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1Department of Physiology, New York Medical College, Valhalla, New York 10595; 2Department of Pathophysiology, Semmelweis University, 1445 Budapest; and 3Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, 4004 Debrecen, Hungary
Submitted 23 March 2004 ; accepted in final form 16 August 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-nitro-L-arginine-methyl-ester (L-NAME) or by endothelium removal, whereas duration of postocclusion dilations were reduced by L-NAME or by endothelium removal only in P+F protocols. Furthermore, in both protocols, catalase significantly reduced the peak but not the duration of reactive dilations. Thus, mechanosensitive mechanisms that are sensitive to deformation, pressure, stretch, and wall shear stress elicit release of NO and H2O2, resulting in reactive dilation of isolated coronary arterioles.
reactive hyperemia; deformation; pressure; stretch; flow/shear stress; myogenic; endothelium
Early studies by Eikens and Wilcken (10) suggested a possible role for pressure-induced myogenic responses in the development of reactive hyperemia by demonstrating that even short periods of occlusions (<1 s) caused reactive hyperemic response in coronary circulation. Also, Schwartz et al. (28) found that coronary occlusion during diastole with a duration of 
100 ms resulted in reactive hyperemia in coronary circulation. In these conditions the role of metabolic factors is likely to be negligible and suggest the involvement of mechanisms that are sensitive to changes in hemodynamic forces during and after release of occlusion. It is plausible that during and after release of occlusions, changes in blood pressure and flow activate myogenic (15) and wall shear stress sensitive mechanisms (18, 19, 21, 31) eliciting the release of vasoactive factors from the vascular wall, which contribute to the development of reactive hyperemia. Although our previous study (17) provided evidence for the role of intrinsic vascular mechanosensitive mechanisms in the development of reactive dilation in isolated rat skeletal muscle arterioles, the role and nature of these mechanisms in coronary arterioles is not known.
We hypothesized that mechanosensitive mechanisms activated by changes in pressure and flow/shear stress during and after release of occlusions elicit reactive dilation in isolated coronary arterioles. Thus we have studied the effects of brief occlusions on the diameter of isolated coronary arterioles and aimed to elucidate the role of mechanosensitive mechanisms in the development of reactive dilation.
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METHODS |
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90 µm diameter) (19, 21). In brief, after overnight fasting, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The heart was excised and placed in a silicone-lined petri dish containing cold (0–4°C) physiological salt solution (PSS) composed of (in mM) 110 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose, and 24.0 NaHCO3 equilibrated with a gas mixture of 10% O2 and 5% CO2, balanced with nitrogen, at pH 7.4. With the use of microsurgical instruments and an operating microscope, the second branch of septal artery (1.5 mm in length) running intramuscularly was isolated and transferred into an organ chamber containing two glass micropipettes filled with PSS. Vessels were cannulated on both ends and micropipettes were connected with silicone tubing to an adjustable PSS reservoir. Inflow and outflow pressures were set to 80 mmHg and continuously measured by a pressure servo control system (Living Systems Instrumentation). Temperature was set at 37°C by a temperature controller (Grant Instruments). The internal arteriolar diameter at the midpoint of the arteriolar segment was measured by videomicroscopy with a microangiometer (Texas Instruments). Changes in arteriolar diameter and intraluminal pressure were continuously recorded with the Biopac-MP100 system connected to a computer and analyzed with AcqKnoweldge data-acquisition software (Biopac Systems). Perfusate flow was measured with a ball flowmeter (Omega).
Experimental protocols.
Arterioles were allowed to develop spontaneous tone in response to intraluminal pressure (80 mmHg) under no flow conditions (equilibration period 1 h). Changes in the diameter of arterioles during and after brief occlusion of the cannulating tube were then continuously measured (Fig. 1, A and B). First, responses were obtained to changes only in intraluminal pressure at a zero-flow condition. During occlusion of perfusion tubes (both input and output) intraluminal pressure decreased from 80 to 10 mmHg for 30 s, then after the release of the occlusion, it increased back to 80 mmHg (within 1–2 s). Between interventions, 15-min equilibration periods were kept. Responses were also obtained after 60 or 120 s occlusions. In one group of experiments, all procedures were repeated after 30 min to obtain time controls (17).
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30 µl/min flow was established by changing the inflow (100 mmHg) and outflow (60 mmHg) pressure to an equal degree, but in opposite directions, to keep midpoint luminal pressure constant (80 mmHg). Arteriolar responses to brief occlusions of the inflow cannula were then obtained. In this condition, both intraluminal pressure and flow were changed. The inflow cannula was occluded for 30, 60, or 120 s, whereas the output pressure was maintained at 10 mmHg. After the occlusion was released and flow reestablished, the output pressure was brought back to 80 mmHg. In these protocols, a 15-min equilibration period was kept between occlusions, and time controls were obtained as well (17).
