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J Physiol. Apr 1, 1998; 508(Pt 1): 199–209.
PMCID: PMC2230857

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 μm) 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 μm (fully dilated, Ca2+ free) to 116 ± 7 μm 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 μm from 196 ± 8 μm 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 μm mV−1, respectively, resulting in a Ca2+ sensitivity of diameter of 1 μm 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.

Intracellular Ca2+ plays a pivotal role in electromechanical coupling in muscle, including the vascular smooth muscle of the arterial wall. However, little is known about the physiological levels of intracellular Ca2+, and its regulation by membrane potential in the smooth muscle cells of small arteries subjected to physiological intravascular pressures. Elevation of intravascular pressure causes a graded membrane potential depolarization of the smooth muscle cells in small (i.e. resistance sized) arteries, and causes a graded constriction (‘myogenic tone’) (Bayliss, 1902; Harder, 1984; Brayden & Nelson, 1992; Meininger & Davis, 1992; Knot & Nelson, 1995). Pressure-induced constrictions of rat cerebral arteries as well as many other types of small arteries does not directly depend on endothelial or neural factors (Meininger & Davis, 1992; Knot, Zimmermann & Nelson, 1996). The constriction in response to pressure, but not the depolarization, in small cerebral arteries, is blocked by inhibitors of L-type voltage-dependent Ca2+channels (Brayden & Nelson, 1992; Knot & Nelson, 1995). At a fixed pressure, arterial diameter is very sensitive to membrane potential, with membrane hyperpolarization causing vasodilatation, a mechanism common to many endogenous and synthetic vasodilator compounds that activate K+ channels (Nelson, Patlak, Worley & Standen, 1990; Nelson & Quayle, 1995). Conversely, many vasoconstrictors have been shown to depolarize arterial smooth muscle. Intravascular pressure has been shown to elevate intracellular [Ca2+] in cremaster muscle arterioles (Meininger, Zawieja, Falcone, Hill & Davey, 1991; D'angelo, Davis & Meininger, 1997). However, the underlying mechanism or precise relationships amongst membrane potential, arterial wall [Ca2+] and blood vessel diameter have not been completely defined in cerebral or other small arteries.

The ionic basis by which pressure depolarizes cerebral arteries is incompletely understood. Inhibitors of voltage-dependent calcium channels, ATP-sensitive potassium channels or calcium-sensitive potassium channels did prevent pressure-induced membrane potential depolarizations (Knot & Nelson, 1995; Knot et al. 1996). Removal of extracellular sodium did not affect pressure-induced responses, arguing against a sodium-permeable channel participating in this response (Nelson, Conway, Knot & Brayden, 1997). Recent evidence suggests that pressure-induced depolarizations involve the activation of chloride channels (Nelson et al. 1997).

The goals of this study were to determine the levels of intracellular Ca2+ in pressurized cerebral arteries, and determine its regulation by intravascular pressure and membrane potential. Further, using organic Ca2+ channel inhibitors, we sought to determine the pathways for Ca2+ entry in myogenic cerebral arteries.

In this study, we provide for the first time the relationship between intravascular pressure in the physiological range, membrane potential and arterial diameter in intact resistance-sized arteries from brain. Further, we provide the relationship between membrane potential, arterial wall [Ca2+] and diameter at a steady pressure, a condition, in which arteries would normally operate, and from which they can dilate or constrict upon demand in response to vasoactive stimuli.

Our results are consistent with the idea that intravascular pressure increases arterial wall [Ca2+] through changes in smooth muscle membrane potential, which activates L-type voltage-dependent Ca2+ channels. Arterial diameter was steeply dependent on membrane potential and arterial wall [Ca2+]. These results support the idea that small changes in membrane potential and intracellular calcium can have profound effects on vessel diameter (Nelson et al. 1990) via changing the activity of voltage-dependent Ca2+ channels.

METHODS

Preparation

Female Sprague-Dawley rats (12–14 weeks, ca 228 g) were killed with pentobarbitone (150 mg (kg body wt)−1 by intraperitoneal injection), followed by thoracotomy, removal of the heart, decapitation and removal of the whole brain. The brain was quickly transferred to an oxygenated physiological salt solution (PSS, for composition see below) at 0–4°C on melting ice. Intact isolated cannulated pressurized distal posterior cerebral arteries were used in this study. All experiments with intact arteries were done under continuous superfusion with PSS (3–6 ml min−1) at 37°C. All experiments involving animals were conducted in accordance with the guide for the care and use of laboratory animals (NIH publication 85–23, 1985) following a protocol approved by the Institutional Animal Use and Care Committee of the University of Vermont, USA.

