Myogenic behavior, prevalent in resistance arteries and arterioles, involves arterial constriction in response to intravascular pressure. This process is often studied in vitro by using cannulated, pressurized arterial segments from different regional circulations. We propose a comprehensive model for myogenicity that consists of three interrelated but dissociable phases:1) the initial development of myogenic tone (MT),2) myogenic reactivity to subsequent changes in pressure (MR), and 3) forced dilatation at high transmural pressures (FD). The three phases span the physiological range of transmural pressures (e.g., MT, 40–60 mmHg; MR, 60–140 mmHg; FD, >140 mmHg in cerebral arteries) and are characterized by distinct changes in cytosolic calcium ([Ca2+]i), which do not parallel arterial diameter or wall tension, and therefore suggest the existence of additional regulatory mechanisms. Specifically, the development of MT is accompanied by a substantial (200%) elevation in [Ca2+]i and a reduction in lumen diameter and wall tension, whereas MR is associated with relatively small [Ca2+]i increments (<20% over the entire pressure range) despite considerable increases in wall tension and force production but little or no change in diameter. FD is characterized by a significant additional elevation in [Ca2+]i (>50%), complete loss of force production, and a rapid increase in wall tension. The utility of this model is that it provides a framework for comparing myogenic behavior of vessels of different size and anatomic origin and for investigating the underlying cellular mechanisms that govern vascular smooth muscle mechanotransduction and contribute to the regulation of peripheral resistance.
- membrane potential
- vascular smooth muscle
in organs such as the brain, heart, and kidney, a significant portion of basal vascular tone (an important determinant of peripheral resistance, blood pressure, and regional blood flow) is thought to be of myogenic origin (4,21). This intrinsic response of vascular smooth muscle to pressure or stretch is often studied in vitro by using cannulated, pressurized arterial segments (e.g., 5, 7, 9, 11, 13, 15, 16, 23, 24). With the use of this approach, the artery can be exposed to the primary stimulus (pressure-stretch) under conditions that are both controlled and devoid of the confounding influences of tissue metabolites, humoral factors, and neurotransmitters released from periarterial nerves. Myogenic behavior has been described in arteries from a number of regional circulations, including the brain, heart, kidney, skeletal muscle, and splanchnic circulation (1-6). Myogenic behavior has also been documented in isolated veins (10) and in pressurized segments of the lymphatic circulation (25). Several reports have attempted to model the myogenic response (1, 14), most recently from the standpoint of wall tension (24); however, a comprehensive model for myogenic behavior in vitro has not been developed to date.
This study describes a three-phase model of in vitro arterial myogenic behavior that may help unify some of the discrepant observations and provide a framework for comparing the myogenic behavior of vessels of different size and origin. It also provides new information on the ionic events underlying forced dilatation (phase 3). Forced dilatation occurs when the ability of a vessel to constrict is overcome by high pressure and is associated with the development of hypertensive encephalopathy in vivo (12). The model is described from the standpoint of concurrent changes in transmural pressure (TMP), lumen diameter (φ), membrane potential, cytosolic calcium ([Ca2+]i), and wall tension. The underlying hypothesis is that myogenic behavior can be partitioned into three distinct phases and that different cellular mechanisms may contribute to different degrees in each phase. For example, phase 1(the development of tone) involves cellular deformation, depolarization, and significant increases in arterial wall calcium. These processes may, in turn, activate enzyme systems that enablephase 2, myogenic reactivity, in which changes in calcium and membrane potential are relatively minor, suggesting a greater role for calcium sensitivity (possibly via PLC/PKC and Rho kinase activation). We understand less about phase 3, forced dilatation, but we hypothesize that it may result from a depletion of G actin and enhanced calcium entry.
Animals and Preparation of Vessels
Adult (16–24 wk old) male Wistar-Kyoto rats (n = 32) were anesthetized by an intraperitoneal injection of methohexital sodium (Brevital, 50 mg/kg) and killed by decapitation. The brain was removed and immersed in a dissection dish filled with physiological salt solution (PSS, see composition underSolutions and Drugs). The entire posterior cerebral artery, including its branches, was removed and carefully dissected free from surrounding connective tissues under a stereo dissection microscope with the use of microdissection tools. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Vermont and were conducted in accordance with The Guide for the Care and Use of Laboratory Animals (National Research Council; Washington, DC).
Measurement of Smooth Muscle Ca2+and Arterial Diameter
To obtain simultaneous measurements of arterial diameter and [Ca2+]i in arterial smooth muscle cells, arteries were loaded with fura 2-AM, a Ca2+-sensitive fluorescent dye. Fura 2-AM (1 mM stock dissolved in DMSO) was premixed with an equal volume of a 25% solution of pluronic acid in DMSO and then diluted in PSS to yield a final concentration of 5 μM. The excised posterior cerebral artery, including its branches, was incubated in the fura 2-AM-PSS loading solution at room temperature in the dark for 60 min. Fura 2-loaded arteries were then washed with PSS and kept refrigerated until use.