Arteriolar responses in the same protocols (Pressure and Pressure+Flow) were obtained after endothelium denudation. The endothelium of the arterioles was removed by perfusion of the vessel with air, as described previously (19). Endothelium denudation was asserted by the loss of dilation to acetylcholine (10–7 M) and the maintained dilation to the NO donor, sodium nitroprusside (10–7 M). The same protocols were repeated in the presence of N-nitro-L-arginine-methyl-ester (L-NAME; 10–4 M for 20 min), an inhibitor of the endothelial NO synthase or after incubation and intraluminal presence of catalase (120 U/ml, for 30 min) (1, 4). Finally, in separate experiments, coronary arteriolar responses to exogenously administered H2O2 (10–8-3 x 10–6 M) were also obtained and during this condition the level of H2O2 in the bath solution was measured by the ISO-HPO-100 electrode tips of a free radical analyzer (model APOLLO 4000, World Precision Instruments).
Data analysis and statistics. Changes in arteriolar diameter were recorded and analyzed during and after occlusions. During occlusion, when first arteriolar diameter decreased and then increased, both the maximum decrease and the maximum increase in diameter were measured. The reactive dilations of arterioles that developed after release of occlusions were characterized by the peak increase in diameter above the baseline and the duration of increase in diameter, which was assessed by the time necessary for the diameter to return to 110% of the baseline diameter. The contribution of flow-sensitive mechanisms in reactive dilation was estimated by the subtraction of diameter data obtained in Pressure protocol from the data obtained in Pressure+Flow protocols. Similar subtractions were performed to estimate the contribution of NO and H2O2 in eliciting reactive dilation.
At the conclusion of each experiment, to obtain the maximum passive diameter, the suffusion solution was changed to a Ca2+-free PSS, which contained EGTA (10–3 M), and the vessel was incubated for 10 min. All drugs were added to the vessel chamber, and final concentrations are reported. All salts and chemicals were obtained from Sigma Aldrich. Solutions were prepared on the day of the experiment. Data are expressed as means ± SE. For statistical analysis, two-way ANOVA, followed by Tukey's post hoc test and Pearson correlations were used as indicated. P < 0.05 was considered statistically significant.
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RESULTS |
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Reactive dilation induced by occlusions when only intraluminal pressure changed.
In the first series of experiments, we obtained changes in diameter when only intraluminal pressure changed (Pressure protocols, Fig. 1A). Original figures show that arteriolar diameter decreased during occlusion (by 29 ± 8 µm), but after 50 s diameter significantly increased close to the diameter obtained before occlusion (Fig. 1C). After the release of 30-, 60-, or 120-s occlusions of cannulating tube, intraluminal pressure increased to 80 mmHg (within 1–2 s), followed by a marked increase in arteriolar diameter, above the initial, baseline value. The arteriole then constricted toward the control diameter (Fig. 1C). This diameter response was designated as "reactive dilation" because it resembles the characteristics and time course of reactive hyperemia observed in vivo (7, 13, 16, 20, 25, 26). Summary data show that the peak of reactive dilation of arterioles significantly increased as a function of the length of occlusion (Fig. 3A). The duration of dilations, although tended to increase as a function of the length of occlusion, did not reached the level of significance (Fig. 3C).
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Effects of L-NAME or endothelium removal on the magnitude of reactive dilation. In the presence of L-NAME or after endothelium removal, during occlusions, when intraluminal pressure decreased from 80 to 10 mmHg, similarly to control, arteriolar diameter decreased (by 19 ± 9 µm). In contrast, however, the diameter remained reduced either in the presence of L-NAME or after endothelium denudation (Fig. 2A). Figure 2B shows that during occlusion there was a significant, positive correlation between the magnitude of decreases in arteriolar diameter and the magnitude of the developed dilations, but no correlation was observed in the presence of L-NAME.
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Effect of catalase on the magnitude of reactive dilation.
In the presence of catalase, during occlusions, changes in arteriolar diameters were similar to those of control responses. However, after release of occlusion, presence of catalase significantly reduced the peak but not the duration of reactive dilations in both Pressure and Pressure+Flow protocols (Fig. 4, A–D). Original record demonstrates that cumulative doses (10–8-3 x 10–6 M) of exogenously administered H2O2 elicited dilation of isolated coronary arterioles (Fig. 4E). Summary data show dose-dependent dilations of arterioles to H2O2 approaching 90% dilations at higher doses (10–6 and 3 x 10–6 M; Fig. 4F).