Recording methods (see also Fig. 1)

Figure 1
Experimental methods

Arterial membrane potential

Membrane potential was recorded in intact pressurized arteries using conventional intracellular glass microelectrodes filled with 3 m KCl solution, and tip resistances of 40–60 MΩ, as described previously (Knot et al. 1996). Cerebral arterial smooth muscle cells were impaled from the cleaned adventitial side of the pressurized artery. Membrane potentials were recorded with an Neuroprobe amplifier (0.5–1 kHz bandwidth; Model 1600, A-M Systems Inc.) at an acquisition rate of 0.5–2 Hz. Criteria for acceptance of recordings were: (1) an abrupt change in potential on impalement of the cell; (2) stable membrane potential for at least 2 min before experimental manipulations; (3) maintained impalement throughout the experimental protocol; (4) unchanged tip resistance before and after impalement; and (5) change in tip potentials of less than 3 mV. In the figures the sudden voltage changes associated with impalement and exit from a cell have been removed for clarity (but see Fig. 1). The analog output voltage of the amplifier was recorded using a data acquisition system (see below).

Arterial wall [Ca2+]

The arteries were loaded with the ratiometric Ca2+ sensitive fluorescent dye fura-2 (Grynkiewicz, Poenie & Tsien, 1985). Fura-2 AM (the acetoxymethyl ester form of fura-2) was dissolved in dry DMSO as a 1 mm stock and frozen in 50 μl aliquots until used. Shortly before use, fura-2 AM was mixed with an equal volume of a 25 % (w/v) solution of pluronic acid in DMSO. The fura-2 AM-pluronic acid mixture was diluted in 500 μl of the physiological salt solution to yield a final concentration of fura-2 AM of 2 μm (see below for the determination of this loading concentration). The ends of the arteries were closed by pinching to prevent the fura-2 AM solution from diffusing into the vessel lumen where it could conceivably load the endothelium (see below for control experiments to exclude this possible measurement interference), and incubated in the 2 μm fura-2 AM in PSS loading solution at room temperature (ca 21°C) in the dark for 45 min (see Results section for determination of the loading conditions). Fura-2 loaded arteries were washed, and kept in ice-cold PSS without dye. Fura-2 loaded arteries were cannulated, and mounted in a specially designed close-working distance arteriograph (H. J. Knot & G. Kennedy, Instrumentation & Model Facility (IMF), University of Vermont, Burlington, VT, USA) allowing the artery to be within 300 μm of the arteriograph's 150 μm-thick glass bottom. The arteriograph, with the cannulated vessel, was placed on the stage of an Nikon Diaphot 200 microscope equipped with a ×20 CF Fluor objective (NA = 0.75). Superfusion was started immediately. After a 20 min equilibration period, intravascular pressure was gradually increased from 2 to 60 mmHg in oxygenated PSS under continuous superfusion (3–6 ml min−1) at 37°C. Arteries that did not constrict to pressure (i.e. did not develop myogenic tone) were not used. A transmural isobaric pressure of 60 mmHg was used in most protocols. This is the estimated half-systolic pressure these vessels experience in situ (McCarron, Osol & Halpern, 1989). Ratio images were obtained at a rate of 0.2 Hz from background-corrected four-frame averaged images of the 510 ± 40 nm emission from the arteries alternately excited at 340 and 380 nm using the Image-1/FL quantitative ratio imaging software (Universal Imaging Corp., West Chester, PA, USA). Averaged ratio values from a selected region of interest (typically the whole artery image, see Fig. 1) were stored on disk and converted in real time via a Data Translation DT2814 digital to analog converter (Data Translation, Marlboro, MA, USA) to an analog output voltage.

