Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Apr 1, 1998; 508(Pt 1): 211–221.
PMCID: PMC2230867

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

Abstract

  1. The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100–200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential.
  2. Ryanodine (10 μm), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nm and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (~22 %). Iberiotoxin (100 nm), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nm, and constricted by 37 μm (~19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels.
  3. Caffeine (10 mm), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nm in control arteries and by 158 ± 12 nm in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nm.
  4. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter.
  5. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+].

Elevation of intravascular pressure causes a graded membrane potential depolarization, elevation of arterial wall [Ca2+] and constriction (‘myogenic tone’) of pressurized cerebral arteries (Bayliss, 1902; Harder 1984; Brayden & Nelson, 1992; Knot & Nelson, 1995). The membrane potential depolarization caused by intravascular pressure opens voltage-dependent Ca2+ channels, increasing Ca2+ entry and thus intracellular [Ca2+] (Nelson, Standen, Brayden & Worley, 1988; Nelson, Patlak, Worley & Standen, 1990; Knot & Nelson, 1998). As cerebral arteries develop myogenic tone, Ca2+-dependent K+ (KCa) channels are activated to cause a tonic hyperpolarization to oppose the depolarization in response to pressure (Brayden & Nelson, 1992). KCa channels appear to be activated by local calcium release events (‘calcium sparks’) through ryanodine-sensitive Ca2+ release channels (also referred to as RyR channels or ryanodine receptors) in the sarcoplasmic reticulum (SR) of arterial smooth muscle cells (Nelson et al. 1995; Kirber, Etter, Singer, Walsh & Fay, 1997). Therefore, Ca2+ sparks via KCa channels appear to represent a negative feedback pathway to regulate arterial membrane potential (Brayden & Nelson, 1992; Nelson et al. 1995; Fig. 8).

Figure 8
Proposed scheme for the regulation of steady-state arterial wall calcium and diameter by ryanodine-sensitive Ca2+-release channels in the sarcoplasmic reticulum

Direct and indirect inhibitors of Ca2+ sparks (ryanodine, thapsigargin and cyclopiazonic acid) have been shown to depolarize (8–10 mV) and constrict (by ~30 %) pressurized (at 60 mmHg) cerebral arteries to the same extent as blockers (iberiotoxin, charybdotoxin) of KCa channels (Miller, Moczydlowski, Latorre & Phillips, 1985; Galvez et al. 1990; Brayden & Nelson, 1992; Knot & Nelson, 1995; Nelson et al. 1995). Furthermore, iberiotoxin has been shown to be without effect on membrane potential or diameter in the presence of ryanodine. These results supported the idea that calcium sparks act to oppose vasoconstriction by activating KCa channels (Nelson et al. 1995), which causes a tonic membrane potential hyperpolarization (Brayden & Nelson, 1992) that closes voltage-dependent calcium channels (Rubart, Patlak & Nelson, 1996), leading to reduction in ‘global’ intracellular [Ca2+].

In this study, we tested further the hypothesis that RyR channels regulate myogenic tone through changing KCa channel activity by directly examining the effects of ryanodine and iberiotoxin on arterial wall [Ca2+], as well as on membrane potential and arterial diameter. Specifically, we tested several key predictions of this hypothesis: (1) ryanodine and iberiotoxin elevate arterial wall calcium to a similar extent, since the induced depolarizations and constrictions have been shown to be of similar magnitude and not additive (Nelson et al. 1995); (2) ryanodine- and iberiotoxin-induced elevations in arterial wall [Ca2+] and vasoconstrictions result from the induced membrane potential depolarization; (3) prior addition of iberiotoxin or ryanodine prevents the actions of the other agent; and (4) ryanodine and iberiotoxin should be without effect in the presence of inhibitors of voltage-dependent calcium channels.

