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Smooth Muscle Physiology Group, Department of Medicine, Manchester Royal Infirmary, Manchester, United Kingdom
Submitted 29 March 2005 ; accepted in final form 13 October 2005
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ABSTRACT |
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contractile function; coronary circulation; vasoconstriction/dilation
In the coronary circulation, therefore, it is clear that small arteries exhibit myogenic regulatory responses to changes in both intra- and extravascular pressure. At any time, therefore, the transmural pressure across the arterial walls is determined by both the intra- and extravascular pressures. As such, similar transmural pressures may be attained by different combinations of intra- and extravascular pressures. Although it is generally assumed that stable levels of myogenic tone are dependent only on transmural pressure, this has not previously been directly proven. It is now recognized that changes in cyclic strain and/or endothelial cell deformation may alter the release and/or synthesis of various endothelial factors, including nitric oxide (1, 13). Because the influence of intra- and extravascular pressures on these endothelial stimuli may vary, and because the endothelium is known to modulate stable levels of coronary artery myogenic tone (e.g., Refs. 4, 14), the possibility exists that the relative contributions of these pressures to the transmural pressure does influence tone. As such, it is essential that this is fully characterized.
Although changes in the extravascular forces experienced by the coronary arteries obviously will be experienced during the normal cardiac cycle, elevated pressures also are evident during disease states associated with elevated ventricular pressures such as congestive heart failure. Although impaired endothelium-dependent responses of the coronary vasculature have been reported in such conditions (e.g., Refs. 17, 19), others have found that if correction is made for the elevated left ventricular diastolic pressure, the responses are similar to normal (16). This finding has led to the suggestion that increased extravascular forces may act to limit endothelium-dependent dilatory responses; however, this possibility has not been directly examined. It also is unknown whether elevated external pressures modulate responses to vasoconstrictor stimuli.
Therefore, the aims of this study were to directly examine the effects of elevated extravascular pressure on responses of isolated coronary arteries to vasoconstrictor and vasodilator stimuli. We examined its influence on myogenic tone, on responses to constrictor stimuli (agonist and depolarization), and on responses to both endothelium-dependent (flow, acetylcholine) and endothelium-independent (isoprenaline) vasodilatory stimuli.
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MATERIALS AND METHODS |
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Male Wistar rats (150–250 g; Charles River) were humanely killed by stunning and cervical dislocation. All procedures were performed in accordance with our institutional guidelines and the United Kingdom Animals (Scientific Procedures) Act of 1986. Experiments were performed with the approval of the Review Board of the University of Manchester and the Home Office. The heart was removed and placed in ice-cold physiological salt solution (PSS) of the following composition (in mM): 119 NaCl, 4.7 KCl, 1.2 MgSO4·7H2O, 25 NaHCO3, 1.17 KHPO4, 0.03 K2EDTA, 5.5 glucose, and 1.6 CaCl2·2H2O (pH 7.4). First-order septal arteries (200–250 µm initial diameter, 1–2 mm in length) were dissected out and mounted on a modified pressure myograph (Living Systems Instrumentation, Burlington, VT). Arteries were initially pressurized to an intraluminal pressure of 60 mmHg by using a pressure servocontrol unit and were checked for leaks. A tightly fitting lid was then placed over the myograph chamber. Extravascular pressure was altered by introduction of gas (95% air-5% CO2; from a standard gas cylinder) from a luer connection in the top part of the chamber above the vessel, as we have described previously (2). Inflow of gas, and thus extravascular pressure, was controlled via a pneumatic valve and recorded with a mercury manometer. All vessels were constantly superfused with PSS at 37°C, pH 7.4, and gassed with 95% air-5% CO2, with the superfusate entering and leaving the chamber via luer connections in the side of the chamber. pH was continuously monitored using a flow-through pH probe (Living Systems Instrumentation) placed immediately after the luer fitting through which the solution left the chamber. We could detect no change in pH with the elevations in extravascular pressure used in this study. Lumen diameters were constantly measured using a video dimension analyzer, with data recorded on computer and a chart recorder for later analysis. Where appropriate, arteries were superfused with calcium-free PSS containing 2 mM EGTA for at least 30 min to obtain passive diameters.