Arterial diameter

Arterial diameter of fura-2 loaded arteries was measured from live video of the background-corrected ratio images or from videotaped Ca2+-fura-2 imaging experiments. Arterial diameter was measured using the length-calibrated edge-detection function of the Image-1/AT imaging software (Universal Imaging Corp.), at a sampling rate of 2 Hz. Arterial diameter measurements of non-fura-2 loaded arteries was performed similarly using the calibrated edge-detection function of the Image-1/AT imaging software at 2 Hz on the video signal from a Hamamatsu SIT or a Javelin CCD camera. Results were written to the imaging computer's hard disk in ASCII file format of diameter (μm) vs. time (s) and converted to an analog output voltage for external recording. A constriction in response to pressure refers to arterial diameter relative to the diameter in Ca2+-free PSS (presumably the passive diameter) at a given pressure.

Combined arterial function

All analog output signals representing physiological parameters of arterial function (intravascular pressure, membrane potential, Ca2+-ratio values and diameter) were recorded using Axotape 2.0 software (Axon Instruments) and an Indec IBX (Indec Systems Inc., Sunnyvale, CA, USA) data acquisition system on a PC (Gateway 386/20DX), thus allowing synchronized recording at 2 Hz.

Experimental protocols and controls

Where applicable, 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 et al. 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 arteries loaded with fura-2, the absence of endothelial loading was also verified by stimulating the arteries with acetylcholine (10 μm) or bradykinin (100 nm) in the presence of a Ca2+ channel blocker. The absence of an endothelial Ca2+ signal was interpreted as a lack of endothelial loading.

To minimize structural alterations of the arteries, most of the experiments done in this study were done in intact arteries with the endothelium present. Removal of the endothelium does not affect either pressure-induced responses in this artery or the actions of ryanodine and iberiotoxin (McCarron et al. 1989; Brayden & Nelson, 1992; Knot & Nelson, 1995; Nelson et al. 1995; Knot et al. 1996). In the current studies, we did test the effects of the removal of the endothelium on elevated potassium, and on ryanodine and iberiotoxin responses (see Fig. 6 in Knot, Standen & Nelson, 1998). Since removal of the endothelium did not alter the responses that we were examining, we chose to use intact arteries for the majority of experiments.

Solutions, compounds and drugs

Composition of PSS

A physiological salt solution (PSS) was used as the bathing solution and had the following composition (mm): NaCl, 119; KCl, 4.7; NaHCO3, 24.0; KH2PO4, 1.2; CaCl2, 1.6; MgSO4, 1.2; EDTA, 0.023; and glucose, 11.0 (pH 7.4). This solution was continuously bubbled with 95 % O2-5 % CO2 and heated to 37°C. The arteries were superfused at 3–6 ml min−1 (bath volume, ca 3 or 6 ml). High external potassium solutions were made by isosmotic replacement of NaCl with KCl in the PSS.

Chemicals and drugs used

Fura-2 AM was purchased from Molecular Probes. Iberiotoxin was obtained from Peptides International (Louisville, KY, USA). All other salts and drugs were obtained from Sigma. Nisoldipine was a gift from Dr A. Scriabine of Miles Laboratories (West Haven, CT, USA).

Data analysis, statistics and presentation

Membrane potential values were determined as the average membrane potential measured over a 30 s, cursor selected data segment. Data segments were taken before and after addition or removal of a substance when membrane potential had reached a new steady level. For this purpose Axotape binary data files were imported into Origin (Microcal Software Inc., Northampton, MA, USA) using the pCLAMP module of this program. For graphical representation, selected data plots were exported from Origin as windows metafiles into CorelDraw (Corel Corp., Ottawa, Canada) and processed further (thickening of the trace and axes labelling). Membrane potential values are expressed in millivolts as means ± sample standard deviation of pooled individual impalements from n different arteries. Arterial [Ca2+] was calculated using the following equation (from Grynkiewicz et al. 1985):

equation image

At the end of every experiment Rmin and Rmax were measured from ionomycin treated arteries (see Results for the composition of this solution), and β determined. For every set of experiments within a protocol, these values were pooled and used to convert the averaged ratio values (R) into a [Ca2+]. Kd was determined separately using an in situ titration of Ca2+ in fura-2 loaded arteries (see Results). The variances between mean Rmax, Rmin and β values were compared within and between experimental protocols. An ANOVA was performed on these values to ensure that all measurements could be compared across experimental protocols. The pooled mean values for Rmax, Rmin and β were 2.31 ± 0.14, 0.43 ± 0.03 and 2.08 ± 0.15 (means ±s.d.), respectively. Since no changes were made to the loading conditions or the optical light path of the imaging equipment, these calibration values remained very similar throughout this study.

