Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure

Abstract

1. The regulation of intracellular [Ca2+] in the smooth muscle cells in the wall of small pressurized cerebral arteries (100-200 micron) of rat was studied using simultaneous digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2. Elevation of intravascular pressure (from 10 to 100 mmHg) caused a membrane depolarization from -63 +/- 1 to -36 +/- 2 mV, increased arterial wall [Ca2+] from 119 +/- 10 to 245 +/- 9 nM, and constricted the arteries from 208 +/- 10 micron (fully dilated, Ca2+ free) to 116 +/- 7 micron or by 45 % ('myogenic tone'). 3. Pressure-induced increases in arterial wall [Ca2+] and vasoconstriction were blocked by inhibitors of voltage-dependent Ca2+ channels (diltiazem and nisoldipine) or to the same extent by removal of external Ca2+. 4. At a steady pressure (i.e. under isobaric conditions at 60 mmHg), the membrane potential was stable at -45 +/- 1 mV, intracellular [Ca2+] was 190 +/- 10 nM, and arteries were constricted by 41 % (to 115 +/- 7 micron from 196 +/- 8 micron fully dilated). Under this condition of -45 +/- 5 mV at 60 mmHg, the voltage sensitivity of wall [Ca2+] and diameter were 7.5 nM mV-1 and 7.5 micron mV-1, respectively, resulting in a Ca2+ sensitivity of diameter of 1 mum nM-1. 5. Membrane potential depolarization from -58 to -23 mV caused pressurized arteries (to 60 mmHg) to constrict over their entire working range, i.e. from maximally dilated to constricted. This depolarization was associated with an elevation of arterial wall [Ca2+] from 124 +/- 7 to 347 +/- 12 nM. These increases in arterial wall [Ca2+] and vasoconstriction were blocked by L-type voltage-dependent Ca2+ channel inhibitors. 6. The relationship between arterial wall [Ca2+] and membrane potential was not significantly different under isobaric (60 mmHg) and non-isobaric conditions (10-100 mmHg), suggesting that intravascular pressure regulates arterial wall [Ca2+] through changes in membrane potential. 7. The results are consistent with the idea that intravascular pressure causes membrane potential depolarization, which opens voltage-dependent Ca2+ channels, acting as 'voltage sensors', thus increasing Ca2+ entry and arterial wall [Ca2+], which leads to vasoconstriction.

Figure 1. Experimental methods

Illustration of the membrane potential impalements into smooth muscle cells in an artery pressurized to 60 and 100 mmHg. The edge detection of the arterial wall and ratio images of the artery loaded with fura-2 at 60 and 100 mmHg (± nisoldipine) are shown. All experiments were done at 37°C.

Figure 6. Steady-state effects of intravascular pressure on arterial wall Ca²⁺ and diameter

The effects of intravascular pressure on arterial wall Ca²⁺ (A) and diameter (B) in PSS (± nisoldipine, ± external calcium) are illustrated. The steady-state increases in arterial wall Ca²⁺ and decreases in arterial diameter (relative to the diameter in Ca²⁺-free PSS) were measured under the following conditions: in PSS (•), in Ca²⁺-free PSS (^), and in PSS with 10 nm nisoldipine (▴).

Figure 2. Determination of the loading condition of fura-2 AM

Effect of the loading concentration of fura-2 AM on diameters of arteries bathed in normal PSS (6 mm K⁺) and in elevated K⁺ (61 mm). The pressurized (60 mmHg) cerebral arteries were incubated with 0, 2 and 10 μm fura-2 AM for 45 min at room temperature (∼21°C). Pressurized cerebral arteries dilate fully in response to blockers of L-type Ca²⁺ channels. This level was taken as 100 % dilatation and is indicated by the horizontal dotted line. *Statistically significant at P < 0.05. R is the ratio between emission signals, when exciting with 340 and 380 nm light; R_min is R under Ca²⁺-free conditions (5 mm EGTA); R_max is R under Ca²⁺-saturated conditions (2.4 mm calcium); β is the ratio between the emission signals when exciting with 380 nm light under Ca²⁺-free and Ca²⁺-saturated conditions.

Figure 3. Determination of the in situ K_d of fura-2

The relationship between free calcium in the buffer and fluorescence ratio of fura-2 loaded pressurized (60 mmHg) cerebral arteries that had been permeabilized to calcium (see Methods and Results; n= 11) is shown.