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DISCUSSION |
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Reactive hyperemia in coronary circulation has been intensively studied (7, 11, 13, 16, 20, 25, 26). Early investigations (14, 16, 20) have shown that longer occlusions prolonged the duration of reactive hyperemia. Therefore, it was logical to propose a primarily role for tissue-derived metabolic factors in mediating reactive hyperemia, especially in working cardiac muscle. Yet, in other studies (10, 28), a possible role for hemodynamic factors, such as myogenic response was also proposed. On the basis of studies investigating flow-dependent dilation of coronary vessels in vivo (13) and in vitro (9, 21), it is also plausible to suggest a role for wall shear stress-dependent mechanism in the development of reactive hyperemia. However, there are no studies demonstrating a clear evidence for the contribution of mechanosensitive mechanisms to the development of reactive hyperemia in coronary circulation. This is due to the fact that all previous investigations of reactive hyperemia were conducted in vivo, a condition, which does not allow separation of the contribution of neural, humoral, tissue-metabolic, or mechanosensitive mechanisms in the development of reactive hyperemia. Thus we aimed to elucidate the contribution of pressure- and shear stress-dependent vascular mechanisms in the development of coronary reactive hyperemia by investigating the effect of brief occlusion-elicited changes in hemodynamic forces on the diameter of isolated coronary arterioles.
Effects of occlusions on coronary arteriolar diameter. In the absence of intraluminal flow (Pressure protocol) in response to occlusion-induced reduction in intraluminal pressure, from 80 to 10 mmHg, arteriolar diameter first decreased, but during longer occlusions (60 and 120 s) the diameter started to increase even before the release of occlusions (Fig. 1C). Similar changes in diameter were observed in Pressure+Flow protocols during occlusions (Fig. 1C). When we analyzed these responses, we found a positive correlation between the maximum decreases in diameter and the maximum increases in diameter developed during occlusions (Fig. 2B). The dilations during occlusions and consequently the correlation were abolished either by inhibition of eNOS or by endothelium denudation (Fig. 2). On the basis of the present and our previous findings (17), we suggest that during occlusion the deformation of endothelial cells, as a result of decreases in diameter, is responsible for the sequential dilations during occlusion. One could argue that reduction in intraluminal pressure eliciting myogenic dilation could be responsible for the dilation during occlusion. The finding, however, that the correlation between the reduction and increase in diameter during occlusion was sensitive to NO synthase inhibition suggests that diameter reduction-induced deformation of vascular tissue, is primarily responsible for the subsequent dilation during occlusion (Fig. 2). Indeed, it has been shown that endothelium deformation that occurs when arteriolar diameter decreases activates NO synthesis by eNOS (30), and thus it is likely that this mechanism contributes to the increases in arteriolar diameter during occlusion.
In the present study, we found that after release of occlusions, increases in diameter increased as a function of the length of the occlusion, regardless whether or not flow was present (Fig. 3, A and B). Interestingly, the time-dependent enhancement of peak dilations in the Pressure protocols was similar in characteristic compared with in vivo hyperemic responses of coronary circulation. However, the exact mechanisms by which the longer occlusions elicit greater post (7, 10, 28) occlusion peak reactive dilation of isolated coronary arterioles are not known. Because inhibition of eNOS or endothelium removal significantly reduced the increments of postocclusion peak dilations, it is likely that a release of endothelium-derived NO is involved (Fig. 3), in part, due to the activation of eNOS by endothelial cell deformations during occlusions (6, 12). That is, longer occlusions elicited greater NO release. This idea is supported by our previous findings (17) that longer occlusions cause a greater deformation-induced activation of eNOS in a tyrosine-kinase inhibition-dependent manner.
Role of NO and H2O2 in peak dilations of coronary arterioles. Previously, it has been shown that inhibition of NO synthesis reduced the peak reactive hyperemia in coronary vessels of dogs although the underlying mechanism was not clarified (33). In the present study, we have found that peak reactive dilations were significantly reduced by eNOS inhibition (Fig. 3, A and B). We propose that after release of an occlusion, a sudden increase in pressure occurs, which, by causing stretch of the endothelial cells could lead to increase in endothelial intracellular [Ca2+], eliciting release of endothelium-derived NO, as suggested previously (5, 22). Collectively, these findings suggest an important role for deformation and pressure/stretch-induced release of NO from the endothelium in the development of the peak reactive dilation.