Diameter values, when recorded seperately, were imported as ASCII files of diameter vs. time into Origin and combined with the Ca2+, pressure and membrane potential data. Deliberate closures of the light path were used to create tags in these data to provide synchronization with the diameter measurements. Data were analysed using Origin. For graphical representation, selected data plots were exported from Origin as windows metafiles into CorelDraw, and processed further (thickening of the trace and axes labelling). Diameter values are expressed in micrometres as means ±s.e.m. for n vessels. Statistical significance was tested at the 95–98 % confidence level using Student's paired or unpaired t test where applicable. Significant difference vs. control values is indicated in graphs by an asterisk or dagger on top or under the bar or data point.

RESULTS

Determination of the loading condition for fura-2 Ca2+ measurements in intact pressurized arteries

The de-esterified fluorescent dye fura-2 introduces an additional mobile Ca2+ buffer to the cytosol of the smooth muscle cells of the arterial wall, and thus could conceivably alter the contractile response of the arteries, if its intracellular concentration were too high. Therefore, a criterion for choosing the appropriate loading concentration of fura-2 would be its lack of effect on the contractile response of cerebral arteries to pressure and external potassium. To explore this issue, arteries were loaded in the presence of different concentrations (0, 1, 2, 5 and 10 μm) of fura-2 AM for 45 min (at room temperature). Pressure (60 mmHg)- and high potassium (61 mm)-induced constrictions were unaffected by 2 μm fura-2 AM (Fig. 2), which was then used as the loading concentration in this study.

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

Estimation of Kd of fura-2 in intact arteries

The apparent dissociation constant (Kd) of fura-2 for calcium was estimated in situ in pressurized (60 mmHg) cerebral arteries. The calcium permeability of the smooth muscle cells in the arterial wall was increased by 10 μm ionomycin, and the arteries were superfused with a bath solution containing 140 mm KCl, 20 mm NaCl, 5 mm Hepes, 5 mm EGTA, 1 mm MgCl2 and 5 μm nigericin, at pH 7.15 (adjusted with KOH) for a minimum of 10 min (Sato, Ozaki & Karaki, 1988; Williams & Fay, 1990; Jensen, Mulvany, Aalkjaer, Nilsson & Yamaguchi, 1993; Khalil, Lajoie & Morgan, 1994; Morgan & Jacob, 1994; Nilsson, Jensen & Mulvany, 1994; Chen & Rembold, 1995). Extracellular free [Ca2+] in this bathing solution was changed by altering the level of total Ca2+, and calculated using an EGTA stability constant of 6.79 (Martell & Smith, 1974; Perrin, 1979). The effect of changing bath free [Ca2+] on arterial fluorescence was then determined (Fig. 3), yielding an apparent Kd of 282 nm (obtained from a linear fit to averaged data points in a double logarithmic plot) (Jensen et al. 1993), which compares well with estimates by others (Sato et al. 1988; Williams & Fay, 1990; Jensen et al. 1993; Nilsson et al. 1994). This Kd value was subsequently used for the estimates of intracellular free [Ca2+] in this study.

Figure 3
Determination of the in situ Kd of fura-2

Intravascular pressure depolarizes, elevates intracellular calcium of and constricts intact pressurized cerebral arteries

Elevation of intravascular pressure causes a graded membrane potential depolarization and vasoconstriction of smooth muscle cells in a number of different types of small myogenic arteries (Harder, 1984, 1985; Brayden & Nelson, 1992; Knot & Nelson, 1995). The pressure-induced constriction, but not the membrane potential depolarization (Knot & Nelson, 1995), was prevented by inhibitors of voltage-dependent calcium channels (Brayden & Nelson, 1992; Knot & Nelson, 1995; Nelson et al. 1995; Knot et al. 1996). Elevating intravascular pressure from 10 to 100 mmHg depolarized posterior cerebral arteries from -63 ± 1 mV (n= 3) to about -36 ± 2 mV (n= 5; Fig. 4). The mean membrane potential at 60 mmHg was -45.2 ± 1.4 mV (n= 27; pooled value from all control measurements at this pressure).