Each arterial segment was cannulated, mounted in an arteriograph, and continuously superfused at 3 ml/min with oxygenated PSS (95% O2-5% CO2
Measurement of Membrane Potential
1) abrupt negative change in voltage on impalement of the cells; 2) a sharp return to zero voltage after withdrawal of a microelectrode tip;3) tip potential of <7 mV; and 4) unchanged resistance of microelectrodes after impalement.
Control of Transmural Pressure
Pressure within a vessel was measured and controlled by means of a pressure servomechanism that consists of an inline transducer attached to an electronic regulator. A potentiometer allows for the automatic adjustment of pressure (manually set), which is produced by a peristaltic pump linked to the cannula via inert silicone tubing (Living Systems Instrumentation). All experiments were conducted under no-flow conditions, hence, pressure was uniform throughout and could be controlled to within 1 mmHg.
After fura 2 loading was completed, arterial segments were cannulated within the chamber of a specialized arteriograph by tying each end onto a glass cannula (tip diameters: 50–75 μm). A more detailed description of this system can be found elsewhere (5,20). Two experimental protocols were used, differing primarily in the pressure at which equilibration occurred.
With the use of the servocontrol, pressure within each segment was elevated to 10 mmHg and the artery allowed to equilibrate for 30 min before the start of the experimentation. The subsequent protocol consisted of imposing stepwise changes in transmural pressure in 10-mmHg increments up to 60 mmHg, and 20- to 40-mmHg increments thereafter, up to 220 mmHg. Sufficient time (3–7 min) was allowed after each pressure step to ensure a stable diameter response.
Vessels were cannulated and transmural pressure was elevated to 60 mmHg before turning on the heat exchanger and beginning equilibration. In this case, a 30-min equilibration period was used as well. In all cases, it took ∼10–15 min for temperature to increase from room to physiological (37°C) temperature.
Arterial wall calcium.
Arterial wall [Ca2+]i was calculated by using the following equation (from Ref. 6): [Ca2+]i =K dβ(R − Rmin)/(Rmax − R), where Rminand Rmax were measured from ionomycin-treated arteries, and β was determined as previously described (6). These values were then pooled and used to convert the ratio values into a [Ca2+]i. The K d was 282 nM as determined by using in situ titration of Ca2+ in fura 2-loaded posterior cerebral arteries (13
Wall tension is defined by the Laplace formulation as the product of pressure and radius (P × r); pressure (measured in mmHg) was converted into dynes per squared centimeter (1 mmHg = 1333.2 dyn/cm2); arterial radius (measured in μm) was converted to centimeters to yield tension in dynes per centimeter.
Solutions and Drugs
The physiological salt solution (PSS) contained (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11.0 glucose equilibrated with a mixture of 95% O2-5% CO2, pH = 7.4. Papaverine (0.1 mM) was administered at the end of an experiment to obtain passive diameters at all pressures studied. All chemicals were purchased from Sigma Chemical (St. Louis, MO), with the exception of fura 2-AM, which was purchased from Molecular Probes (Eugene, OR), dissolved in DMSO as a 1 mM stock solution, and frozen in 2-μl aliquots until used.
All data are expressed as means ± SE, where eachn equals the number of arterial segments studied. One-way ANOVA, followed by a multiple-comparison test (Student-Newman-Keuls) was used for comparing more than two groups, whereas the Student'st-test was used to determine the significance of differences between two groups of data (e.g., large vs. small branches). In all cases, P values <0.05 were considered significant.
Phase 1: Development of Myogenic Tone
Stepwise elevation of transmural pressure from 10 to 40 mmHg resulted in passive arterial distension. Pressures between 40 and 60 mmHg triggered the development of myogenic tone, as evidenced by significant elevations in calcium, followed by substantial reductions in diameter. An example of a tracing from one experiment, relating changes in pressure and diameter, and showing the appearance of tone is shown in Fig. 1. Vascular smooth muscle [Ca2+]i averaged 63 ± 11 nM before versus 198 ± 10 nM after tone development; i.e., an increase of ∼200% over baseline values (P < 0.01).
Although we were unable to follow the changes in membrane potential during the development of tone (because of difficulty in maintaining vascular smooth muscle impalement in a vessel undergoing constriction), there was clearly a sizeable membrane depolarization between 10 and 60 mmHg with values averaging −67 ± 1 mV at 10 mmHg and −38 ± 1 mV at 60 mmHg (P < 0.05; Fig. 7). The development of tone was often associated with small oscillations in diameter. As shown in Fig. 2, these were highly correlated with parallel oscillations in [Ca2+]i and membrane potential, both of which were slightly preceded changes in diameter and were on the order of 30–35 oscillations per minute.