We also tested two alternative hypotheses that could explain the effects of ryanodine (or thapsigargin) in cerebral arteries. First, the superficial buffer barrier (SSB) hypothesis proposes that the (superficial) SR, just below the plasma membrane, takes up entering calcium continuously, and thus lowers average available Ca2+ near the myofilaments, and thereby acts as a ‘buffer barrier’ (Chen, Cannell & Van Breemen, 1992). The second hypothesis is that depletion of SR calcium content leads to the activation of a sarcolemmal calcium entry pathway that is independent of the voltage-dependent calcium channel, i.e. ‘store-depletion activated calcium entry’ as observed in many non-excitable cells (Putney, 1990; Hoth & Penner, 1992; Randriamampita & Tsien, 1993).

Here, we demonstrate that ryanodine and iberiotoxin elevated arterial wall [Ca2+] to the same extent (~50 nm). The effects of ryanodine and iberiotoxin were not additive, and could be quantitatively explained by the induced membrane potential depolarization. The lack of effect of ryanodine in the presence of iberiotoxin argues for a major role of RyR channels in the regulation of KCa channels. Furthermore, L-type Ca2+ channel blockers prevented the elevation of arterial wall [Ca2+] and vasoconstriction in response to iberiotoxin (see also Brayden & Nelson, 1992; Knot & Nelson, 1995) and to ryanodine and thapsigargin, arguing against ‘store-depletion’ activating Ca2+ entry in this preparation. These results support the hypothesis that RyR channels play an important role in regulating myogenic tone through activation of KCa channels, which in turn regulates Ca2+ entry through voltage-dependent calcium channels.

METHODS

The methods used in this paper are identical to those described in Knot & Nelson (1998), with the following additions.

Experimental protocols

In some experiments, as indicated in the text and figure legends, the endothelium was removed by placing an air bubble in the lumen of the artery for 1 min followed by a 30 s wash with distilled water (Brayden & Nelson, 1992; Knot & Nelson, 1995; Nelson et al. 1995; Knot, Zimmermann & Nelson, 1996). The absence of a functional endothelium was verified by the absence of a dilator response to application of 10 μm acetylcholine or 100 nm bradykinin. In the absence of external Ca2+ or in the presence of a Ca2+ channel inhibitor, elevation of intravascular pressure causes a graded increase in diameter (e.g. Fig. 2), which presumably reflects the passive properties of the arteries. In contrast, in physiological salt solution (PSS), arterial diameter often changes little with a pressure elevation (Figs 3 and and4),4), indicating that the artery has constricted in response to pressure. Pressure-induced constriction is defined as the difference in diameter in PSS and Ca2+-free PSS at a given pressure.

Figure 2
Iberiotoxin increases pressure-induced constrictions
Figure 3
Ryanodine increases arterial wall Ca2+ and constricts pressurized cerebral arteries
Figure 4
Iberiotoxin increases arterial wall Ca2+ and constricts pressurized cerebral arteries

Membrane potential measurements

The effects of ryanodine and iberiotoxin on the measured arterial responses were slow and took up to 5 min to reach a steady state. In the case of membrane potential measurements impalements were often lost during the initial application of these compounds. Cursor selected data segments of > 3 min were taken after the drug effects had reached steady state. In many cases multiple impalements (≥ 3 min) at the new steady state (> 5 min after application) were averaged in the same artery to describe accurately the effect of the drugs. In describing membrane potentials, the values are means ±s.d. of data from independent experiments, i.e. different arteries.

Solutions, compounds and drugs

Chemicals and drugs used

Iberiotoxin was obtained from Peptides International (Louisville, KY, USA). Ryanodine was obtained from Calbiochem (La Jolla, CA, USA). All other salts and drugs were obtained from Sigma. Nimodipine was a gift from Dr A. Scriabine of Miles Laboratories (West Haven, CT, USA).

