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. 2002 Oct 25;545(Pt 2):615–627. doi: 10.1113/jphysiol.2002.027904

Differential activation of ion channels by inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive calcium stores in rat basilar artery vasomotion

R E Haddock 1, C E Hill 1
PMCID: PMC2290697  PMID: 12456838

Abstract

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura-2 AM to study changes in [Ca2+]i in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca2+]i were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca2+]i oscillations in the arterial wall became reduced and irregular, while [Ca2+]i oscillations in adjacent SMCs were no longer synchronized. BAPTA-AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM-34, but not iberiotoxin, 4-aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca2+]i oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP3 (inositol 1,4,5,-trisphosphate)-sensitive stores which activates calcium-dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage-dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.

Contraction of vascular smooth muscle is ultimately dependent on an increase in cytoplasmic calcium concentration and activation of the contractile apparatus. The underlying mechanisms culminating in contraction have been described as resulting from voltage-dependent events and influx of extracellular calcium, as well as voltage-independent events, which rely on calcium release from intracellular stores, often following the activation of intracellular second messenger pathways (Hirst & Edwards, 1989; Somlyo & Somlyo, 1994). More recently the discovery of localized calcium signalling events, termed calcium sparks and calcium waves, has challenged the traditional ideology that homogeneous changes in cytoplasmic calcium control vessel diameter (Iino et al. 1994; Nelson et al., 1995; Mironneau et al. 1996; Imaizumi et al. 1998).

Rhythmical contraction of blood vessels, or vasomotion, has been observed in both small vessels of the microcirculation and larger vessels in vivo and in vitro (Auer & Gallfhoffer, 1981; Hundley et al. 1988; Fujii et al. 1990b; Morita-Tsuzuki et al. 1992; Bertuglia et al. 1994). Vasomotion can occur spontaneously (Bouskela & Grampp, 1992; Hill et al. 1999) or in response to agonist stimulation (Gustafsson, 1993; Peng et al. 2001) and is considered to have both physiological and pathophysiological relevance (Gratton et al. 1998; Hudetz et al. 1998; Shimamura et al. 1999; Rucker et al. 2000).

Vasomotion has been shown to be associated with underlying oscillations in calcium and with rhythmical changes in membrane potential (Segal & Beny, 1992; von der Weid & Beny, 1993; Hill et al. 1999; Peng et al. 2001; Haddock et al. 2002). Indeed, it has recently been demonstrated in mesenteric arteries that agonist-induced vasomotion is the result of synchronization of calcium oscillations in individual smooth muscle cells (SMCs) (Mauban et al. 2001; Peng et al. 2001). These calcium oscillations are suggested to result from the intermittent release of calcium from ryanodine stores in the sarcoplasmic reticulum (SR) in individual cells, while synchronicity occurs following the activation of voltage-dependent calcium channels (VDCCs), assumed to occur following a depolarization (Peng et al. 2001).

Vasomotion recorded in the adult rat basilar artery in vivo has been shown to be dependent on voltage-activated mechanisms involving calcium-dependent potassium (KCa) channels (Fujii et al. 1990a). Paradoxically, studies of cultured SMCs and of pressurized cerebral arteries have demonstrated an important role for intracellular ryanodine sensitive calcium stores and potassium channels in relaxation of cerebral vessels (Nelson et al. 1995; Bonev et al. 1997; Gollasch et al. 1998; Jaggar & Nelson, 2000; Jaggar, 2001; Perez et al. 2001). Modulation of agonist-induced vasomotion by potassium conductances has also been demonstrated in the rat mesenteric artery (Gustafsson & Nilsson, 1994). Since control of blood flow through cerebral vessels varies in several respects from that in systemic vessels (Faraci & Heistad, 1998) we were interested to investigate the role of intracellular stores in the mechanisms underlying cerebral vasomotion. By correlating changes in membrane voltage with changes in calcium in individual SMCs, and more globally in the arterial wall, we show that vasomotion in the juvenile rat basilar artery depends on calcium release from inositol 1,4,5-trisphosphate (IP3) stores which leads to depolarization via calcium-dependent chloride (ClCa) channels. This depolarization permits cyclical voltage oscillations due to calcium influx through VDCCs, calcium-induced calcium release from intracellular ryanodine stores and hyperpolarization via activation of intermediate conductance calcium-dependent potassium (IKCa) channels.

Methods

All experiments were performed in accordance with the guidelines of the Animal Experimentation Ethics Committee of the Australian National University. Male Wistar rats aged 14-17 days postnatal were anaesthetized with ether and decapitated. The brain was removed and placed in cold (5-7 °C) dissection buffer containing (mm): 3 3-(N-morpholino)propanesulphonic acid (Mops); 1.2 NaH2PO4; 4.6 glucose; 2 pyruvate; 0.02 EDTA(Na); 0.15 albumin; 145 NaCl; 4.7 KCl; 2 CaCl2; 1.2 MgSO4. A rectangular section of the meninges, containing the basilar artery and its primary and secondary branches was isolated and pinned firmly to the bottom of a Sylgard (Dow Corning Corporation, USA)-coated coverslip which formed the base of a 1 ml recording chamber. In all cases, the endothelial cell layer remained intact.