Figure 4
Intravascular pressure depolarizes pressurized cerebral arteries

The observed membrane potential depolarization caused by an elevation of intravascular pressure should increase intracellular [Ca2+] through activation of voltage-dependent calcium channels (Nelson, Standen, Braydon & Worley, 1988; Nelson et al. 1990; Fleischmann, Murray & Kotlikoff, 1994; Rubart, Patlak & Nelson, 1996). Elevating intravascular pressure from 10 to 60 mmHg increased intracellular [Ca2+] from 119 ± 10 to 190 ± 10 nm (n= 7), and caused a transient distention of the artery followed by a maintained constriction (Fig. 5A). The step increase and decrease in pressure occurred in less than 3 s. The time course of the increase and decrease in [Ca2+] in response to the pressure steps between 10 and 60 mmHg could be fitted by exponentials, with time constants of (increase, single exponential) 36.9 ± 8.0 s (n= 12), and (decay, two exponentials) 26.8 ± 8 and 123.0 ± 12.0 s (n= 12), respectively. The constriction in response to pressure that followed the rapid passive distention could be fitted with a time constant of 38.4 ± 11.0 s (n= 12), indicating a close relationship between the rise in [Ca2+] and the rate of force development under these conditions. The voltage-dependent calcium channel blocker, diltiazem (30 μm), inhibited the pressure-induced increase in intracellular calcium as well as the vasoconstriction (Fig. 5B).

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

To explore and understand further the role of voltage-dependent Ca2+ channels in the pressure-induced constriction, the effects of the dihydropyridine Ca2+ channel antagonist nisoldipine (10 nm) on arterial wall [Ca2+] were explored at different pressure levels under steady-state conditions. The apparent half-inhibition concentration of nisoldipine for calcium channels in vascular smooth muscle is < 1 nm, at −40 mV (Nelson & Worley, 1989). Elevating intravascular pressure from 10 to 100 mmHg increased intracellular wall [Ca2+] from 119 ± 10 to 245 ± 9 nm (n= 7; Fig. 6A). However, in the presence of nisoldipine or PSS with no added external Ca2+, pressure did not increase intracellular [Ca2+]. Intracellular [Ca2+] at 80 mmHg was the same in arteries bathed in 0 Ca2+ (93 ± 9 nm, n= 7) or superfused with nisoldipine (80 ± 12 nm, n= 7). Increasing intravascular pressure from 10 to 100 mmHg caused the arteries to constrict by almost 45 % relative to the diameter in Ca2+-free PSS (diameters in 0 external Ca2+ were 208 ± 10 μm, n= 8, vs. 116 ± 7 μm, n= 8, in physiological Ca2+; Fig. 6B). The Ca2+ channel inhibitor was as effective (198 ± 11 μm in 10 nm nisoldipine, n= 8) as 0 external Ca2+ in inhibiting pressure-induced constrictions (Fig. 6B). These results are consistent with the idea that the pressure-induced elevations in intracellular [Ca2+] and constrictions depend on Ca2+ entry through voltage-dependent Ca2+ channels (see Fig. 8 of Knot et al. 1998 for scheme of the proposed sequence of events).

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

Membrane potential dependence of arterial wall calcium and arterial diameter

To study the effect of the membrane potential (Vm) of smooth muscle cells in the intact pressurized (60 mmHg) artery we changed the external K+ concentration in the bath solution by isosmotic replacement of NaCl. Increasing external potassium from 6 to 16 mm caused a membrane hyperpolarization from -45 ± 1.4 to -58 ± 1.4 mV (n= 9), bringing the membrane potential very close to the equilibrium potential (EK) for K+, due to activation of inward rectifier K+ channels (Edwards, Hirst & Silverberg, 1988; Quayle et al. 1993b; Knot et al. 1996; see also Fig. 8). This hyperpolarization was associated with a decrease in arterial wall Ca2+ from 192 ± 14 to 126 ± 7 nm (n= 4). Increasing external K+ further from 16 to 61 mm caused a membrane potential depolarization to -23 ± 1.8 mV (n= 5), and arterial wall [Ca2+] to increase to 349 ± 12 nm (n= 4; Fig. 7). In the presence of 81 mm K+, the dihydropyridine calcium channel inhibitor nisoldipine (10 nm) lowered arterial wall [Ca2+] to 77 ± 17 nm (n= 4). The relationship between extracellular K+ and Vm above 16 mm K+ was Nernstian (Fig. 8). Therefore membrane potentials for extracellular [K+] (> 16 mm) were estimated from this relationship.