RESULTS

Ryanodine and iberiotoxin cause membrane potential depolarization and vasoconstriction of small cerebral arteries

Inhibitors of RyR channels (ryanodine and thapsigargin) or KCa channels (iberiotoxin and charybdotoxin) have been shown to depolarize (by 9 mV) and constrict (by about 30 %) pressurized (60 mmHg) rabbit and rat cerebral arteries (Brayden & Nelson, 1992; Knot & Nelson, 1995; Nelson et al. 1995). Figure 1A and B illustrates the membrane potential depolarization of pressurized (60 mmHg) posterior cerebral arteries caused by iberiotoxin and ryanodine, and that the effects of these agents were not additive. The results of these experiments are summarized in Fig. 1C. The pressurized cerebral arteries (to 60 mmHg) used in these experiments had a resting membrane potential of -45.2 ± 1.4 mV (n= 27). Ryanodine (10 μm) and iberiotoxin (100 nm) depolarized the membrane potential of the arteries by 8.8 ± 1.1 mV (n= 10) and 10.0 ± 2.0 mV (n= 6), respectively (Fig. 1C). Addition of iberiotoxin (100 nm) to arteries bathed in ryanodine (10 μm) or vice versa did not result in an additional effect (membrane potential depolarizations under these conditions were 9.7 ± 1.4 mV (n= 4) and 10.2 ± 2.1 mV (n= 3), respectively). In the presence of ryanodine and iberiotoxin, the arteries were able to depolarize further, since elevation of external potassium to 61 mm depolarized arteries to -22.8 ± 1.8 mV (n= 5; Fig. 1C).

Figure 1
Inhibitors of ryanodine-sensitive calcium release channels (ryanodine) and KCa channels (iberiotoxin) depolarize pressurized cerebral arteries

The effect of iberiotoxin on myogenic tone at different intravascular pressures

To investigate the role of KCa channels in the myogenic response, we studied the effect of iberiotoxin on arterial diameter as a function of intravascular pressure. Figure 2 illustrates the effect of increasing intravascular pressure from 2 to 100 mmHg on arterial diameter, with and without KCa channels blocked by iberiotoxin, and with and without nimodipine which blocks voltage-dependent Ca2+ channels. Iberiotoxin caused a statistically significant vasoconstriction at an intravascular pressure of 60 mmHg and above (P < 0.05; Fig. 2). Nimodipine prevented pressure-induced constrictions, and iberiotoxin was ineffective in the presence of nimodipine and in the absence of external calcium (Fig. 2). These results are consistent with the idea that the negative feedback regulation of myogenic tone by KCa channels increases with the elevation of calcium entry caused by pressure-induced membrane depolarization (Brayden & Nelson, 1992; see Fig. 8).

Ryanodine and iberiotoxin cause elevation of arterial wall calcium and vasoconstriction

Based on the voltage dependence of arterial wall calcium and diameter at 60 mmHg (Knot & Nelson, 1998), the depolarization (8–10 mV) induced by iberiotoxin or ryanodine would be expected to elevate arterial wall Ca2+ by about 45 nm and constrict the arteries by about 50 μm or 25 %, if the effects of these two substances were solely mediated by membrane potential depolarization. Ryanodine increased arterial wall [calcium] in pressurized (60 mmHg) cerebral arteries from 186 ± 14 to 232 ± 20 nm or by 46 nm, and constricted the arteries from 119 ± 18 to 75 ± 20 μm or by 44 μm (~22 %; n= 6; Fig. 3A and B). As expected from the membrane potential and diameter experiments, in the presence of ryanodine, iberiotoxin did not alter arterial wall [Ca2+] or arterial diameter (Fig. 3A and B). These results are consistent with the idea that ryanodine increases steady-state arterial wall calcium and decreases diameter through membrane potential depolarization caused by inhibition of KCa channels.