Measurements of contraction and membrane voltage

The recording chamber was perfused at a constant flow rate (3 ml min−1) with Krebs solution (mm): 120 NaCl; 5 KCl; 25 NaHCO3; 1 NaH2PO4; 2.5 CaCl2; 2 MgCl2; 11 glucose; gassed with 95 % O2 and 5 % CO2 and maintained at 33-34 °C. All preparations were equilibrated for 20 min by which time spontaneous contractions were routinely recorded from the entire vascular tree. A section of a primary or secondary branch of the basilar artery was visualized using video microscopy and the vessel diameter was continuously measured using the DIAMTRAK computer tracking program (Neild, 1989). SMCs were impaled with sharp microelectrodes, filled with 0.5 m KCl (120-220 MΩ; Flaming Brown micropipette puller, Sutter Instrument Co.) and membrane potential records were low-pass filtered (cut off frequency 1 kHz) and amplified with an Axoclamp 2B (Axon Instruments). Simultaneous changes in membrane potential and changes in vessel diameter in the region where the cell was impaled were acquired with sample rates of greater than 100 Hz. Following equilibration, preparations were incubated in drug solutions for a maximum period of 30 min before being returned to control Krebs solution.

Measurements of intracellular calcium

Measurements of changes in intracellular calcium concentration were made using the ratiometric fluorescent dye, Fura-2 acetoxymethyl ester (Fura-2 AM), whereby calcium release is expressed as the ratio of the fluorescence emission recorded at 510 nm following sequential excitation of the preparation with 340 and 380 nm light (F340/380) (Polychrome II illumination system; T.I.L.L. Photonics GMBH, Germany). The preparation was set up as described above, except that a small area of the meninges surrounding the primary and secondary branches was gently removed to expose the artery to the Fura solution. The preparations were then incubated at room temperature (22 °C) in modified Krebs solution containing reduced calcium (0.5 mm CaCl2), the detergent pluronic F-127 (0.01 %) and Fura-2 AM (5 μm) for 40 min. After loading, preparations were superfused with warmed (34 °C) Krebs solution, containing 2.5 mm CaCl2 for 20 min.

In order to study changes in arterial wall calcium, fluorescence emission from a region of the loaded artery was recorded at 30 Hz, with a photometry system (T.I.L.L. Photonics) and pCLAMP 8 software (Axon Instruments). In order to study changes in individual smooth muscle cell calcium, an intensified cooled CCD camera (Princeton Instruments, USA) and Axon Imaging Workbench software were used (Axon Instruments). Preparations were excited with alternating 340 and 380 nm light at a frequency of 3.7 ± 0.1 Hz. In all cases, care was taken to limit exposure to the fluorescent light. Simultaneous measurements of vessel diameter were made by illuminating the preparation with infrared light (775 nm) and collecting continuous images with a Hamamatsu Performance Vidicon camera. Measurements of arterial wall calcium and individual smooth muscle cell calcium were recorded from the same preparation, while electrophysiological data were collected from a different animal.

Analysis of results

Changes in intracellular calcium due to drug intervention were expressed as a percentage of the fluorescence ratio recorded in control, where the F340/380 in control is defined as 100 %. Control measurements were made for 5 min immediately following the equilibration period and at the time when drugs would have been present in the tissue bath for 20 min. In the majority of cases, responses to drugs were observed within a 5 min exposure period. If responses to drugs had not stabilized by 20 min, measurements were continued until drugs had been present for 30 min. Changes in vessel diameter were expressed as a percentage of the control resting vessel diameter (RVD). Membrane potential values of spontaneously active preparations were determined from the most negative value recorded. Data were calculated as the means ± s.e.m. of n preparations, where each preparation was from a different rat. Statistical analysis was determined using 95 % confidence limits (P < 0.05). Student's paired t test was used when comparing drug effects to their respective control measurements. Data analysis was performed using the scientific statistical package GraphPad Prism (GraphPad Software Inc., USA).

Drugs and solutions

The following drugs were used: (1,2-bis(o-aminophenoxy)ethane-N,N,N‘,N‘-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)-hexyl)-1H-pyrrole-2,5-dione (U73122), 1-(6-((17β-3- methoxy- estra- 1,3,5(10)-trien-17-yl)amino)-hexyl)-2,5-pyrrolidine-dione (U73343), 4,4′-diisocthiocyanatostilbene-2,2′-disulphonic acid (DIDS), nifedipine, tetraethylammonium (TEA), 2-hydroxyethanesulfonic acid (sodium isethionate), niflumic acid (NFA), charybdotoxin (CbTX), apamin, 4-aminopyridine, 5-hydroxytryptamine (5-HT) (Sigma Chemical Co., USA); thapsigargin (Calbiochem, Germany); Fura-2 AM, pluronic acid (Molecular Probes, USA); iberiotoxin (IbTX) (Tocris, UK); ryanodine (Biomol, USA); caffeine (Alomone Labs Ltd, Israel). TRAM-34 (kindly supplied by Dr Heike Wulff) was synthesized from clotrimazole (Sigma) as described previously (Wulff et al. 2000). Stock solutions of U73122, U73343, BAPTA-AM, DIDS, NFA, TRAM-34 and thapsigargin were dissolved in DMSO (0.0001 %); nifedipine was dissolved in ethanol (0.0001 %) and charybdotoxin was dissolved in PBS containing 0.1 % BSA. All other drugs were made up as × 1000 stock solutions in distilled water and diluted into Krebs solution.