In protocol 2, arteries were allowed to equilibrate at 60 mmHg, a pressure just above the myogenic threshold. Under these conditions, myogenic tone developed some time (usually 5–15 min) after temperature stabilized at 37°C (Fig.3) but was otherwise similar in the magnitude of both calcium and diameter change. The development tone was a reversible phenomenon, both in terms of calcium elevation and diameter reduction; i.e., if pressure was lowered below the myogenic range (e.g., 10 mmHg, see Fig. 3), [Ca2+]idecreased to baseline conditions, and the artery underwent complete dilation. Reelevation of pressure resulted in calcium elevation and the redevelopment of tone (Fig. 3).
Wall tension was calculated simultaneously from the pressure and diameter data and is included in Figs. 1 and 3. In the step protocol (protocol 1, Fig. 1), wall tension increased substantially before the development of tone, because both transmural pressure and arterial radius were increasing with each pressure step. On the other hand, the development of tone per se was associated with a significant reduction in wall tension. This was most clearly evident inprotocol 2 (equilibration at 60 mmHg), where pressure was unchanged as tone developed, and diameter (and, by definition, wall tension) was reduced accordingly (by 20–35%, e.g., see Fig. 3).
Phase 2: Myogenic Reactivity
As shown in Fig. 4, once tone had developed, subsequent increases in transmural pressure between 60 and 140 mmHg were associated with the maintenance of a relatively constant diameter or slight constriction. In contrast to the marked elevation in calcium coincident with the appearance of tone (Figs. 1 and 3), small but consistent increases were evident between 60 and 140 mmHg (e.g., Fig. 4). Calcium concentrations were 198 ± 10 nM at 60 mmHg and 246 ± 7 nM at 140 mmHg, increasing ∼0.6 nM/mmHg. Depolarization was measurable between 60 and 100 mmHg: −39 ± 1 mV at 60 and −30 ± 1 mV at 100 mmHg (P < 0.05). No depolarization could be detected with further increases in pressure (see Fig. 7).
By definition, wall tension increased in proportion to the increase in pressure in vessels maintaining a constant diameter. Although constriction tended to decrease tension somewhat, the magnitude of this effect was minor relative to that induced by pressure (Fig. 4).
Phase 3: Forced Dilatation
At transmural pressures above 140 mmHg, arterial behavior became more variable. Approximately one-third of the vessels were able to withstand elevations up to 180 or even 200 mmHg for some time without any evidence of instability, whereas others began to lose tone and undergo spontaneous dilations with only partial recovery of constriction at 160 or 180 mmHg (Fig. 5). Once instability appeared, further increases in pressure led to a complete loss of tone characterized by rapid dilation, which was accompanied by significant additional elevation in cytosolic calcium (246 ± 7 to 328 ± 22 nM; P < 0.05). Little or no additional depolarization was measured (at 220 mmHg, membrane potential = −29 ± 1 mV; P > 0.05). Wall tension was also significantly increased due to the combination of high transmural pressure and dilatation. A tracing showing the simultaneous changes in pressure, tension, calcium, and diameter range is shown in Fig. 5.
Overview of 3 Phase Model
Figure 6 shows a tracing from an experiment in which transmural pressure, arterial diameter, and smooth muscle [Ca2+]i were measured and recorded continuously in one arterial segment. Changes in wall tension, calcium, and diameter over the entire pressure range studied (10–220 mmHg) are summarized in Fig. 7. Similarly, changes in calcium and membrane potential are summarized in in Fig.8. It is clear that wall tension increases progressively and that the effect of tone or of myogenic reactivity is offset by the effects of pressure. In terms of calcium, the largest increases were associated with the development of tone (phase 1) and with forced dilatation (phase 3), with small but consistent increases over the range of pressures in which myogenic reactivity (phase 2) was observed.
Consideration of Methodology and Utility of Model
Myogenic behavior has been documented in arteries, arterioles, veins, and lymphatic vessels. Some studies have utilized isometric wire-mounted ring preparations (10, 19), although the majority of recent investigations have used isolated pressurized vessels as the model of choice (e.g., 2, 4, 8, 9, 11, 13, 15, 16, 18, 22–25). This preparation offers the advantages of maintaining normal geometry of the vascular wall and of eliminating confounding neural and metabolic influences so as to allow quantitative and precise measurement of pressure-induced diameter responses.
The experimental protocol used in a particular study varies from investigator to investigator, complicating direct comparisons. For example, some investigators evaluate myogenic or pressure-induced behavior by imposing a single large pressure step, whereas others use pressure steps of varying amplitude and delivered at varying rates of pressure change (4, 15, 22).