Experimental Protocols
Do relative contributions of intra- and extravascular pressure to transmural pressure influence stable levels of myogenic tone? Arteries were initially pressurized to an intravascular pressure of 60 mmHg and allowed to develop spontaneous myogenic tone. All those arteries that did not develop tone within 90 min or that did not develop tone of at least 25% of original diameter were excluded from the study. We wanted to determine whether stable levels of myogenic tone were influenced by the relative levels of intra- and extravascular pressure or were purely dependent on the overall transmural pressure. To investigate this, we examined the effects on tone of similar transmural pressures produced by differing combinations of intra- and extravascular pressures. For example, transmural pressures of 40 mmHg were obtained by the following combinations of intra- and extravascular pressures: intravascular pressure 60 mmHg/extravascular pressure 20 mmHg; intravascular pressure 40 mmHg/extravascular pressure 0 mmHg; and intravascular pressure 100 mmHg/extravascular pressure 60 mmHg (see Fig. 1 for other combinations used). In all cases, zero additional extravascular pressure is defined as atmospheric pressure. The ways in which these stable combinations of extra- and intravascular pressure were attained were varied.
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Does changing the relative contributions of intra- and extravascular pressures to overall transmural pressure modulate constrictor responses to agonist addition or depolarization? Constrictor responses to addition of the thromboxane analog U-46619 (0.25 µM) or by depolarization (40 mM KCl) were investigated at a transmural pressure of 60 mmHg, obtained using the following combinations of intra- and extravascular pressure: intravascular pressure 60 mmHg/extravascular pressure 0 mmHg; intravascular pressure 100 mmHg/extravascular pressure 40 mmHg; and intravascular pressure 120 mmHg/extravascular pressure 60 mmHg. At an intravascular pressure of 60 mmHg, these stimuli produced submaximal levels of constriction.
Does changing the relative contributions of intra- and extravascular pressure to overall transmural pressure modulate responses to vasodilatory stimuli? INTRALUMINAL FLOW.
We have previously shown that isolated rat coronary arteries possessing myogenic tone (at intravascular pressure of 60 mmHg/extravascular pressure 0 mmHg) exhibit vasodilatory responses to introduction of intraluminal flow. Responses are maximal and repeatable at flow rates of 
5 µl/min (maximal dilations = 35 ± 6, 25 ± 4, and 25 ± 4% of tone with 3 successive exposures to 5 µl/min flow, n = 3). As such, we investigated the influence of differing combinations of intra- and extravascular pressure (see above), to give a combined transmural pressure of 60 mmHg, on responses of isolated rat coronary arteries to this flow rate. Vessels were initially allowed to develop myogenic tone at an intravascular pressure of 60 mmHg (additional extravascular pressure = 0 mmHg). We have previously shown that responses of isolated rat coronary arteries to intraluminal flow are entirely dependent on the presence of a functionally intact endothelium (unpublished observations).
DILATOR AGONISTS.
In a separate series of experiments, the influence of the different pressure combinations (intravascular pressure 60 mmHg/extravascular pressure 0 mmHg; intravascular pressure 120 mmHg/extravascular pressure 60 mmHg), designed to maintain transmural pressure at 60 mmHg, on dilatory responses to the endothelium-dependent agonist ACh (1 µM) were examined. The influence on dilatory responses to the 
-adrenergic agonist isoprenaline (0.5 µM) also were studied. In coronary arteries, isoprenaline has been shown to produce dilatory responses that are independent of the endothelium (20); these responses are readily reversible.
Data Analysis
All results are presented as means ± SE, with n representing the number of animals. One artery was studied from each animal. Statistically significant differences between groups were assessed using ANOVA or t-tests as appropriate. Myogenic tone was calculated as the difference between active (in calcium-containing PSS) and passive (in calcium-free PSS) diameters at each transmural pressure. To allow direct comparison between the different experimental protocols, we normalized diameters to the stable diameter at an intravascular pressure of 60 mmHg (extravascular pressure 0 mmHg) as appropriate. Transmural pressure was calculated as the difference between intra- and extravascular pressures.
Drugs and Chemicals
All drugs and chemicals were obtained from Sigma and were dissolved in PSS.
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RESULTS |
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Arteries used in these studies had an initial mean diameter of 237 ± 5 µm (n = 52). All arteries began to develop myogenic tone within 60 min of mounting and attained mean stable active diameters of 172 ± 8 µm. All arteries rapidly dilated when placed in calcium-free PSS.