Results

Vasomotion in control preparations

Spontaneous, rhythmical contractions were observed in the rat basilar artery and its branches and they could be reliably recorded for more than 2 h. These contractions occurred at a rate of 16.5 ± 0.7 min−1 and had an average amplitude in control Krebs solution of 6.2 ± 0.6 % RVD (n = 84 animals). In those preparations used for electrophysiology, the mean resting vessel diameter was larger due to the tighter stretching required for long-term impalements in actively contracting vessels (57.1 ± 2.2 μm, n = 40), compared to those preparations used for measuring intracellular calcium (33.0 ± 1.0 μm, n = 44).

When intracellular recordings were made from SMCs, spontaneous rhythmical depolarizations were found to precede the spontaneous contractions by 1.6 ± 0.1 s (peak depolarization to peak contraction; Fig. 1A). These typically ranged from 5 to 25 mV in amplitude (10.3 ± 0.9 mV, n = 40) and the most negative membrane potential reached was −43.0 ± 0.9 mV (n = 40), a membrane potential similar to that observed in pressurized cerebral arteries (Knot & Nelson, 1998).

Figure 1. Spontaneous rhythmical contractions recorded in control Krebs solution.

Figure 1

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A, intracellular recordings show that spontaneous depolarizations precede spontaneous contractions. Using photometry and calcium imaging, calcium oscillations were recorded from the arterial wall and from individual SMCs and these preceded spontaneous contractions (B and C, respectively). Constriction is represented by a downward deflection while increases in [Ca2+]i are represented by an upward deflection. D, image of SMCs (arrows) loaded with Fura-2 AM, illuminated with 380 nm light. The black lines highlight the vessel edge bordering the area from which changes in vessel diameter were recorded. The scale bar is 10 μm.

Using photometry, calcium oscillations were recorded from the vessel wall and these oscillations preceded the spontaneous contractions by 0.9 ± 0.1 s (Fig. 1B; n = 5). With time, the basal calcium level increased (107.1 ± 2.5 % F340/380, P > 0.05; n = 5) and the vessel diameter decreased (94.4 ± 1.1 % RVD, P > 0.05, n = 5). Imaging of individual SMCs recorded after the onset of rhythmical contractions, revealed that calcium propagated in a wave-like manner from one end of the cell to the other (not shown). When averaged over the entire cell, these waves were recorded as oscillations similar to those recorded with photometry from the arterial wall (Fig. 1C). In a proportion of preparations, large regular contractions (11.9 ± 0.7 % RVD; n = 4) and well synchronized calcium oscillations were recorded (Fig. 2A). In others, contractions were smaller and irregular (5.1 ± 0.4 % RVD; n = 4), although the calcium oscillations in individual cells were of similar amplitudes to those seen in the previous group, but were more asynchronous (Fig. 2B). In both cases, calcium oscillations showed variations in amplitude between cells, as did sequential oscillations in individual cells (Fig. 2C).

Figure 2. Calcium oscillations in individual smooth muscle cells (SMCs).

Figure 2

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A, adjacent SMC profiles during vasomotion in which calcium oscillations were well synchronized and contractions robust, compared to preparations in which calcium oscillations were more asynchronous (B). C, calcium oscillations recorded from groups of adjacent cells were well synchronized, but showed variations in amplitude and in temporal characteristics (e.g. cell 2). Variations in the amplitude of calcium oscillations were also observed amongst the cells (boxed area). Increases in [Ca2+]i are represented as an upward deflection.

In a number of preparations, periodic variations in the amplitude of the spontaneous contractions, depolarizations (Fig. 3A) and oscillations in arterial wall calcium (Fig. 3B) were observed. In these preparations the amplitude of the contractions tended to be similar to the amplitude of the underlying depolarizations and calcium oscillations. Imaging of individual SMCs in preparations displaying this periodic pattern showed that calcium oscillations in individual cells were synchronized within small groups of two to three adjacent cells, while calcium oscillations in adjacent groups of cells behaved in an unsynchronized manner (Fig. 3C).

Figure 3. Spontaneous depolarizations (A) and oscillations in arterial wall calcium (B) show periodic variations in amplitude.

Figure 3

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The amplitude of the voltage changes and the calcium oscillations is proportional to the amplitude of the following contraction (A and B). Under these conditions (C), calcium oscillations in individual SMCs are synchronized within small groups of adjacent cells, while oscillations in adjacent groups of cells behave in an unsynchronized manner. Increases in [Ca2+]i are represented as an upward deflection.

