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

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 [Ca(2+)](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 [Ca(2+)](i) were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca(2+)](i) oscillations in the arterial wall became reduced and irregular, while [Ca(2+)](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 [Ca(2+)](i) oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP(3) (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.

Figure 1. Spontaneous rhythmical contractions recorded in control Krebs solution

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.

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

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.

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

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.

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

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.

Figure 5. Thapsigargin abolishes spontaneous rhythmical activity

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.

Figure 6. The effect of U73122 on spontaneous rhythmical activity

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.

Figure 7. Effect of ryanodine on spontaneous rhythmical activity

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.

Figure 8. Effect of chloride substitution on rhythmical activity

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.

Figure 9. Charybdotoxin increases the frequency of spontaneous rhythmical contractions

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.