In myocytes, L-type Ca2+ channels and the sarcoplasmic reticulum (SR) are closely linked in terms of structure and function. The SR may buffer influxed Ca2+(1) and may participate in Ca2+ extrusion to avoid an excess increase in cytosolic Ca2+ in arterial myocytes (2-4). In our previous study (5), ryanodine increased the tone of skeletal muscle small arteries with increasing cytosolic Ca2+, but not that of mesenteric small arteries. The myogenic tone also was strong in skeletal muscle small arteries, but weak in mesenteric small arteries (6). Because the myogenic activity required extracellular Ca2+(5,7), was abolished by dihydropyridine Ca2+ channel blockers, (5,8), and was accompanied by depolarization of the membrane potential (9), we presumed that there are some differences in the Ca2+ metabolism between these two small arteries that may be related to the ryanodine-induced contraction as well as the myogenic activity.
Several mechanisms have been proposed concerning the role of the SR in the regulation of intracellular Ca2+, including the capacitative Ca2+ entry theory (10), the superficial buffer barrier theory (1-3), and the calcium influx factor (11). The purpose of the present study was to elucidate how the SR functions in cellular Ca2+ metabolism in arterial myocytes using skeletal muscle small arteries where the ryanodine-induced contraction is strong and in mesenteric small arteries where the contraction is weak. The SR may participate in the regulation of the tone in skeletal muscle small arteries (5,12), but not in mesenteric small arteries. We hypothesized that the activity of voltage-dependent Ca2+ channels causes the difference. To test the hypothesis, we examined the following issues: (a) The membrane potential of the smooth muscle cells may be different between these small arteries under identical transmural pressure; (b) Increased Ca2+ channel activity may enhance the ryanodine-induced contraction in the mesenteric small arteries; (c) Furthermore, to elucidate the mechanism of the ryanodine-induced contraction, the vasodilatory effects of a Ca2+ channel blocker on ryanodine-induced constriction were examined.
Vessel isolation and diameter measurements
This experiment was approved by the Institutional Committee for Animal Experiments. The methods for isolation and diameter measurement have been described elsewhere (13). In brief, Wistar rats (6-9 weeks old) were anesthetized with ether and killed by head blow. The small arteries (3-5 mm in length) were carefully dissected from the branches of the femoral artery and the mesenteric artery. The branches for the gracilis muscle and the second-order branch of the mesenteric artery were used. Both ends of the small artery were cannulated with polyethylene or glass microtubes (tip inner diameter, ∼80 μm) in a tissue bath. The chamber was mounted on a transillumination system or an inverted microscope, and the lumen diameter was measured with a video-monitored microscopic system (SMZ-2T, Nikon Corporation, Tokyo, Japan; Model KP-115, Hitachi Denshi Ltd., Tokyo, Japan). The vessel was pressurized from one end, and the lumen pressure was measured from the side-arms of both ends. Small branches were tied down with fine silk strands (∼7 μm), when necessary. That no leaks were present was confirmed through the absence of a pressure difference between the two ends.
The video signal from the CCD camera was digitalized on a video memory (512 × 512 pixels, eight-bit depth) installed in a personal computer (PC-9801; Nihon Electric Company, Tokyo, Japan). The diameter was measured automatically every 15 s. Additionally, the steady-state data were obtained manually (i.e., they were measured on a CRT with a caliper manipulated by a pointing device). The calibration of the measurement was performed using an 80-μm tungsten wire. The minimal resolution of the system including the optics and the video-monitor system was 1.5 μm.
Membrane potential measurements
The membrane potential of the vascular smooth muscle cell was measured using a conventional glass microelectrode filled with 3 M KCl. The tip resistance of the micropipette was usually ≥40 MΩ. The vessel was cannulated at both ends with glass micropipettes. Perivascular connective tissue was carefully removed with forceps under a vital microscope. At least 5 min after the transmural pressure changes, the microelectrode was vertically advanced to the vessel. We accepted data when the following conditions were satisfied. The measured values of the membrane potential showed sharp changes when the pipette was withdrawn. No zero-shift (<3 mV) and no change in tip resistance were found between before and after the measurement. In our setup, because it was impossible to measure the membrane potential continuously over the pressure changes, we obtained the data in steady state (≥5 min after the intervention) and repeated for three successful measurements in nearly identical areas of the vessels. We adopted the middle value for use as data.