Involvement of voltage-dependent calcium channels

To assess the involvement of extracellular calcium entering the SMCs via L-type VDCCs, preparations were exposed to the antagonist nifedipine (1 μm). Nifedipine resulted in a loss of the rhythmical contractions and the appearance of uncoordinated movements without apparent loss of vessel tone (96.9 ± 1.7 % RVD, P > 0.05, n = 12, Fig. 4A) within 4 min of the drug entering the recording chamber. Spontaneous depolarizations were rapidly abolished (P < 0.05) and the membrane potential depolarized (control: −42.7 ± 3.3 mV; nifedipine: −32.7 ± 2.8 mV; P < 0.05, n = 4). Measurement of arterial wall calcium revealed the presence of irregular calcium oscillations. Basal calcium levels were unaltered (101.0 ± 13.1 % F340/380; P > 0.05, n = 8, Fig. 4B). Imaging of SMCs showed that small calcium oscillations were present in individual cells, but these were unsynchronized (Fig. 4C). Superfusion with Ca2+-free Krebs solution led to a loss of spontaneous contractions in about 6 min. On washout, uncoordinated contractions were observed within 3 min and coordinated contractions were recorded by 6 min.

Figure 4. The effect of the voltage-dependent calcium channel antagonist nifedipine on rhythmical activity.

Figure 4

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A, Nifedipine (1 μm) abolished rhythmical depolarizations and contractions and depolarized SMCs (trace 4 min in drug). B, calcium oscillations recorded from the arterial wall became irregular (trace 20 min in drug). C, calcium oscillations recorded in individual, adjacent SMCs were reduced in amplitude and became asynchronous. Increases in [Ca2+]i are represented as an upward deflection.

Involvement of intracellular calcium stores

The calcium-ATPase inhibitor thapsigargin (2 μm; Fig. 5) and the intracellular calcium chelator BAPTA-AM (10 μm) were used to examine the role of intracellular calcium stores and intracellular signalling pathways. Both of these drugs abolished spontaneous contractions, rhythmical depolarizations and oscillations in both arterial wall calcium and in individual SMCs (P < 0.05), although the time course of action of the two drugs differed. The initial effect of thapsigargin was an increase in the amplitude of the voltage oscillations and contractions along with a small constriction, consistent with cessation of calcium clearance from the cytoplasm. This was followed sequentially by a decrease in the amplitude of the voltage oscillations and contractions, a loss of vasomotion, then hyperpolarization and relaxation. The effects of BAPTA-AM, on the other hand, were initially to reduce and abolish rhythmical depolarizations and contractions. At this point the membrane remained depolarized for several minutes before a hyperpolarization was associated with relaxation. The final end point for the two drugs, however, was that the membrane potential hyperpolarized (control: −40.0 ± 2.0 mV, thapsigargin: −50.5 ± 0.5 mV; control: −42.7 ± 3.7 mV, BAPTA-AM: −64.2 ± 3.1 mV; P < 0.05, n = 4), calcium levels across the vessel wall decreased (thapsigargin: 93.1 ± 1.7 %; BAPTA: 88.6 ± 4.5 % F340/380; P < 0.05, n = 4) and the artery relaxed (thapsigargin: 107.6 ± 1.4 % RVD; BAPTA: 111.9 ± 2.8 % RVD; P < 0.05, n = 8).

Figure 5. Thapsigargin abolishes spontaneous rhythmical activity.

Figure 5

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A, Ca2+-ATPase inhibition by thapsigargin (2 μm) hyperpolarized SMCs and abolished spontaneous depolarizations and contractions (trace 20 min in drug). B, calcium oscillations in the vessel wall were inhibited and basal calcium levels decreased (trace 20 min in drug). C, calcium oscillations in individual SMCs were also abolished. Increases in [Ca2+]i are represented as an upward deflection.

The role of phospholipase C and the IP3 pathway was tested using U73122 (10 μm), which reduced and then abolished the spontaneous contractions (P < 0.05, n = 4) and caused a relaxation of the vessel (U73122: 107.6 ± 1.2 % RVD, P < 0.05, n = 8, Fig. 6). Electrophysiological recordings revealed that voltage oscillations were also reduced in amplitude and abolished, and the membrane potential became significantly hyperpolarized (control: −43 ± 6.1 mV, U73312: −50.0 ± 8.1 mV; P < 0.05, n = 4, Fig. 6A). Measurements of arterial wall calcium showed that the calcium oscillations were abolished (P < 0.05) and the basal level of calcium was decreased (U73122: 92.7 ± 1.1 % F340/380, P < 0.05, n = 4, Fig. 6B). Calcium oscillations in individual SMCs were also abolished (Fig. 6C). The inactive isomer of U73122, U73343 (10 μm) had no effect on rhythmical activity (basal calcium: 110.7 ± 2.1 % F340/380; vessel diameter: 96.8 % RVD, P > 0.05, n = 4).

Figure 6. The effect of U73122 on spontaneous rhythmical activity.

Figure 6

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Phospholipase C inhibition by U73122 (10 μm) hyperpolarized SMCs and abolished spontaneous depolarizations and contractions (A, trace 9 min in drug), inhibited calcium oscillations in wall calcium (B, trace 20 min in drug) and in individual SMCs (C). Increases in [Ca2+]i are represented as an upward deflection.