Transmural pressures of 20–60 mmHg resulted in stable diameters that were not significantly different from one another, confirming our previous findings that there is active myogenic regulation over this pressure range (2). The results of the present study, however, show that stable levels of tone were independent of the relative contributions of intra- and extravascular pressure to overall transmural pressure (n = 12) (Fig. 1). Although endothelial denudation did significantly reduce active diameter (P < 0.01 at intravascular pressure 60 mmHg/extravascular pressure 0 mmHg), stable levels of myogenic tone once again appeared to be dependent only on the transmural pressure (n = 7) (Fig. 1); this also was observed in the presence of NOS and cyclooxygenase inhibition (n = 4) (Fig. 1). In calcium-free solution, arteries dilated when transmural pressure was increased; diameters were again independent of the relative contributions of intra- and extravascular pressures to overall pressure (n = 4) (Fig. 1).
Does the Changing Relative Contributions of Intra- and Extravascular Pressures to Overall Transmural Pressure Modulate Constrictor Responses to Agonist Addition or Depolarization?
In a separate set of experiments, the effects of different intra- and extravascular pressure combinations on constrictor responses to addition of the thromboxane analog U-46619 and depolarization were examined. Responses to addition of U-46619 were unaffected by modulation of the intra- and extravascular pressure ratio at a transmural pressure of 60 mmHg (n = 4). Similarly, responses to high-K+ solution were unchanged (Fig. 2).
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Intraluminal flow. Introduction of intraluminal flow (5 µl/min) resulted in a dilation of all vessels by 55 ± 13% of developed tone (maximal dilation). At transmural pressures of 60 mmHg, produced by elevating both extra- and intravascular pressure (to 40 and 100 mmHg, respectively), however, dilation to flow was significantly reduced (P < 0.04) and was virtually abolished when extra- and intravascular pressures were elevated further to 60 and 120 mmHg, respectively (P < 0.03). Responses to flow were restored (to 86 ± 17% of original) when any extravascular pressure was removed and intravascular pressure was returned to 60 mmHg. As above, myogenic tone was independent of the relative contributions of intra- and extravascular pressure to it (tone = 66 ± 8 µm at extravascular pressure 40 mmHg/intravascular pressure 100 mmHg and 65 ± 10 µm at extravascular pressure 60 mmHg/intravascular pressure 120 mmHg) (Fig. 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Although it has generally been assumed that myogenic tone is governed by the absolute level of transmural pressure, it had previously only been demonstrated in isolated pressurized coronary arteries in responses to changes in intravascular pressure (5, 9, 8). Although this may be valid for many vascular beds, in those embedded in contracting muscle, as discussed previously, internal pressures will be opposed by external compressive forces. We are the first to have shown that isolated coronary arteries also exhibit regulatory myogenic responses to elevations of external pressure (2); as such, extravascular pressure will contribute to stable levels of tone in these vessels. In the present study we have demonstrated that stable levels of myogenic tone are dependent only on the absolute value of transmural pressure, irrespective of the relative values of intra- and extravascular pressure, at least over the range of transmural pressures of 20–80 mmHg. This is in support of our earlier findings over a smaller pressure range (2). Interestingly, although we observed active regulatory responses to changes in intra- and extravascular pressure over this pressure range, stable diameters are maintained, rather than reduced, as we have found in other vascular beds (10, 11). This is in accordance with our previous findings and those of others in our group (Ref. 2; unpublished observations). The reasons for this are unclear but may reflect differences in their physiological roles and perhaps the relative importance of, and interactions with, other local regulatory mechanisms. Differences in arterial size/anatomical location and, indeed, species also may be important. In support of this, porcine subepicardial arteries have been shown to exhibit more pronounced myogenic constrictory responses than observed in the present study; however, porcine subendocardial arteries showed significantly less myogenic responsiveness (5). We present, however, direct evidence that stable levels of myogenic tone are dependent only on the overall transmural pressure, irrespective of the relative contributions of intra- and extravascular pressures to this.
