arteries (n = 11), respectively; P < 0.05]. Values are means ± SE; n values for inactivation curves were 16 (large arterioles), 14 (small arteries), and 16 (conduit arteries) cells. Data were obtained from 4 to 5 animals.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of sustained depolarization on inward Ca2+ current. I-V relationships for whole cell VGCC current in smooth muscle cells from conduit arteries (n = 12), small arteries (n = 11), and large arterioles (n = 11) are shown with 10 mM Ba2+ as external charge carrier. Current is plotted as peak inward current measured during a 330-ms step depolarization to Vm indicated from holding potential of 30 mV. VGCC current is normalized to cell membrane capacitance (pA/pF). I-V relationships for all arteries were significantly reduced compared with those obtained with holding potential of 80 mV (Fig. 1B). However, threshold of activation and Vm at peak inward current were unchanged, consistent with a predominant L-type VGCC current in all arteries. Data were obtained from 4 to 5 animals.

	DISCUSSION

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Despite numerous studies demonstrating a functional heterogeneity within the coronary circulation (20-23, 26), the cellular basis for this differential responsiveness is poorly understood. The present study provides novel information regarding the heterogeneous distribution of Ca²⁺ current density within the coronary circulation. An overall pattern demonstrating an inverse relationship between smooth muscle Ca²⁺ current density and arterial diameter was found. Based on the well-documented importance of voltage-gated Ca²⁺ channels in regulation of vascular tone (29, 33), the direct electrophysiological findings of the present study provide a foundation for better understanding the cellular mechanisms responsible for functional heterogeneity within the coronary circulation.

Coronary vessels respond to physiological and pharmacological stimuli in a heterogeneous, size-dependent manner (8, 23, 26). In a recent review, Jones et al. (21) provided a microdomain scheme to categorize the size-dependent heterogeneity of the coronary circulation. In general, as arterial diameter decreases, responsiveness to pressure (myogenic tone) and vasoactive metabolites increases, whereas sensitivity to flow-induced dilation is greatest in large arterioles. Although the cellular mechanisms responsible for this functional heterogeneity are unclear, recent evidence suggests that a heterogeneous distribution of ion channels and/or pumps in smooth muscle may contribute. Quayle et al. (36) recently demonstrated a heterogeneous distribution of inward rectifier potassium (K_ir) channels in the coronary circulation. They reported a K_ir current density more than twofold greater in smooth muscle cells from fourth-order branches (93-290 µm) of the anterior descending artery compared with cells from the main artery (1.8-2.5 mm). Similarly, voltage-gated Ca²⁺ channel density has been reported to be greater in precapillary arterioles of the cerebral circulation compared with basilar arterial smooth muscle (30). In the pulmonary circulation, Ca²⁺ current densities approximately twofold greater in resistance compared with conduit arteries were reported in the rabbit (13). A similar longitudinal distribution of Ca²⁺ current density was found in the present study of the coronary circulation. With Ba²⁺ as a charge carrier, peak inward currents were ~2.5- and ~1.5-fold greater in large arteriole and small artery, respectively, compared with conduit smooth muscle cells. In physiological Ca²⁺ concentrations (2 mM), peak inward Ca²⁺ current density was approximately sixfold greater in large arterioles and approximately threefold greater in small arteries compared with cells from conduit arteries. Increased inward Ca²⁺ current in the smaller arteries was also resolved at 40 mV, well within the range of physiological membrane potentials of arterial smooth muscle (33). It is also important to note that apparent thresholds for Ca²⁺ current activation in whole cell voltage clamp are really detection thresholds and that voltage-gated Ca²⁺ channel activity is a continuous function of membrane potential with no threshold (16). Therefore, it is highly probable that the size-dependent differences in whole cell Ca²⁺ current extend to more negative membrane potentials. In addition to an increased channel density, Ca²⁺ channels in cerebral arterioles activate at more negative membrane potentials than larger basilar arterial cells (30). We report a similar finding in that the membrane potential for half-maximal activation of Ca²⁺ current in cells from large arterioles was significantly more negative than that in small and conduit arteries. This may serve to further enhance voltage-gated Ca²⁺ influx in large arterioles compared with larger arteries.

Smooth muscle contains two distinct types of voltage-gated Ca²⁺ current (2, 5, 19, 41). T-type channels are activated by small depolarizations and inactivate quickly, whereas L-type channels require greater depolarization for activation and inactivate slowly (5). The channel type responsible for whole cell currents can be distinguished by several characteristics: T-type channels are approximately equally permeable to Ba²⁺ and Ca²⁺, whereas the L-type channel has a greater permeability to Ba²⁺, and L-type channels are highly sensitive to dihydropyridines, whereas T-type channels are insensitive to this class of drugs (16). In coronary smooth muscle, L-type Ca²⁺ current has half-maximal activation and inactivation at 4.4 mV and 27.9 mV, respectively (28). The Ca²⁺ channels responsible for whole cell currents in the present study possessed similar half-maximal activation and inactivation values and a similar peak I-V relationship at holding potentials of 30 and 80 mV and were completely abolished by the dihydropyridine nifedipine, all of which are strongly indicative of L-type Ca²⁺ channels being the predominant channel type in all arterial sizes of the present study. This conclusion is in agreement with other studies showing a predominance of L-type Ca²⁺ channels in vascular smooth muscle (19, 28, 30).

Various vasoactive stimuli, including norepinephrine (32), serotonin (14), endothelin (17), and pressure (25, 29), all depolarize arterial smooth muscle and activate, directly or indirectly, voltage-gated Ca²⁺ channels as a mechanism for vasoconstriction. In addition, vasodilators such as endothelium-derived hyperpolarizing factor (6), nitric oxide (4), and adenosine (10) have been shown to activate, directly or indirectly, K⁺ channels to hyperpolarize arterial smooth muscle, inactivating Ca²⁺ channels and resulting in vasodilation. Thus a heterogeneous distribution of voltage-gated Ca²⁺ channels could contribute to the increased sensitivity of smaller arteries to both vasodilators and vasoconstrictors.

In conclusion, the present study demonstrates a heterogeneous distribution of L-type Ca²⁺ current density in the coronary circulation. In arterial smooth muscle, Ca²⁺ current density appears to be distributed longitudinally in an inverse relationship to arterial size, i.e., large arterioles > small arteries > conduit arteries. As recently emphasized in a National Heart, Lung, and Blood Institute (NHLBI) microcirculation workshop (7), understanding the mechanisms responsible for heterogeneity within the microcirculation is imperative for complete comprehension of coronary microvascular structure and function in health and disease. The present finding of a differential distribution of L-type Ca²⁺ channels may provide one such cellular mechanism underlying the heterogeneous control of blood flow within the coronary circulation.