One advantage of the present experimental-based model is that it segregates the events associated with in vitro myogenic reactivity into distinct phases, each of which may be associated with specific mechanisms. Hence, the behavior of vessels of different size or anatomic origin can be compared more directly. For example, one might expect that smaller vessels would both develop tone and undergo forced dilatation at lower transmural pressures due to a reduction in vascular smooth muscle mass and, therefore, force generating ability. If viewed from the standpoint of wall tension, however, this may be a fallacy because wall tension would be reduced due to the smaller radius.
The efficiency of the myogenic response has been evaluated in various ways, most often by calculating the “gain” or percent change in diameter per millimeter of mercury or pressure (20); clearly, this type of analysis pertains only to arterial reactivity within the myogenic range (phase 2, myogenic reactivity). Including the development of tone in the gain calculation is both nonphysiological and misleading because the gain of the response is significantly overestimated. In this sense, the three phases are dissociable. For example, in some myogenic studies, the vessels used do not spontaneously develop pressure-induced tone and must be preconstricted before active responses to pressure become evident. Hence, phase 2 can be observed in the absence of phase 1, and its characteristics are affected by the degree of activation and nature of constricting stimulus (e.g., depolarizing potassium solution vs. receptor-mediated responses, e.g., catecholeamines; 16, 24).
Relationship Among Membrane Potential, Calcium, Diameter, and Wall Tension as a Function of Transmural Pressure
Graded membrane depolarization has been measured in isolated cerebral (7, 13) arteries at pressures below the threshold for tone development (<40 mmHg). As seen in Figs. 1 B and2 A, however, step increases in pressure do not result in a progressive elevation in calcium; rather, calcium increases spontaneously and significant only when sufficient pressure is attained. Furthermore, the comparable increases in both intracellular calcium and arterial constriction observed in protocol 1versus 2 indicate that the principal stimulus for tone development is the ambient level of pressure, rather than an acute stretch of the wall due to an acute increase in pressure.
Phase 2, myogenic reactivity is characterized by relatively minor but consistent changes in calcium and diameter despite a substantial elevation in wall tension. If smooth muscle force increases in proportion to the increase in pressure (and tension), as might be predicted, it is likely that other mechanisms come into play, e.g., increases in calcium sensitivity or the induction of a “latch” state, in which pressure-induced distension is resisted by load-bearing (but not necessarily force producing) cellular elements (26).
As pressure is increased further, vessels begin to undergo some instability, typically evidenced by rapid dilations with only partial reconstriction. If pressure is not lowered, forced dilatation (phase 3), defined as a complete loss of tone, occurs. The point at which dilatation occurs may be determined by the distending force alone (wall tension or, more accurately, cell stress) or by a secondary event such as a depletion of G actin. One hypothesis is that pressure stimulates actin polymerization and that cytoskeletal “remodeling” results in the formation of additional filaments available for interaction with myosin (2, 3). As pressure increases, the available pool of actin decreases until there is no reserve; any further increase in pressure will then result in forced dilatation. Whereas attractive and substantiated by some data (2,3, 17), this hypothesis of pressure-induced vascular smooth muscle actin dynamics requires additional study before verification, because simultaneous measurements of both G and F actin have yet to be reported in myogenic vessels.
In summary, cerebral artery responses to transmural pressure may be partitioned into three distinct phases. The first, development of tone (mygenic tone; 40–60 mmHg), is characterized by substantial membrane depolarization, elevation of cytosolic calcium, constriction, accompanied by a reduction in wall tension. Phase 2, myogenic reactivity, involves the maintenance of tone in the face of elevated transmural pressure. Between 60 and 140 mmHg, little change in diameter is evident despite significant increases in wall tension; some membrane depolarization and calcium elevation is observed, although changes are relatively minor. At pressures above 140 mHg, vessels become unstable and, if pressure is increased further, undergo forced dilatation (forced dilatation, phase 3). This event is associated with further significant increases in calcium and wall tension and a complete loss of myogenic tone. If pressure is reduced to be within the myogenic range, recovery of tone and a reduction in calcium is observed.
The authors acknowledge the National Heart, Lung, and Blood Institute (Grant RO1 HL-59406) in supporting these studies.
Address for reprint requests and other correspondence: G. Osol, Dept. of Obstetrics and Gynecology, Univ. of Vermont College of Medicine, Given C-217A, Burlington, VT 05405 (E-mail:).
This article belongs to a collection of papers accepted in response to the Editor's special call for papers entitled “Mechanisms of vascular myogenic tone.”
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August 22, 2002;10.1152/ajpheart.00634.2002
- Copyright © 2002 the American Physiological Society