Recent studies by Gutterman and colleagues (23, 24) and Faraci and colleagues (29) suggested a significant role for H2O2 in mediating mechanosensitive responses of coronary and cerebral microvessels. In human atrial coronary arterioles, it has been found that H2O2 mediates flow-induced dilation (23, 24). Thus it seemed logical to hypothesize that H2O2 may contribute to hemodynamic force-induced reactive dilations of coronary arterioles. Indeed, we have found that incubation with and presence of catalase, is known to rapidly convert H2O2 to H2O, significantly reduced the peak reactive dilations in both Pressure and Pressure+Flow protocols (Fig. 4, A and B). Furthermore, in this study we have also found that in coronary arterioles, low concentrations (10–7-10–6 M) of exogenously administered H2O2 elicited substantial dilations (Fig. 4, E and F), suggesting that coronary arterioles are sensitive to H2O2. Together, these findings indicate that H2O2 released endogenously within the vascular wall to changes in hemodynamic forces during and after occlusions contributes, at least in part, to the development of reactive dilations of coronary arterioles. Extrapolating these findings, we propose a physiological role for NO and H2O2 in the development of reactive hyperemia in the coronary circulation.
Interaction of pressure-induced myogenic constriction and flow/shear stress determining the duration of reactive dilation. After the release of an occlusion, increases in pressure can activate a myogenic mechanism eliciting vasoconstriction (15). In this study, we have found that greater peak dilation most likely induces a greater activation of arteriolar myogenic mechanism, because in Pressure protocols the duration of reactive dilations did not increase substantially (Fig. 3C). In contrast, in Pressure+Flow protocols, after release of occlusions, the duration of reactive dilation increased significantly (Fig. 3D), suggesting that increases in flow by activating shear stress-dependent mechanisms determine primarily the duration of the reactive dilations. It is of note, however, that although in the Pressure protocol, only pressure had been changed, but because the cross-sectional area of vessel increases when pressure increases, there is fluid moving into the vessel. This expansion is termed a capacitance-related flow, the effect of which cannot be completely discounted in the observed responses.
In previous studies, we (3, 32) and others (21) have shown that in coronary arterioles, flow-induced dilation is mediated by endothelial release of NO. In this study, we have found that inhibition of eNOS or endothelium removal significantly decreased the duration of reactive dilation of coronary arterioles (Fig. 3D), whereas presence of catalase affected primarily the peak of reactive dilations (Fig. 4, A and B). Collectively, these findings suggest that H2O2 contributes to the development of the early phase, whereas flow/shear stress-induced release of NO prolongs the later phase of reactive dilation.
It is logical to assume that the flow/shear stress-dependent mechanism has an even more substantial role in the development of reactive hyperemia in vivo, because the dilation of the distal arteriolar network in coronary microcirculation allows a greater increase in blood flow velocity, hence, wall shear stress after release of occlusions (in the present experiments after release of occlusions, intraluminal flow only returned to the initial flow rate).
The time-dependent contribution of mechanical forces-activated, NO- and H2O2-mediated mechanisms in the development of reactive dilation of coronary arterioles is depicted in Fig. 5 by subtracting the diameter response curves obtained in Pressure protocols from those obtained in Pressure+Flow protocols in the presence of L-NAME or catalase (Fig. 5). It shows that during occlusion, there is a substantial release of NO, likely due to endothelial deformation, which is further enhanced when occlusion is released, allowing increases in pressure and flow, all of them contributing to the peak and duration of reactive dilations (dashed line, Fig. 5). Obviously, during occlusion intraluminal flow has no role in changes in diameter; however, after occlusion, increases in flow (with a delay characteristic of shear stress-dependent mechanism) contribute significantly to the later phase of reactive dilation (dotted line, Fig. 5). On the basis of catalase experiments, H2O2 does not seem to be released during occlusion (dash-dotted line, Fig. 5), but after release of occlusion, there is a significant release of H2O2, which contributes primarily to the early phase of reactive dilation (dash-dotted line; Fig. 5).
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In summary, the present findings indicate that in isolated coronary arterioles changes in endothelial cell shape, intraluminal pressure, and flow/shear stress, as result of brief occlusions and release of perfusion, activate mechanosensitive signaling mechanisms in the arteriolar wall, which via release of NO and H2O2, elicit reactive dilation. Thus, we propose that in addition to previously described factors, activation of mechanosensitive mechanisms contribute substantially to the development of reactive hyperemia in the coronary circulation.
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ACKNOWLEDGMENTS |
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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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