All vessels were incubated in normal physiologic salt solution (PSS) at 37 ± 1°C for ≥1 h before the experimental measurements, with the transmural pressure maintained at 40 mm Hg. At the end of the experiment, the vessels were maximally dilated by 10 μM nisoldipine. Additional papaverine did not cause further dilation.
The membrane potentials of the skeletal muscle and mesenteric small arteries were measured. The transmural pressure varied from 20 to 100 mm Hg in a stepwise fashion. The order of the pressure changes was randomized. The steady-state lumen diameter was also measured.
1. Using mesenteric small arteries, the effects of the Ca2+ channel agonist Bay K 8644 on ryanodine-induced contraction were examined while maintaining the transmural pressure at 40 mm Hg. Two series of experiments were performed. In series I, Bay K 8644 was cumulatively applied from 1 nM to 1 μM, and then, ryanodine (1 μM) was added. In series II, the vessels were pretreated with ryanodine (1 μM, ≥15 min before), and Bay K 8644 was applied in the same manner. Before these agents, 40 mM KCl-induced contraction was tested as control. The pharmacologic interventions were advanced with ≥5-min intervals to confirm the steady state. In some cases, KCl-induced contraction was tested before and after ryanodine (1 μM).
2. Using skeletal muscle small arteries, we tested the effects of the L-type Ca2+ channel blocker, nisoldipine, on the ryanodine- and phenylephrine-induced constriction and on the myogenic tone. At first, all vessels were challenged with 40 mM KCl. Ryanodine (1 μM) and phenylephrine (1 μM)-induced contraction was tested at a transmural pressure of 40 mm Hg. To achieve high myogenic vascular tone, the transmural pressure was increased from 40 to 100 mm Hg. In each experimental run, nisoldipine was cumulatively applied from 0.1 nM to 10 μM. The pharmacologic intervention was advanced after confirming the steady state (usually >5 min).
Reagents and solutions
The ionic composition of the PSS was NaCl, 143; KCl, 4.7; NaHPO4, 1.18; MgSO4-7H2O, 1.17; CaCl2, 1.6; glucose, 11; and N-2-hydroxy-ethylpiperazine-N-2-ethylsulfonic acid (HEPES), 5 mM. The PSS was bubbled with 100% O2 and maintained at 37 ± 1°C, and the pH was adjusted to 7.4 with NaOH. Bay K 8644 and phenylephrine were purchased from Sigma. Nisoldipine was a gift from Bayer Pharmaceutical Company.
All data are expressed as mean ± SEM. One-way analysis of variance and the method of Bonferroni were used for multiple comparisons. Student's t test was used to compare the paired data. A value of p < 0.05 was considered significant.