In the majority of preparations, ryanodine (10 μm), which acts by inhibiting ryanodine receptors, abolished rhythmical contractions (P < 0.05) but did not alter vessel tone (ryanodine: 96.4 ± 1.1 % RVD, P > 0.05, n = 9, Fig. 7). However, in some preparations, small contractions were still observed (Fig. 7B). The spontaneous depolarizations were increased in frequency, but decreased in amplitude, due to a reduction in the hyperpolarizing phase of the voltage oscillations (control: −41.0 ± 3.0 mV, ryanodine: −32.7 ± 2.8 mV; P < 0.05, n = 5; Fig. 7A). Measurements of arterial wall calcium mirrored the effect of ryanodine on rhythmical depolarizations in that the oscillations were also increased in frequency (control: 21.0 ± 1.2 min−1, ryanodine: 29 ± 1.0 min−1, P < 0.05), but significantly decreased in amplitude (ryanodine: 38.1 ± 3.3 % of amplitude of control F340/380 oscillation, P < 0.05, n = 4) although the basal level of calcium was not different from control (ryanodine: 109.2 ± 4.9 % F340/380, n = 4, Fig. 7B). The response to ryanodine was observed within a 5-10 min period after exposure to the drug. Calcium oscillations in individual SMCs were not present (Fig. 7C), despite the fact that weak calcium waves could be observed directly with the ICCD camera. Addition of ryanodine, after nifedipine, led to the abolition of the asynchronous calcium waves, which were seen in nifedipine in individual cells (Fig. 4C), but had no further significant effect on arterial wall calcium levels or on vessel tone (112.1 ± 3.0 % F340/380, 95.0 ± 1.5 % RVD, P > 0.05, n = 4).

Figure 7. Effect of ryanodine on spontaneous rhythmical activity.

Figure 7

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A, ryanodine (10 μm) depolarized the SMCs and abolished rhythmical contractions (trace 15 min in drug). Spontaneous depolarizations and calcium oscillations recorded from the vessel wall were increased in frequency but decreased in amplitude (A and B). Ryanodine generally had no effect on vessel tone and in some preparations small contractions could also be recorded (B, trace 20 min in drug). C, calcium oscillations in individual SMCs were not recorded. Increases in [Ca2+]i are represented as an upward deflection.

In order to determine whether the IP3- and ryanodine-sensitive SR calcium stores spatially overlap or represent separate discrete calcium pools, preparations were exposed to 5-HT (1 μm), to release calcium from the IP3-sensitive calcium stores. This was carried out in the presence of both nifedipine (1 μm) and caffeine (1 mm), to prevent influx of calcium through VDCCs and to cause depletion from the ryanodine-sensitive store, respectively. In the presence of both nifedipine and caffeine, spontaneous contractions were abolished (P < 0.05) and the vessel relaxed (111.3 ± 4.1 % RVD; P < 0.05, n = 4). Addition of 5-HT caused the vessel to constrict (86.7 ± 4.1 % RVD recorded in nifedipine and caffeine; P < 0.05, n = 4) but had no effect on re-establishing rhythmical contractions.

Involvement of calcium-dependent chloride channels

In the presence of the calcium-dependent chloride channel inhibitors NFA (50 μm) and DIDS (100 μm), spontaneous depolarizations and contractions became smaller and then were abolished (P < 0.05, n = 4). DIDS subsequently hyperpolarized the membrane potential (control: −42.7 ± 1.6 mV, DIDS: −55.2 ± 5.7 mV) and both DIDS and NFA caused the vessel to relax (DIDS: 108.3 ± 2.8 % RVD; NFA: 103.5 ± 3.7 % RVD, P < 0.05, n = 4). Both inhibitors abolished the calcium oscillations in the arterial wall (P < 0.05, n = 4) and in individual SMCs. NFA had no significant effect on the basal calcium levels (NFA: 106.7 ± 7.5 % F340/380, P > 0.05, n = 4) while on the other hand, DIDS significantly decreased basal calcium levels in the arterial wall (86.8 ± 3.4 % F340/380, P < 0.05, n = 4). The effects of these inhibitors on vasomotion were observed 3-5 min after the drugs entered the recording chamber, and effects on hyperpolarization and relaxation were seen at about 10 min. Following chloride substitution, in which NaCl (120 mm) was replaced by equimolar sodium isethionate (120 mm + 3.3 mm CaCl2), the vessel constricted (isethionate: 86.1 ± 2.4 % RVD, P < 0.05, n = 10) and all rhythmical activity was abolished (P < 0.05, Fig. 8). Furthermore, the membrane potential depolarized (control: −40.6 ± 2.5 mV; isethionate: −30.6 ± 0.5 mV; P < 0.05, n = 5, Fig. 8A) and the voltage oscillations became smaller and were also abolished. The basal calcium levels in the arterial wall increased significantly (121.0 ± 6.6 % F340/380, P < 0.05, n = 5, Fig. 8B) and calcium oscillations in individual SMCs were also inhibited (Fig. 8C).

Figure 8. Effect of chloride substitution on rhythmical activity.