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

Arterial wall Ca2+ increased and vessel diameter decreased steeply with membrane potential depolarization in pressurized (60 mmHg) cerebral arteries (Fig. 9A and B). Around the resting membrane potential (± 5 mV) of −45 mV at 60 mmHg, the voltage sensitivity of Ca2+ and diameter were 7.5 nm mV−1 and 7.5 μm mV−1, and the Ca2+ sensitivity of diameter was 1 μm nm−1. The entire range of arterial diameters (i.e. fully dilated to almost fully constricted, no lumen) is associated with a membrane potential change of about 35 mV and arterial wall Ca2+ change of ~250 nm.

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

The pressure-induced membrane potential depolarization was not affected by an inhibitor of voltage-dependent calcium channels: the membrane potential in the presence of nisoldipine (10 nm) at 60 mmHg was -44.6 ± 1.8 mV (n= 7). Similarly, the membrane potential depolarization in response to elevated external potassium (61 mm) was also unaffected by nisoldipine at this pressure. The membrane potential of the arteries in 61 mm K+ PSS was -22.9 ± 1.7 mV in the presence of 10 nm nisoldipine at 60 mmHg intravascular pressure (Fig. 9A and B). However, the elevation in Ca2+ and constriction of pressurized (60 mmHg) cerebral arteries in response to membrane depolarization were completely inhibited by nisoldipine (10 nm) (Fig. 9A and B). These relationships indicate that a 9 mV depolarization from the membrane potential with physiological external potassium (PSS with 6 mm K+) would cause a ~45 nm elevation in arterial wall calcium, and ~50 μm (25 %) constriction. Figure 10 illustrates the relationship between arterial diameter and [Ca2+] obtained through cross-correlation of the data in Fig. 9 in pressurized (60 mmHg) cerebral arteries.

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

If pressure is acting to elevate intracellular [Ca2+] solely through the change in membrane potential, then arterial wall [Ca2+] should be related to the membrane potential and not to the level of intravascular pressure. Figure 11 shows that the relationship between membrane potential and arterial wall Ca2+ was the same under isobaric conditions (continuous line) or non-isobaric conditions (filled circles). In the presence of 10 nm nisoldipine pressurization does not elevate Ca2+ (open circles), which is similar to what was observed under isobaric conditions (dashed line). These results suggest that membrane potential, not intravascular pressure per se, determines the level of intracellular [Ca2+], in the absence of exogenous agonists and antagonists.

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

The results are consistent with the idea that intravascular pressure causes membrane potential depolarization, which opens voltage-dependent Ca2+ channels, increasing Ca2+ entry and arterial wall [Ca2+], which leads to vasoconstriction.

DISCUSSION

This study provides the first information on the regulation of arterial wall [Ca2+] in intact cerebral arteries, and its regulation of arterial diameter. Our data are consistent with the following mechanism (see scheme in Fig. 8 in Knot et al. 1998). A rise of intravascular pressure causes a graded membrane potential depolarization, and this leads to a graded increase in the steady open probability of L-type voltage-dependent Ca2+ channels (Nelson et al. 1990; Langton & Standen, 1993; Quayle et al. 1993a; Fleischmann et al. 1994; Rubart et al. 1996). This elevates Ca2+entry, and thus steady-state intracellular [Ca2+]. An elevation of arterial wall [Ca2+] leads to force development, cell shortening and vasoconstriction. Our data suggest that the elevation of arterial wall calcium in response to intravascular pressure (range, 10–100 mmHg) can be explained entirely by the induced change in membrane potential (range, -63 to −36 mV), and hence calcium influx through voltage-dependent calcium channels (see Fig. 11). At membrane potentials positive to about 0 mV, pressure may also activate dihydropyridine-sensitive, voltage-dependent calcium channels, independent of membrane potential changes (McCarron, Crichton, Langton, MacKenzie & Smith, 1997). Elevation in intravascular pressure has been shown to elevate steady-state calcium in arterioles of the hamster cheek pouch, and this led to an elevation in myogenic constriction (D'Angelo et al. 1997).