Membrane potential of the vascular smooth muscles of skeletal muscle and mesenteric small arteries
Figure 1 shows typical tracings of the membrane potential of the small arteries. When the transmural pressure increased from 40 to 100 mm Hg, the skeletal muscle small artery depolarized from −33 to −12 mV, and the mesenteric small artery from −50 to −30 mV. At each transmural pressure, the membrane potential was much more depolarized in skeletal muscle small artery than in the mesenteric small arteries. Figure 2 summarizes the changes in internal diameter (Fig. 2A) and the membrane potential (Fig. 2B) when the transmural pressure was varied from 20 to 100 mm Hg. Both parameters were measured ≥5 min after the pressure changes. In skeletal muscle small arteries, the membrane potentials were more depolarized than in mesenteric small arteries at each transmural pressure. The membrane potential became depolarized with increased transmural pressure. The internal diameter at steady state did not increase despite an increase in transmural pressure, indicating that the myogenic tone had increased. The internal diameter of mesenteric small arteries significantly increased with the transmural pressure increase, although the membrane potential was also significantly depolarized. When the skeletal muscle small arteries were maximally dilated by 10 μM nisoldipine at a transmural pressure of 100 mm Hg, the internal diameter was 190 ± 9 μm. Thus, when the transmural pressure was 100 mm Hg, the vessels significantly constricted by ∼36%, probably through the myogenic mechanism. Conversely, the dilated diameter of mesenteric small arteries was 229 ± 15 μm at 100 mm Hg, which was nearly identical to the value before the application of nisoldipine. Thus, the myogenic tone of the mesenteric small arteries of this study was quite weak at 100 mm Hg of transmural pressure. KCl (40 mM) constricted the skeletal muscle small arteries by 46 ± 2% while depolarizing the membrane potential to −17 ± 2 mV, and mesenteric small arteries by 42 ± 3% with a depolarization of the membrane potential to −20 ± 2 mV. There was no difference in the response to 40 mM KCl in terms of the internal diameter and the membrane potential.
Interaction between ryanodine and Bay K 8644 in mesenteric small arteries
Figure 3 shows representative tracings of the internal diameter when mesenteric small arteries were challenged with Bay K 8644 and/or ryanodine. The KCl-response (40 mM) is shown on the left. In Fig. 3A, Bay K 8644 was cumulatively applied from 1 nM to 1 μM, which did not significantly constrict the vessels. In the presence of Bay K 8644 (1 μM), ryanodine (1 μM) substantially constricted the vessels. In Fig. 3B, the vessels were treated with ryanodine (1 μM). Bay K 8644 constricted the ryanodine-treated vessels in a dose-dependent manner. Figure 4 summarizes these data in which the internal diameter was normalized by the maximally relaxed diameter at a transmural pressure of 100 mm Hg. In the presence of ryanodine, Bay K 8644 (0.1 and 1 μM) significantly constricted the mesenteric small arteries. Without ryanodine, Bay K 8644 did not constrict the vessels. Figure 5 shows the effects of ryanodine on the KCl-induced constriction of the mesenteric small arteries. Ryanodine (1 μM) substantially augmented the KCl constriction. The normalized diameter constricted by 20 mM KCl was 83 ± 6% before ryanodine and 42 ± 17% (n = 4, p < 0.05) after ryanodine.
Effects of the CA2+ channel blocker nisoldipine on the ryanodine-induced tone in skeletal muscle small arteries
Figure 6 shows representative tracings of the internal diameter when the L-type Ca2+ channel blocker nisoldipine was cumulatively applied. The skeletal small arteries were preconstricted by the myogenic activity (Fig. 6A), by 1 μM of phenylephrine (Fig. 6B), and 10 μM of ryanodine (Fig. 6C). In Fig. 6A, the transmural pressure increased from 40 to 100 mm Hg, and the internal diameter passively increased and then constricted to the previous level. Nisoldipine substantially decreased this myogenic tone at 0.01 nM, and the dilatory response reached nearly a plateau level at 0.1 nM. In Fig. 6B, the vessels were preconstricted by phenylephrine (1 μM) at a transmural pressure of 40 mm Hg. Phenylephrine induced phasic constriction followed by tonic constriction. Nisoldipine (0.01 nM) dilated the vessels, and the effects reached nearly a plateau level at 0.1 nM. This case showed that phentramine (1 μM) further dilated the vessels to the control level (before phenylephrine). In Fig. 6C, the vessels were preconstricted by ryanodine (10 μM) at a transmural pressure of 40 mm Hg; nisoldipine also dilated the vessels, but its vasodilatory effects were clearly weak compared with those shown in Fig. 6A and B.