Figure 8

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A, chloride substitution with sodium isethionate (120 mm) depolarized SMCs and abolished rhythmical depolarizations and contractions (trace 11 min in drug). B, calcium oscillations in the arterial wall were abolished and the basal calcium level increased (trace 20 min in drug). C, calcium oscillations in individual SMCs were also abolished. Increases in [Ca2+]i are represented as an upward deflection.

Involvement of calcium-dependent potassium channels

Incubation in the large conductance calcium-activated potassium (BKCa) channel antagonists, iberiotoxin (100 nm) and TEA (1 mm), had no significant effect on the presence of vasomotion, depolarizations, resting membrane potential or calcium oscillations (P > 0.05, n = 4). However, in the presence of iberiotoxin, the basal calcium levels were significantly increased (126.3 ± 2.7 % F340/380, P < 0.05, n = 4) and in the presence of TEA, the amplitude of the calcium oscillations recorded in the arterial wall (122.6 ± 3.7 % F340/380 control oscillation, P < 0.05, n = 4) and the amplitude of the contractions were increased (119.2 ± 6.5 % control contraction, P < 0.05, n = 4). In the presence of TEA, oscillations in individual cells became more synchronized (n = 4).

Inhibition of BKCa and IKCa channels with charybdotoxin (60 nm) and IKCa channels with TRAM-34 (50 nm) rapidly decreased the vessel diameter (CbTX: 87.2 ± 1.9 % RVD, n = 8, Fig. 9; TRAM-34: 92.0 ± 1.0 % RVD; P < 0.05, n = 5) and depolarized the membrane potential (control: −41.7 ± 2.1 mV, CbTX: −30.5 ± 1.2 mV, Fig. 9A; control: −40.0 ± 1.7 mV, TRAM-34: −31.0 ± 1.9 mV; P < 0.05, n = 4). In addition, the frequency of the rhythmical contractions, depolarizations and calcium oscillations, in both the arterial wall and individual SMCs, was increased (control: 16.5 ± 2.2 min−1, CbTX: 24.7 ± 3.9 min−1, n = 8; control: 12.2 ± 0.7 min−1, TRAM-34: 17.1 ± 1.0 min−1, n = 5; P < 0.05) but decreased in amplitude (CbTX: 51.95 ± 9.9 % control amplitude, n = 8, Fig. 9A and B; TRAM-34: 46.7 ± 2.7 % control amplitude, n = 5; P < 0.05). Basal calcium levels in the arterial wall were also increased (CbTX: 123.9 ± 3.6 % F340/380, n = 4, Fig. 9B; TRAM-34: 123.3 ± 5.0 % F340/380, n = 5; P < 0.05). The effect of charybdotoxin and TRAM-34 on rhythmical activity was observed by 5 min after exposure to drug. Apamin (0.5 μm), an inhibitor of small conductance calcium-activated potassium (SKCa) channels, and 4-aminopyridine (100 μm), the voltage-gated potassium (Kv) channel inhibitor, had no significant effect on rhythmical activity (control: 12.0 ± 0.7 min−1, apamin: 12.2 ± 0.9 min−1; control: 15.0 ± 1.4 min−1, 4-AP: 14.4 ± 1.2 min−1; n = 4, P > 0.05), vessel diameter, membrane potential (P > 0.05), or basal calcium levels in the arterial wall (apamin: 106.6 ± 2.2 % F340/380, 4-AP: 110.5 ± 6.1 % F340/380; P > 0.05, n = 4).

Figure 9. Charybdotoxin increases the frequency of spontaneous rhythmical contractions.

Figure 9

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A, charybdotoxin (60 nm) increased the frequency of the rhythmical contractions and depolarizations and depolarized the SMCs (trace 5 min in drug). B, calcium oscillations in the arterial wall were also increased in frequency and the basal calcium level increased (trace 20 min in drug). C, calcium oscillations in individual SMCs were increased in frequency. Increases in [Ca2+]i are represented as an upward deflection.

Discussion

During spontaneous vasomotion of juvenile rat basilar arteries rhythmical oscillations in both membrane potential and SMC calcium were found to precede the rhythmical contractions. We have further shown that calcium release from intracellular IP3-sensitive stores is primarily responsible for the vasomotion since it leads to depolarization of the membrane through activation of calcium-dependent chloride channels. The depolarization in turn produces a cyclical voltage oscillation through calcium influx via VDCCs, calcium-induced calcium release from ryanodine stores and subsequent hyperpolarization through the activation of IKCa channels. The selectivity of the initial phase with IP3-sensitive stores and chloride channels, and the secondary phase involving ryanodine-sensitive stores, VDCC and IKCa provides support for compartmentalization and co-localization of different calcium stores with particular types of ion channels. This mechanism thus differs from that previously described for agonist-induced vasomotion in systemic vessels (Peng et al. 2001).