To exclude the possibility that the Ca2+ and diameter response of the artery had changed over the course of the experiment, we alternately tested the response to elevation of external K+ to 61 mm before and after addition of ryanodine or iberiotoxin (see Figs 3A and and4A4A for an example of each protocol). In this series of experiments, increasing extracellular potassium to 61 mm, which would depolarize to −22 mV, raised [Ca2+] to 363 ± 14 nm (n= 3) in the absence of ryanodine, and to 347 ± 59 nm (n= 3) in the presence of ryanodine and iberiotoxin (Fig. 3C). Extracellular K+ (61 mm) constricted the arteries to a diameter of 37 ± 7 μm before ryanodine, and to 37 ± 10 μm in the presence of ryanodine and iberiotoxin (n= 6; Fig. 3D). The lack of effect of iberiotoxin or ryanodine on arterial wall [Ca2+] of arteries bathed in high K+ is consistent with the idea that both agents act through K+ channels, since in high K+, the membrane potential and the potassium equilibrium potential should be similar.

If ryanodine elevated arterial wall Ca2+ through activation of voltage-dependent calcium channels by membrane depolarization, then calcium channel inhibitors (e.g. nimodipine) should decrease arterial wall Ca2+, and cause vasodilatation. In further support of this mechanism, in the presence of ryanodine and iberiotoxin, nimodipine (10 nm) lowered arterial wall calcium to 76 ± 17 nm (n= 5) and dilated the arteries to 197 ± 10 μm (n= 6; Fig. 3), a level similar to that observed in the absence of extracellular calcium (Fig. 2).

These results indicate that inhibition of Ca2+ release through ryanodine-sensitive Ca2+-release channels in the SR (i.e. inhibition of Ca2+ sparks) prevented the effect of iberiotoxin on membrane potential, arterial wall Ca2+ and diameter, lending further support to the hypothesis that Ca2+ sparks activate KCa channels in these arteries (Nelson et al. 1995). This mechanism (see Fig. 8) would also predict that blockers of KCa channels (e.g. iberiotoxin) should elevate arterial wall calcium by about 45 nm, and prevent at least part of the effect of inhibitors of Ca2+ sparks (ryanodine). Iberiotoxin (100 nm) increased arterial wall calcium from 197 ± 21 to 248 ± 31 nm or by 51 nm, and constricted the arteries from 116 ± 14 to 79 ± 6 μm or by 37 μm (~19 %) (n= 7) (Fig. 4A and B), similar to the effect of 10 μm ryanodine or an ~9 mV depolarization (see Fig. 3A and B, and Fig. 9 in Knot & Nelson, 1998). In the presence of iberiotoxin, ryanodine (10 μm) was without effect on arterial membrane potential (Fig. 1B), arterial wall [Ca2+] and diameter (Fig. 4). However, in the presence of iberiotoxin and ryanodine, high potassium (61 mm K+) still elevated calcium further (to 365 ± 38 nm, n= 5) and constricted the arteries further (from 79 to 39 ± 8 μm, n= 5; Fig. 4). In the presence of iberiotoxin, ryanodine and 61 mm K+, nimodipine (10 nm) lowered calcium (to 78 ± 13 nm, n= 7) and fully dilated (to 202 ± 12 μm, n= 7) the arteries.

If ryanodine and iberiotoxin elevated calcium and caused vasoconstriction largely through the induced membrane potential depolarization (about 9 mV), then an equivalent membrane depolarization by another means should elevate [Ca2+] and constrict to a similar extent. Based on the voltage dependence of arterial wall Ca2+ and diameter (Fig. 9 in Knot & Nelson, 1998), depolarization by 9 mV should increase arterial wall Ca2+ by 45 nm and constrict the arteries by 50 μm (Fig. 5A). Ryanodine (10 μm), iberiotoxin (100 nm) or the combination of the two also depolarized by 8–10 mV, elevated intracellular calcium by 45 nm and constricted by 50 μm. Further, if the effects of these two agents are solely mediated via changes in membrane potential, then the effects of these compounds on arterial wall [Ca2+] and diameter, alone or together, in PSS, in high K+ PSS or in the presence of a Ca2+ channel blocker, should be predicted by the relationship between [Ca2+] and diameter via changing membrane potential (see Fig. 10 in Knot & Nelson, 1998). This is shown to be true in Fig. 5B. Together these results are consistent with the idea that ryanodine and iberiotoxin elevate arterial wall [Ca2+] and constrict through membrane potential depolarization.