Figure 7 summarizes the vasodilatory effects of nisoldipine. Figure 7A shows the dose-dependent Δdilation (μm). At the end of each experimental run, the vessels were maximally dilated by papaverine, 0.1 mM, nisoldipine, 10 μM, and phentramine, 1 μM (only in the phenylephrine series). In the phenylephrine series, nisoldipine alone did not cause maximal dilation. Conversely, the myogenic tone and the ryanodine-induced constriction were almost fully relaxed by nisoldipine. To assess the sensitivity to nisoldipine in these constrictions, the percentage dilation due to nisoldipine is shown in Fig. 7B. In the ryanodine-induced constriction, the dose-response curves significantly shifted to the right.
We found that the membrane potential of vascular smooth muscle was more depolarized in skeletal muscle small arteries than in mesenteric small arteries over transmural pressures ranging from 20 to 100 mm Hg. The myogenic tone was significant in skeletal muscle small arteries but not in mesenteric small arteries. An agonist of voltage-dependent Ca2+ channels, Bay K 8644 (≤1 μM), did not constrict the mesenteric small arteries in control. After the ryanodine treatment, however, Bay K 8644-induced constriction was clearly observed. The KCl (20 mM)-induced constriction was also enhanced by ryanodine in the mesenteric small arteries. In skeletal small arteries, the voltage-dependent Ca2+ channel blocker nisoldipine fully dilated the ryanodine-induced constriction. However, the susceptibility to nisoldipine appeared weaker in the ryanodine-induced constriction than in the myogenic or phenylephrine-induced constriction.
Membrane potentials in skeletal muscle and mesenteric small arteries
The membrane potentials of skeletal muscle small arteries were more depolarized than those of the mesenteric small arteries over transmural pressures of 20-100 mm Hg (Fig. 2B). When the transmural pressure was elevated, the steady-state internal diameter of mesenteric small arteries significantly increased, but that of skeletal muscle small arteries did not. Thus, skeletal muscle small arteries developed greater myogenic tone with the membrane depolarization. Although the membrane potential was significantly depolarized after the pressure changes, the myogenic tone was not clearly observed in the mesenteric small arteries. This difference may be explained, at least in part, by the absolute values of the membrane potential after the pressure changes. The membrane potential changed from −39 to −14 mV in skeletal muscle small arteries, and the voltage-dependent Ca2+ channels of vascular smooth muscle are substantially sensitive to the membrane potential in this range. Conversely, the membrane potential changed from −57 to −36 mV in mesenteric small arteries. Calcium current through the voltage-dependent Ca2+ channels may be less sensitive to membrane potentials in the latter range (14,15). The depolarization and the constriction caused by KCl (40 mM) were nearly identical in both vessels. Therefore, the absolute level of the membrane depolarization may affect the myogenic tone in these small arteries.
Harder et al. (16) reported that canine small renal arteries depolarized from −57 ± 2 to −38 ± 2 mV when the transmural pressure increased from 20 to 120 mm Hg, and cat small cerebral arteries depolarized from −66 to −44 mV when the transmural pressure increased from 20 to 140 mm Hg. The depolarization by the transmural pressure should be an important factor for the development of the myogenic contraction. However, dog renal and cat cerebral arteries developed the myogenic contraction when the membrane potential was at −38 ± 2 and −44 mV, respectively (9,16). Therefore, the absolute value of the membrane potential may not be a critical factor that determines the myogenic tone. The relation between the membrane potential and the myogenic tone must be examined in various arteries of various species.
The combined effects of ryanodine and Bay K 8644 on the tone of mesenteric small arteries
Neither Bay K 8644 (1 nM-1 μM) nor ryanodine (1 μM) constricted mesenteric small arteries when they were applied separately. After ryanodine treatment, however, Bay K 8644 significantly constricted these vessels. The KCl (20 mM)-induced constriction was also augmented by ryanodine. Further, it was observed that ryanodine substantially constricted the Bay K 8644-treated vessels (Fig. 3). Therefore, the activity of the voltage-dependent Ca2+ channels is a critical determinant of the ryanodine-induced constriction in mesenteric small arteries.