In the present study, the intracellular calcium chelator BAPTA-AM and the Ca2+-ATPase inhibitor thapsigargin abolished the spontaneous depolarizations, calcium oscillations and contractions, and hyperpolarized and relaxed the arteries, suggesting that the rhythmical events depend on intracellular signalling pathways and the cyclical release of calcium from an internal store. Calcium waves in individual SMCs were also abolished supporting previous data that calcium waves result from regenerative calcium release from the SR network (Iino et al. 1994; Ruehlmann et al. 2000; Peng et al. 2001). Since inhibition of phospholipase C with U73122 mimicked the effects of BAPTA-AM and thapsigargin, we suggest that IP3 and subsequent calcium release from the IP3-sensitive store is responsible for the initiation and maintenance of vasomotion. In juvenile rats, this pathway may be constitutively active in a manner similar to that previously described in juvenile rat iris arterioles (Haddock et al. 2002) or it may be activated by the stretch imposed during preparation of the vessels for the in vitro experiments.

In contrast to the effects of U73122, BAPTA-AM and thapsigargin, ryanodine caused an increase in frequency and decrease in amplitude of both the spontaneous depolarizations and calcium oscillations, and depolarized the cell membrane. These results suggest that calcium released from the ryanodine-sensitive store is not involved in the initiation of vasomotion but rather is involved in a negative feedback loop of the oscillatory cycle by activating a hyperpolarizing current. Such negative feedback has been described in cultured SMCs and pressurized cerebral arteries where ryanodine receptor channels are co-localized with VDCCs and BKCa channels, forming a functional unit to control levels of intracellular calcium and promote relaxation (see Jaggar et al. 2000 for review). Interestingly, however, calcium from the ryanodine store seems to be also essential for the contractile phase since rhythmical contractions, but not depolarizations, were usually abolished after treatment with ryanodine.

Inhibition of calcium influx through VDCCs abolished the rhythmical contractions, rhythmical depolarizations and calcium oscillations in the arterial wall and depolarized the cell membrane. Calcium oscillations in individual SMCs, although reduced in amplitude, were no longer synchronized. Thus, calcium entering via VDCCs is essential for coordinated vasomotion, but not for individual intracellular calcium oscillations. In the rat mesenteric artery, inhibition of VDCCs also led to a loss of synchronicity of intracellular calcium waves (Peng et al. 2001). On the other hand, in the present study, vessel tone and basal calcium levels were not affected.

The depolarization seen in the presence of nifedipine, like that seen in ryanodine, suggests that calcium influx through VDCCs is responsible for the ryanodine activation of a hyperpolarizing current, while the maintenance of basal calcium levels and vessel tone supports the suggestion that it is calcium from the IP3-sensitive store which contributes to basal tone. Voltage dependence in the activation of ryanodine stores has been reported previously in cerebral vessels (Jaggar et al. 1998). Co-localization of VDCCs and ryanodine-sensitive receptors, but spatial separation of the ryanodine-sensitive and IP3-sensitive stores, is supported by the observation of BAPTA-AM initially abolishing voltage oscillations and contractions, and secondarily hyperpolarizing the cell membrane and relaxing the vessels. On the other hand, the loss of the asynchronous calcium oscillations in individual cells following the addition of ryanodine after VDCC influx has been blocked by nifedipine suggests that a component of calcium released from the IP3-sensitive store could be involved in activating calcium-induced calcium release from ryanodine-sensitive stores. The maintenance of tone under these conditions further supports the contribution of calcium released from the IP3-sensitive store to this phenomenon. Our data support the existence of spatially discrete IP3-sensitive and ryanodine-sensitive calcium stores, since release of calcium from the IP3-sensitive store by 5-HT, in the presence of both nifedipine to block voltage-dependent calcium influx and caffeine to deplete ryanodine-sensitive stores, still caused vessel constriction.

Under physiological conditions, cerebral arteries are subjected to intraluminal pressure which has been shown to cause SMC membrane depolarization to approximately −40 mV, activation of VDCCs, increased intracellular calcium levels and vasoconstriction (Harder, 1987; Knot & Nelson, 1998). Activation of a calcium-dependent chloride channel has been implicated in the depolarization of the membrane potential and opening of VDCCs in response to agonist stimulation (Large & Wang, 1996) and in the depolarization associated with myogenic contraction to pressurization (Doughty & Langton, 2001). In the present study, in which the SMCs were depolarized to a similar value, chloride (Cl−) substitution by channel-impermeant isethionate abolished all rhythmical activity and produced vasoconstriction in association with further membrane depolarization and increased arterial wall calcium levels. Given that Cl− substitution would move the equilibrium potential for Cl− toward more positive values, we suggest that the results are consistent with the depolarization resulting from the activation of a calcium-dependent chloride channel. In support of this finding, the calcium-dependent chloride channel antagonists, NFA and DIDS, abolished spontaneous depolarizations, calcium oscillations in the arterial wall and rhythmical contractions, and hyperpolarized the cell membrane. Interestingly, exposure to DIDS caused the basal calcium levels in the arterial wall to decrease, suggesting that this drug may have additional effects on intracellular calcium stores (see also Haddock et al. 2002). On the other hand, NFA had no effect on basal calcium levels suggesting that its effects were selective on calcium-dependent chloride channels as described previously in vascular smooth muscle cells (Hogg et al. 1994; Criddle et al. 1997).