The physiological range of intracellular calcium in pressurized arteries

Steady-state arterial wall [calcium] is presumably determined by Ca2+ influx and extrusion across the plasma membrane of the smooth muscle cells. At 10 mmHg and in physiological salt solutions, steady-state arterial wall [Ca2+] was about 120 nm, and the membrane potential was about −63 mV. The addition of a calcium channel inhibitor (e.g. nisoldipine) or removal of external calcium reduced arterial wall Ca2+ to about 90 nm, and prevented elevation in Ca2+ and constriction in response to pressure and to membrane potential depolarization (Figs 6, ,7,7, ,99 and and11).11). These results suggest that only one type of calcium channel (dihydropyridine-sensitive, voltage-dependent calcium channels) regulates Ca2+ entry over the entire pressure (10–100 mmHg) and membrane potential (-63 to −23 mV) range examined. The accompanying manuscript (Knot et al. 1998) provides evidence that ryanodine receptors in the SR can alter steady-state arterial wall calcium by regulating KCa channels, which affects calcium entry through voltage- dependent calcium channels through membrane potential changes. However, under non-steady-state conditions, rapid calcium release from the SR, for example by caffeine, can cause transient increases in Ca2+ and decreases in diameter (Knot et al. 1998).

The diameter of pressurized (60 mmHg) cerebral artery traverses its entire dynamic range (fully dilated to maximally constricted) when the smooth muscle membrane potential depolarizes from -65 to −25 mV. This constriction is caused by an elevation of intracellular [Ca2+] from about 120 to 350 nm. Thus, small changes in membrane potential and intracellular [Ca2+] lead to significant changes in arterial diameter. For example, membrane depolarization of a pressurized (60 mmHg) cerebral artery from −45 mV by ~9 mV would elevate global [calcium] by ~45 nm and constrict the vessel by about 25 %. In accord with our study, raising bath [calcium] from 100 to 200 nm constricted permeabilized, pressurized (70 mmHg) cerebral arteries to a diameter observed in the intact pressurized artery (McCarron et al. 1997). Elevating bath [calcium] to 400 nm maximally constricted these permeabilized, pressurized arteries (McCarron et al. 1997), very similar to our intact artery data. Therefore, our data and the data of McCarron et al. (1997) strongly support the idea that the major calcium entry pathway in these cerebral arteries is the dihydropyridine-sensitive, voltage-dependent calcium channel and that the dynamic range of steady arterial wall calcium is 100–400 nm.

The steady-state voltage dependence of calcium channels determines the voltage dependence of intracellular calcium and diameter

Our results strongly suggest that voltage dependence of arterial wall [Ca2+] and diameter reflect the voltage dependence of Ca2+ channels, since selective Ca2+ channel blockers (e.g. nisoldipine and diltiazem) prevent elevations in wall [Ca2+] and prevent or reverse vasoconstriction. Calcium channels in cerebral arteries exhibit a steep steady-state voltage dependence over the physiological range of membrane potentials (Nelson et al. 1988, 1990; Langton & Standen, 1993; Quayle et al. 1993a; Rubart et al. 1996). This is consistent with their role in determining the voltage dependence of arterial wall [Ca2+] and diameter. The changes in intracellular [Ca2+] in response to a graded membrane depolarization measured here in intact pressurized arteries were similar to those measured in isolated myocytes (Fleischmann, Wang, Pring & Kotlikoff, 1996). A steady increase in intracellular [Ca2+] of about 100 nm appears to be caused by an increase of about 0.5 pA Ca2+ current through L-type voltage-dependent Ca2+ channels, measured in isolated myocytes from these arteries (Rubart et al. 1996), as well as in other tissues (Fleischmann et al. 1994).

Small myogenic arteries normally operate in a partially constricted state (‘myogenic tone’) from which they can dilate or contract further upon demand in response to external stimuli. In this study, we describe the voltage dependence of the changes in arterial wall [Ca2+] and the resulting change in arterial diameter (Figs 9 and and10).10). These relationships should aid in further understanding the effect of vasoactive hormones and compounds whose actions may involve biochemical pathways altering the Ca2+-diameter (i.e. force development) relationship aside from changes in membrane potential, since it allows the dissociation of these two pathways.

In conclusion, these results support the idea that steady Ca2+ influx through voltage-dependent calcium channels regulates intracellular [Ca2+], and hence myogenic tone in responses to changes in intravascular pressure. Further, this study underscores the key role that arterial smooth muscle membrane potential plays in the regulation of arterial diameter.

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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