In the previous study (4), we observed that ryanodine and cyclopiazonic acid constricted skeletal muscle small arteries with increased cytosolic Ca2+, but not mesenteric small arteries. The present study may indicate that this difference is caused by the activity of the voltage-dependent Ca2+ channels of these two vessels. The membrane-potential data of the study also supported this view. That is, skeletal muscle small arteries have depolarized membrane potential.
Asano and Nomura (17) also reported that an impairment of SR function augmented Bay K 8644-induced contraction in rat femoral arterial strips. Thus, the SR function not only stores Ca2+ but also actively participates in the vascular tone formation. The present data and our previous data supported that the SR function may buffer the influxed Ca2+ to prevent an increase in cytosolic Ca2+(2), because ryanodine did not increase cytosolic Ca2+(5) but augmented the Bay K 8644 and KCl-induced contraction in mesenteric small arteries (Figs. 3-5).
It is possible that the combination of ryanodine and Bay K 8644 causes greater depolarization than Bay K 8644 by itself because ryanodine can enhance the membrane depolarization (18). The effects of SR dysfunction on membrane potentials must be systematically examined in further studies.
The susceptibility of ryanodine-induced constriction to nisoldipine in skeletal muscle small arteries
The vasodilatory effects of nisoldipine were tested in skeletal muscle small arteries. Figures 6 and 7 demonstrate that the myogenic constriction and the ryanodine-induced constriction were fully relaxed by nisoldipine. In phenylephrine-induced constriction, ∼30% of the constriction was relaxed by phentramine. The susceptibility to nisoldipine of the ryanodine-induced constriction was significantly lower than that of the myogenic and phenylephrine-induced constriction where increased Ca2+ influx plays a major role. Therefore, the ryanodine-induced constriction is caused not only by increased Ca2+ influx through L-type channels. We speculate that impaired Ca2+ extrusion and/or nisoldipine-insensitive channels may also play a role.
Nelson et al. (18) reported that the Ca2+ sparks from SR caused hyperpolarization through Ca2+-dependent K channels. This may also explain why an impairment in SR function can increase the vascular contractility.
Ryanodine and Bay K 8644 are highly specific agonists of ryanodine receptors (calcium-induced calcium release channels on SR) and L-type voltage-dependent Ca2+ channels, respectively. It was reported that ryanodine affected neither the Ca2+ channels (19), the plasma membrane Ca2+ pump (20), nor the contractile apparatus (21). Therefore, ryanodine-induced contraction, which was accompanied by increased cytosolic Ca2+(5), is probably caused by a dysfunction of the SR.
The present preparations had intact endothelium that may have affected the vascular tone. However, it has been reported that the myogenic response and basal diameter were not dependent on the presence of endothelium in isolated preparations of rat small arteries (22,23). In our preliminary studies, we removed the endothelium mechanically using a method previously described (24) and observed no significant change in the myogenic response and ryanodine-induced constriction.
In summary, the present results indicate the following: (a) the membrane potential of skeletal muscle small arteries was more depolarized than that of mesenteric small arteries; (b) Augmented L-type Ca2+ channel activity by Bay K 8644 or KCl-depolarization elicited ryanodine-induced contraction in mesenteric small arteries; (c) Ryanodine-induced contraction is fully relaxed by nisoldipine, but its susceptibility to nisoldipine was lower than that of the myogenic tone or phenylephrine-induced contraction. In conclusion, the contribution of the SR function to Ca2+ metabolism depends on the activity of L-type Ca2+ channels. SR may be able to promote the Ca2+ extrusion mechanism in rat small arteries.
Acknowledgment: We thank Mr. Brent Bell for reading the manuscript.
This study was supported by grants 05670591 and 06670690 (J.W.) from the Science Research Fund of Ministry of Education, Science and Culture, Tokyo, Japan.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
Skeletal muscle small arteries; Mesenteric small arteries; Myogenic tone; Membrane potential; Nisoldipine