Figure 5
The effects of ryanodine and iberiotoxin on arterial Ca2+ and diameter are mediated via changes in membrane potential

Ryanodine decreases, whereas iberiotoxin increases, SR Ca2+ content

The lack of effect of ryanodine in the presence of iberiotoxin suggests that the major role of Ca2 + sparks in these pressurized arteries is to activate KCa channels. Another explanation is that iberiotoxin somehow depleted ryanodine-sensitive calcium stores. To examine this possibility, the effect of caffeine (10 mm), which activates ryanodine receptors, was tested on arterial wall [Ca2+] in the presence of iberiotoxin or ryanodine and compared with control conditions. Caffeine transiently increased arterial wall calcium in pressurized arteries (to 60 mmHg) with myogenic tone to 325 ± 9 nm or by 136 ± 9 nm (n= 9) (Fig. 6A). In the presence of iberiotoxin (100 nm), caffeine transiently increased arterial wall calcium to 404 ± 12 nm or by 158 ± 12 nm (n= 5), indicating that iberiotoxin did not deplete SR calcium stores. Instead, iberiotoxin slightly increased the caffeine response (Fig. 6A; P < 0.02) consistent with a somewhat elevated SR Ca2+ load due to elevated cytoplasmic [Ca2+] (Fig. 6B). In contrast, in the presence of 10 μm ryanodine, which elevated ‘global’ cytoplasmic [Ca2+] to a similar level to that given by iberiotoxin (Fig. 6B), caffeine (10 mm) had little effect on arterial wall calcium (increased by 18 ± 5 nm; n= 4, n.s.; Fig. 6A).

Figure 6
Effect of ryanodine and iberiotoxin on arterial wall [Ca2+] and caffeine-induced Ca2+ release from the sarcoplasmic reticulum in pressurized cerebral arteries with and without endothelium

The effects of ryanodine and iberiotoxin on arterial wall calcium are independent of the endothelium

Intravascular pressure, ryanodine and iberiotoxin could conceivably have an effect on the endothelium, which could release a factor that would alter the Ca2+ or diameter response of the artery. We therefore studied the effects of pressure, ryanodine and iberiotoxin in arteries from which the endothelium had been removed (open bars in Fig. 6). Basal arterial wall [Ca2+] levels were similar in intact and denuded arteries pressurized to 60 mmHg. Arterial wall [Ca2+] levels were 190 ± 12 (n= 9) and 186 ± 16 nm (n= 14), respectively. This is consistent with previous studies that found no change in arterial diameter in these arteries when the endothelium had been removed (Knot et al. 1996). In denuded arteries, ryanodine (10 μm) increased arterial wall calcium from 186 ± 16 to 229 ± 8 nm or by 48 nm (n= 5; P < 0.05), indicating that endothelial removal had no effect on basal [Ca2+] or the response to ryanodine. Similarly, the effect of iberiotoxin (100 nm) was unaltered by removing the endothelium. In denuded arteries, iberiotoxin increased arterial wall calcium from 186 ± 16 to 232 ± 20 nm or by 47 nm (n= 9; P < 0.05).