Although it is well established that myogenic tone is not dependent on the presence of a functionally intact endothelium, it may be modulated by the release of endothelial factors in many vascular beds, including the coronary circulation (e.g., Ref. 4); this has been confirmed in the present study. Although shear stress is a major regulator of endothelial factor synthesis and/or release (6, 18), it is now recognized that cyclic strain, attributed, for example, to changes in arterial diameter, also may modulate the synthesis and/or release of these factors (1, 12). Such effects, at least upon the release of nitric oxide, have been attributed to deformation of the endothelial cells themselves (12). Because changes in intravascular pressure, which act directly on the endothelial cells, and changes in extravascular pressure, which act directly on the outer surface of the blood vessel, may be expected to have different influences on endothelial cell shape (and/or stresses and strains), we postulated that, via this mechanism, they may have different effects on stable levels of myogenic tone. We found, however, that neither mechanical removal of the endothelium nor inhibition of NOS and cyclooxygenase modulated the relative importance of intra- and extravascular pressures in determining stable levels of myogenic tone, suggesting that this is not the case.
Thus we are the first group to provide direct evidence that stable levels of myogenic tone are dependent only on the overall transmural pressure; this also appears to be true for constrictor responses to application of agonists or depolarization (at least for submaximal constrictions). In contrast, however, we have demonstrated that responses to dilatory stimuli do appear to be influenced by the relative contributions of intra- and extravascular pressure to overall transmural pressure. Responses to introduction of intraluminal flow are reduced in a stepwise manner as extravascular pressure is increased. Although we have previously shown (in agreement with many other studies in different vascular beds) that dilatory responses to intraluminal flow are entirely dependent on the presence of a functionally intact endothelium, similar effects on dilatory responses to the endothelium-dependent dilator isoprenaline suggest that this is not due to modulation of endothelial factor release but is more likely a nonspecific physical effect of elevated external pressure opposing dilation. Indeed, responses were restored when extravascular pressure was removed. In support of this, Traverse et al. (16) demonstrated that in dogs with experimentally induced heart failure, reduced dilatory responses to addition of ACh were normalized after correction for the evident elevated left ventricular pressure. This led the authors to propose that endothelium-mediated vasodilation is preserved in congestive heart failure but that increased extravascular compressive force acts to limit the increase in coronary blood flow (16). Although it must be noted that endothelial dysfunction has been observed in peripheral arteries (i.e., those not exposed to elevated external pressures) of patients and animal models of heart failure (e.g., Ref. 3), it is possible that in vivo elevated external pressures may limit responses in coronary vessels. The results of the present study are consistent with this idea and further show that responses to more physiological stimuli of endothelial factor release (i.e., flow) and to endothelium-dependent dilatory agonists are reduced as external pressure is elevated. We cannot totally discount the possibility that these effects may be related to the concurrent increase in intravascular pressure (to maintain transmural pressure), although we think this is unlikely. This is difficult to test, however, because increasing intravascular pressure to 100 and 120 mmHg alone will change diameter (2); this itself will influence responses to flow and other stimuli (7). We have, however, shown that elevating intravascular pressure alone (at zero extravascular pressure) does not inhibit dilatory responses to ACh, making it unlikely to be responsible for the reduced dilatory responses we observed when extravascular pressure also is elevated. A direct inhibitory effect of extravascular pressure on dilator responses will have important implications for our understanding of the underlying mechanisms, and indeed the treatment options, associated with conditions such as heart failure.
Thus we have shown, for the first time, that modulating the relative combinations of intra- and extravascular pressures at a constant transmural pressure has no effect on stable levels of myogenic tone or on responses of isolated rat isobarically mounted coronary arteries to constrictor agonist addition or to depolarization. Conversely, the relative contribution of internal and external pressures does influence responses to vasodilatory stimuli when transmural pressure remains constant. In agreement with a previous in vivo study (16), we suggest that these effects are due to the elevated extravascular pressure opposing dilation. We do, of course, recognize that our simple experimental procedures do not directly represent the more complex role played by elevated extravascular pressure in modulating coronary arterial diameter in vivo, where differences in the absolute and relative values of intra- and extravascular pressures, and indeed transmural pressures, will be observed throughout the cardiac cycle. Similarly, the magnitude of the external pressures required to oppose dilation will likely depend on many factors, including compression of the venular system (and thus elevation of intravascular pressure) and possibly different forces the vessels themselves may have when embedded in intact muscle (e.g., a compressive force of the adventitia). Although these clearly warrant further study, our observation will nevertheless have important implications for our understanding of coronary blood flow in disease states such as congestive heart failure associated with elevated left ventricular pressure.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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