Previous studies in cerebral vessels have shown that calcium release from ryanodine-sensitive stores can directly activate BKCa channels to cause membrane hyperpolarization and vessel relaxation (Benham & Bolton, 1986; Nelson et al. 1995). However, in the present study, despite an increase in the contraction amplitude in the presence of TEA and an overall increase in the basal calcium levels and vessel tone in the presence of iberiotoxin, blocking BKCa channels had little effect on rhythmical activity or membrane potential. In fact, it has been proposed that TEA can non-specifically activate VDCCs (Gustafsson & Nilsson, 1994; Hirst et al. 1996), which is suggested in the present study by the synchronization of calcium oscillations in individual SMCs. Inhibition of SKCa channels or Kv channels also had no effect on membrane potential or rhythmical activity. On the other hand, charybdotoxin, an antagonist of both BKCa and IKCa channels (Neylon et al. 1999) caused rapid depolarization of the membrane potential and vessel constriction. Charybdotoxin also increased the frequency, but decreased the amplitude of spontaneous depolarizations, calcium oscillations and contractions in a manner similar to that observed in the presence of ryanodine, suggesting that the residual oscillations result from the continued opening and closing of VDCCs. As the calcium oscillations were larger after inhibition of IKCa channels than after ryanodine, it appears that the voltage-dependent calcium influx is amplified by calcium-induced calcium release from the ryanodine-sensitive store.

The results above suggested that IKCa channels, but not BKCa channels, were involved in the basilar artery rhythmical activity. Moreover, these data provided support for a sequential relationship between the release of calcium from the ryanodine-sensitive calcium store and activation of this channel. To confirm the involvement of IKCa channels, we tested TRAM-34, a potent and selective blocker of IKCa channels. This drug has been derived from clotrimazole, but lacks the inhibitory effect on cytochrome P450 enzymes (Wulff et al. 2000). As TRAM- 34 mimicked the effect observed in the presence of charybdotoxin, our results show that activation of IKCa, following release of calcium from the ryanodine-sensitive stores and subsequent membrane hyperpolarization, is essential to negatively modulate both vessel tone and rhythmical activity of the juvenile rat basilar artery. It is interesting to note that smooth muscle IKCa channels, but not BKCa channels, have recently been shown to play a role in regulating the functional properties of immature smooth muscle cells (Neylon et al. 1999).

During spontaneous vasomotion we have been able to record calcium waves in individual SMCs similar to those observed in small mesenteric arteries following agonist stimulation (Mauban et al. 2001). When averaged for a single cell, these waves appeared as an oscillation. These oscillations frequently showed variations in amplitude and in temporal characteristics, suggesting that movement of calcium through SMC gap junctions is not occurring in this preparation, as suggested previously (Miriel et al. 1999). Calcium oscillations within individual cells during vasomotion appeared to be approximately synchronized, although the absolute degree was variable. In those arteries exhibiting more robust contractions, calcium oscillations between adjacent cells appeared to be well synchronised. In addition, a number of preparations showed periodic variations in the amplitude of the rhythmical contractions and depolarizations. In these preparations, similar oscillatory patterns of arterial wall calcium were recorded and calcium oscillations in individual SMCs appeared to be synchronised within only small groups of adjacent cells, while calcium oscillations in adjacent groups of cells were out of phase. Thus, in the present study, there appear to be variations in the degree of cell coupling, presumably through differences in the regulation of gap junctions, which affect the spread of current along the vessel, resulting in differential patterns of calcium release, and the appearance of coordinated contraction.

In summary, spontaneous vasomotion of basilar arteries of juvenile rats results from an interaction between voltage-independent and voltage-dependent events. Constitutive or stretch-induced activity of the phospholipase C pathway leads to an increase in basal calcium levels and a depolarizing current through the opening of ClCa channels in the membrane. The depolarization in turn leads to the influx of extracellular calcium through VDCCs activating ryanodine receptors to cause further release of calcium and the activation of IKCa channels. The subsequent hyperpolarizing current inactivates VDCCs promoting relaxation. The coordinate loss of activation of ryanodine-sensitive stores leads to closure of the IKCa channels and reopening of VDCCs to repeat the cycle. Gap junctions coordinate the spread of the depolarizing current and the resulting calcium influx through VDCCs entrains the ryanodine stores to synchronize the contractile activity. While a role for the endothelium in basilar artery vasomotion cannot be excluded, our preliminary experiments show that removal of the vascular endothelium results in asynchronous calcium oscillations in individual SMCs, without affecting the amplitude of the oscillations or the basal calcium in the arterial wall. This would suggest that the endothelium plays an important role in coordinating calcium fluxes in individual SMCs, presumably through electrical coupling, rather than contributing to them. Interestingly, myoendothelial gap junctions and large endothelial cell gap junctions are present in the juvenile rat basilar artery (S. L. Sandow, unpublished observations) providing the pathway for such electrical coupling.

Acknowledgments

We appreciate the valuable discussions with Professor G. D. S. Hirst concerning the manuscript and thank Dr H. Wulff for the kind gift of TRAM-34.

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