Ryanodine does not activate a secondary calcium entry pathway

Ryanodine (or thapsigargin and cyclopiazonic acid) could conceivably elevate arterial wall [Ca2+] and lead to constriction by opening a calcium entry pathway activated in response to SR Ca2+ depletion, as has been reported in several non-excitable cell types including amphibian gastric smooth muscle cells (Guerrero, Fay & Singer, 1994). Although there is no precedent for such a pathway in cerebral arteries, this possibility was examined by testing the effects of ryanodine, thapsigargin and cyclopiazonic acid, which all inhibit Ca2+ sparks (Nelson et al. 1995), in the presence of a maximally effective (> 10–100 ×Kd) concentrations of the inhibitors of voltage-dependent Ca2+ channels used in our studies (100 nm nimodipine or 30 μm diltiazem). For completeness, iberiotoxin was also tested in the presence of these drugs. In these series of experiments in pressurized (60 mmHg) arteries, nimodipine (100 nm) and diltiazem (30 μm) lowered arterial wall calcium from 170 ± 13 (n= 10) to 65 ± 19 (n= 6) and 73 ± 20 nm (n= 4), respectively, consistent with previous results. The arteries dilated from 116 ± 8 to 197 ± 11 and 197 ± 12 μm, respectively. Ryanodine, thapsigargin, cyclopiazonic acid and iberiotoxin were without effect on arterial wall calcium and diameter in the presence of nimodipine or diltiazem. Arterial wall [Ca2+] and diameter values in the presence of these drugs were, respectively: ryanodine, 69 ± 20 nm and 202 ± 9 μm (n= 7); thapsigargin, 63 ± 20 nm and 203 ± 9 μm (n= 6); cyclopiazonic acid, 76 ± 9 nm and 194 ± 7 μm (n= 4); iberiotoxin, 65 ± 15 nm and 198 ± 10 μm (n= 8; see Fig. 7 for selected traces; see also Figs 3 and and4).4). As previously shown, iberiotoxin was without effect in the presence of a calcium channel blocker (n= 8; Fig. 7B; Brayden & Nelson, 1992; Knot & Nelson, 1995). These results further support the idea that voltage-dependent calcium channels are the major calcium entry pathway determining cytosolic [Ca2+] in cerebral arteries (see Worley, Quayle, Standen & Nelson, 1991; Quayle, McCarron, Asbury & Nelson, 1993; Rubart et al. 1996), and argue against the existence of a ‘store depletion’ activated Ca2+ entry pathway in this preparation.

Figure 7
Ryanodine and thapsigargin do not increase arterial wall [calcium] or decrease arterial diameter in the presence of L-type Ca2+ channel inhibitors

DISCUSSION

This study provides the first direct information on the regulation of arterial wall calcium in cerebral arteries by ryanodine-sensitive Ca2+ release channels and KCa channels. Our data are consistent with the following mechanism (see Fig. 8). Intravascular pressure causes a graded membrane potential depolarization, which causes a graded increase in the steady open probability of L-type voltage-dependent Ca2+ channels, and this elevates steady Ca2+ entry (Nelson et al. 1990; Langton & Standen, 1993; Quayle et al. 1993; Fleischmann, Murray & Kotlikoff, 1994; Rubart et al. 1996) and intracellular [Ca2+] (Knot & Nelson, 1998). An elevation of steady arterial wall [Ca2+] leads to increased tone, presumably through activation of myosin light chain kinase (MLCK). The opening rate of RyR channels (Ca2+ spark frequency) should increase in response to elevated cytoplasmic calcium (global and local) (Cannell, Cheng & Lederer, 1995; Lopez-Lopez, Shacklock, Balke & Wier, 1995; Santana, Cheng, Gomez, Cannell & Lederer, 1996) and to increased SR calcium load (Fig. 6A; Nelson et al. 1995; Lukyanenko, Gyorke & Gyorke, 1996). Increased steady-state Ca2+ spark frequency, by itself, should have little direct effect on arterial wall [Ca2+], and hence on MLCK activity. Ca2+ sparks, however, activate KCa channels, which leads to membrane potential hyperpolarization and closure of L-type Ca2+ channels (Brayden & Nelson, 1992; Nelson et al. 1995), thus forming a negative feedback element to limit Ca2+ influx.