Figure 4. Intravascular pressure depolarizes pressurized cerebral arteries

The relationship between membrane potential and intravascular pressure is illustrated. Each point represents one measurement of membrane potential. The continuous line represents a polynomial fit to the data. Data were collected from a total of 14 arteries.

Figure 5. Intravascular pressure elevates intracellular Ca²⁺ and constricts pressurized arteries

The effects of pressure steps from 10 to 60 mmHg on arterial wall calcium and diameter, in the absence (A) and presence (B) of the voltage-dependent calcium channel inhibitor diltiazem.

Figure 8. The membrane potential of pressurized posterior cerebral arteries is close to the theoretical K⁺ equilibrium potential when K⁺ is elevated above 16 mm

Effect of elevated K⁺ on the membrane potential recorded from pressurized (60 mmHg) cerebral arteries at different external [K⁺]. □, data ±s.e.m. The dotted line is added to the graph representing the theoretical (Nernst) K⁺ equilibrium potential (E_K) as a function of external [K⁺], and assuming an intracellular [K⁺] of 145 mm (at 37°C). Membrane potential of arteries bathed in external potassium lower than 11 mm deviated from the Nernst prediction.

Figure 7. The effects of external potassium on arterial wall Ca²⁺ and diameter at a constant (isobaric at 60 mmHg) pressure

Original trace showing the effect of elevation of external K⁺ from 6 to 81 mm on arterial wall Ca²⁺ (A) and arterial diameter (C). Elevation of K⁺ (from 6 to 11 mm) causes a membrane potential hyperpolarization through activation of inward rectifier potassium channels, which leads to a decrease in arterial wall Ca²⁺ and vasodilatation (Knot et al. 1996). The horizontal dotted line indicates the calcium and diameter level of arteries pressurized to 60 mmHg and bathed in PSS (6 mm external potassium). B, bar graph indicating mean ±s.e.m. arterial Ca²⁺ as a function of the external [K⁺]. The effect of external [K⁺] on arterial wall Ca²⁺ and diameter was completely reversed by the addition of 10 nm nisoldipine to the superfusing buffer at 81 mm K⁺. D, bar graph indicating mean ±s.e.m. arterial diameter as a function of the external [K⁺].

Figure 9. Membrane potential dependence of arterial wall [Ca²⁺] and diameter in pressurized cerebral arteries at 60 mmHg

Membrane potential dependence of arterial wall Ca²⁺(A) and diameter (B). Open symbols represent values measured in the presence of nisoldipine (Nis). Measured membrane potentials are indicated by open and filled circles. Filled squares represent Ca²⁺ values plotted against the calculated membrane potential (from Fig. 8). The horizontal dashed line indicates the level of Ca²⁺ or diameter in the presence of 10 nm nisoldipine. The vertical dashed line indicates the membrane potential, [calcium] and diameter of arteries pressurized to 60 mmHg in physiological salt solutions. Also annotated are the hyperpolarized condition in 16 mm K⁺ (16K) and the depolarized condition in 61 mm K⁺ (61K).

Figure 10. Arterial diameter as a function of arterial wall [Ca²⁺] in pressurized cerebral arteries at 60 mmHg

Data are derived from cross-correlation of measured data points taken from the relationships in Fig. 9A and B. The [calcium]-diameter relationship of pressurized arteries bathed in physiological salt solution is indicated. This relationship should describe the behaviour of pressurized (to 60 mmHg) cerebral arteries in response to changes in intracellular calcium. Any agent that increased or decreased the calcium sensitivity of the arteries would shift this relationship to the left or the right.

Figure 11. Elevation of arterial wall [Ca²⁺] in response to increased intravascular pressure depends on the membrane potential

[Ca²⁺] levels at 20, 40, 60, 80, 100 and 110 mmHg are plotted against the measured membrane potentials at those pressures. Also plotted are the measurements of Ca²⁺ as a function of membrane potential under isobaric conditions at 60 mmHg (the continuous line is a fit through the data in Fig. 9A). Nisoldipine (10 nm) prevents the increase in Ca²⁺ to changes in membrane potential under isobaric (via external K⁺, horizontal dashed line from Fig. 9A) and non-isobaric (through changes in pressure) conditions. The two dashed lines indicate the boundaries of the 95 % confidence interval for the fit.