Calcium sparks activate KCa channels to regulate myogenic tone

Steady arterial wall calcium, which regulates arterial tone, should be determined in the steady state by calcium influx and extrusion across the plasma membrane (e.g. see Chen et al. 1992). The primary calcium influx pathway in these cerebral arteries is through voltage-dependent calcium channels, which regulates steady calcium influx over the range of physiological membrane potentials (-60 to −35 mV) (Nelson et al. 1990; Langton & Standen, 1993; Quayle et al. 1993; Rubart et al. 1996; Knot & Nelson, 1998). Our results suggest that steady-state activity of ryanodine-sensitive Ca2+ release channels (‘Ca2+ sparks’) (Nelson et al. 1995) regulates steady arterial wall calcium, and hence myogenic tone, through modulation of Ca2+ influx through voltage-dependent calcium channels by altering membrane potential through KCa channels (see Fig. 8). Furthermore, the following observations support the idea that a major role of calcium sparks in pressurized cerebral arteries is to regulate KCa channel activity, and thus the membrane potential. (1) Calcium sparks activate KCa channels in cerebral artery myocytes (Nelson et al. 1995). (2) Inhibitors of calcium sparks (ryanodine, thapsigargin, cyclopiazonic acid) depolarize, elevate arterial wall [calcium] and constrict pressurized (denuded) cerebral arteries (Figs 1, ,2,2, ,3,3, ,4,4, ,55 in this study; see also Nelson et al. 1995). (3) Iberiotoxin is without effect in the presence of ryanodine or thapsigargin (Figs 1A and and3).3). In contrast, in normal PSS, increasing pressure, which depolarizes and increases [Ca2+], the effect of iberiotoxin increases at higher pressures (Fig. 2; see also Brayden & Nelson, 1992). Since iberiotoxin had no effect in the presence of ryanodine or thapsigargin, these results suggest that KCa channels do not contribute significantly to the membrane conductance of the smooth muscle cells in the intact pressurized arteries in the absence of calcium sparks (see Nelson et al. 1995). (4) A potent and selective blocker of KCa channels, iberiotoxin, prevents the effects of ryanodine on steady-state membrane potential, arterial wall calcium and diameter (Figs 1B and and4).4). Under this condition, the SR and ryanodine receptors are functional, as verified by caffeine-induced calcium transients (Fig. 6). This result argues strongly against a major direct contribution of spark calcium to steady arterial wall [Ca2+] as well as against significant steady-state calcium-induced inactivation of voltage-dependent calcium channels, and indicates that the major function of calcium sparks is to activate KCa channels. (5) The depolarizations in response to ryanodine and iberiotoxin were similar and not additive, and could account for the elevation in intracellular calcium and the subsequent vasoconstriction (Fig. 5A and B). This result suggests that ryanodine- and iberiotoxin-induced depolarizations are sufficient to explain the observed elevations in intracellular [calcium] and increases in arterial tone.

Our experiments indicate that the steady-state activity of ryanodine receptors decreases steady arterial wall [calcium] and tone by decreasing calcium entry through membrane hyperpolarization via KCa channels. However, calcium sparks are not dissociated from global calcium. It is an issue of signalling strength (see Berridge, 1997). A single calcium spark has a much more pronounced effect on KCa channels than it does on MLCK. At steady-state conditions that would be appropriate for a pressurized cerebral artery with tone (−40 mV), calcium sparks would have a very small effect on global calcium, and thereby MLCK activity. The observed Ca2+ spark frequency in cerebral artery myocytes (< 5 s−1 cell−1) even at steady depolarized levels is consistent with a very small direct contribution to global calcium (< 5 nm; Nelson et al. 1995). However, caffeine causes a very significant activation of ryanodine receptors in a short time period, which leads to a global change in calcium and contraction (Fig. 6A). Presumably the spatially and temporally co-ordinated activation of calcium sparks by caffeine effectively sends a greater signal to MLCK than to the KCa channels (see Nelson et al. 1995; Berridge, 1997), i.e. in this case the release of calcium dominates transiently over the decrease in calcium influx caused by membrane hyperpolarization. The final outcome will be determined by the amplitude and frequency modulation of calcium sparks as well as their location relative to KCa channels, MLCK, and other calcium-sensitive processes.

Activation of L-type voltage-dependent Ca2+ channels by membrane depolarization underlies the effects of ryanodine and iberiotoxin on arterial calcium and diameter

Our data are consistent with the hypothesis that ryanodine (or thapsigargin and cyclopiazonic acid) through membrane potential depolarization activates voltage-dependent Ca2+ channels leading to increased cytosolic Ca2+. In support of this hypothesis, nimodipine and diltiazem reverse or prevent all the effects of ryanodine (also thapsigargin and cyclopiazonic acid) on arterial wall Ca2+ and arterial diameter (Figs 3, ,44 and and7).7). Two alternative possible explanations for the observed effects of ryanodine (or thapsigargin) are as follows. (1) The superficial buffer barrier (SSB) hypothesis proposes that the (superficial) SR, just below the plasma membrane, takes up entering calcium continuously, and thus lowers average available Ca2+ near the myofilaments, and thereby acts as a ‘buffer barrier’ (Chen et al. 1992). According to this proposal, blocking SR function would lead to an increase in average Ca2+ and contraction because the buffer barrier or calcium sink has been removed. Since steady-state calcium should depend on sarcolemmal calcium entry and extrusion, the SSB model requires direct calcium transfer from the SR to the extracellular solution, bypassing the bulk cytoplasm (Chen et al. 1992). (2) The second possibility is that depletion of SR calcium content leads to the activation of a sarcolemmal calcium entry pathway that is independent of the voltage-dependent calcium channel, i.e. ‘store depletion activated calcium entry’ observed in many non-excitable cells (Putney, 1990; Hoth & Penner, 1992; Randriamampita & Tsien, 1993).

The following evidence argues against these two possibilities in our preparation. (1) There is no evidence for a SSB or ‘store depletion activated calcium entry’ in pressurized arteries including cerebral arteries. (2) Ryanodine and thapsigargin cause membrane potential depolarization that can quantitatively account for the increase in arterial wall calcium and the decrease in arterial diameter (Fig. 5A and B). The SSB hypothesis cannot account for the observed depolarization. (3) Ryanodine and thapsigargin prevented the effects of iberiotoxin on membrane potential, arterial wall Ca2+and diameter, indicating that ryanodine-sensitive SR calcium release activates KCa channels. (4) Furthermore, iberiotoxin prevented the elevation in arterial wall calcium and vasoconstriction to ryanodine, a result incompatible with the SSB or store hypotheses. (5) Ryanodine, thapsigargin and iberiotoxin were without effect in arteries dilated by L-type calcium channel blockers, arguing against the existence of a second alternative ‘store depletion activated calcium entry’ pathway. Therefore, our data do not support, and in fact argue against, both the SSB and store hypotheses in this preparation, and are consistent with the scheme shown in Fig. 8.

Conclusion

In conclusion, this study provides further evidence that RyR channels in the SR regulate cerebral artery diameter through changes in KCa channel activity (Fig. 8) at the level of the intact artery. Inhibition of KCa channels would lead to membrane potential depolarization (Fig. 1), activation of voltage-dependent Ca2+ channels (see Figs 2, ,3,3, ,44 and and7),7), increased Ca2+ influx (Fleischmann et al. 1994; Rubart et al. 1996), increased arterial wall [Ca2+] (Figs 3, ,44 and and5),5), and vasoconstriction (Figs 2, ,3,3, ,44 and and5).5). The proposed mechanism in this study underscores the key role that arterial smooth muscle membrane potential plays in the regulation of arterial diameter and would predict that the effect of other vasoactive stimuli that work via changes in ryanodine-sensitive Ca2+ release from the SR (e.g. by increasing the SR Ca2+ load) may be mediated, at least in part, through changes in membrane potential via the activity of KCa channels. Consistent with this proposal is that activation of Ca2+ sparks, and thus KCa channels, would lead to hyperpolarization and vasorelaxation as has recently been suggested (Porter, Bonev, Kleppisch, Lederer & Nelson, 1997a; Porter, Stevenson, Knot & Nelson, 1997b). These results support the idea that Ca2+ sparks act as important signals to the plasma membrane to regulate cell function.

Acknowledgments

We would like to thank Dr Christian Walther for comments on the manuscript. This work was supported by the National Science Foundation grant IBM-9631416 and National Heart, Lung and Blood Institute grants 44455 and 51728.

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