Intrinsic Tone as Potential Vascular Reserve in Conductance and Resistance Vessels
Background The purpose of this study was to define the degree of intrinsic tone in conductance and resistance vessels, to define the calcium dependency of intrinsic tone in these vascular preparations, and to investigate the efficacy of vasodilatory agents on the level of intrinsic tone in these vascular preparations.
Methods and Results All vessels were deendothelialized. Isometric force was recorded from strips of ferret aorta, ferret pulmonary artery, and human coronary artery. Vessel diameter was recorded from the ferret epicardial coronary artery and from ferret coronary microvessel in a pressurized no-flow state. Intrinsic tone was defined as the active increase in force or decrease in diameter with warming from 6°C to 37°C. Changes in force or diameter with various pharmacological agents were expressed as a percentage of intrinsic tone. Our results indicate that intrinsic tone accounts for ≈35% to 40% of total tone in all vascular preparations studied and is not dependent on extracellular calcium. Agents that increased cAMP levels (eg, forskolin, milrinone) and agents that decreased protein kinase C activity (eg, staurosporine) were partially effective in decreasing intrinsic tone. Nitroprusside, adenosine, hydralazine, and nifedipine had no significant effect.
Conclusions Our results indicate that intrinsic tone represents a significant component of vascular tone that has not been previously recognized and remains largely unexploited by current pharmacological therapies.
Vascular tone can be considered the sum of resting tone and agonist-induced tone. Interest in the former can be traced to Gaskell,1 who in 1881 described a “state of tonicity” in denervated vascular tissue. The vasoconstrictive reaction of isolated and denervated segments of arterial wall in response to the stimulus of a change in pressure was noted by Bayliss2 and defined as the “myogenic state” of vascular smooth muscle. Further investigation demonstrated that basal tone was at least partially dependent on stretch and extracellular calcium and involved activation of the muscle contractile apparatus.3 4 This basal tone is independent of circulating vasoactive agents and neurally or endothelially released substances5 6 and is thought to be a major factor in the regulation of regional blood flow in response to changes in blood pressure.7 In the absence of such basal vascular tone, cardiac output would be insufficient to maintain circulation, and peripheral collapse may occur.8 Review of the literature in this area, however, reveals that often different terms (eg, myogenic, basal, intrinsic) are used interchangeably, although they probably represent different processes.
Previous studies reporting a truly intrinsic tone have focused on the aorta of the ferret, a vessel that does not display a myogenic response. In that vessel, it was noted that cooling the preparation at slack length to ≈5°C abolished a truly intrinsic tone and separated it from passive tone.9 The decrease in active tone to cooling, ie, the temperature-dependent intrinsic tone, amounted to ≈30% of maximal agonist-induced tone in this vessel10 and was not affected by the presence of endothelium, nor was it dependent on intracellular or extracellular calcium.9 Previous work by Keatinge11 has shown vasodilation to cold in the precontracted state but did not examine the resting state. Thus, these novel results led us to hypothesize that calcium-independent intrinsic tone may represent a degree of vascular reserve that if defined mechanistically and exploited pharmacologically may be clinically important. It is essential, however, to first demonstrate that temperature-dependent intrinsic tone represents vascular reserve in tissue other than large conduit vessels such as the aorta. Thus, in the present study, we defined the degree of intrinsic tone in a number of vascular preparations, including both conductance and resistance vessels. We further investigated the effect of vasodilatory agents on the level of intrinsic tone in an effort to gain insight into its subcellular mechanism.
General Methods and Vascular Strip Preparations
Male ferrets ≈12 weeks old were anesthetized with chloroform. The appropriate tissue was removed and placed immediately into physiological saline solution (PSS) as previously described.10 All procedures were approved by the Institutional Animal Care and Use Committee of the Beth Israel Hospital (Boston, Mass). For preparation of the strips, the adventitia was dissected away, and strips were obtained. The endothelium was removed through gentle rubbing with a blunt probe. Final strips were 5 to 6 mm long and 1 to 2 mm wide and weighed an average of 1.3 mg.
Strips were anchored at one end in a muscle bath, and the other end was attached to a Gould UC2 force transducer. Strips were stretched to 1.4 slack length, which, in preliminary studies, has been shown to approximate the length for optimal force production (Lo). A circulating waterjacket attached to a temperature-controlled bath circulator (Lauda RM3) maintained temperature ±0.5°C. Unless indicated otherwise, all experiments were performed in oxygenated PSS. Experiments were begun at 37°C, and adequate time was allowed for equilibration (45 minutes). Viability was demonstrated by maximal force response to elevated potassium PSS. After washing and equilibration, the tissue was cooled to 6°C and allowed to achieve steady state. The increase in active tone with rewarming expressed as a percentage of total vascular tone is defined as percent intrinsic tone. On the tissue attaining steady state, pharmacological agents were added, and any decrease in force was expressed as a percentage decrease in intrinsic tone. For calcium-free experiments with EGTA, the entire procedure was repeated in calcium-free solution.
Ferret Coronary Artery and Microvessel Preparation
Segments of left anterior descending coronary artery (LAD) (average diameter, 380 μm) and microarterial vessels emanating from the LAD were carefully dissected with a ×10 to 60 dissecting microscope (Olympus, Optical Co). Microvascular segments varied in size from 90 to 170 μm and from 1 to 2 mm long. Endothelium was removed with the use of a human hair followed by an air bubble.12 13 Vessels were placed into an isolated Plexiglas organ chamber, cannulated with dual glass micropipettes, and secured with 10-0 nylon microfilament suture. Oxygenated PSS was continuously circulated through the organ chamber. The microvessels were pressurized to 40 mm Hg (except in high-pressure experiments) in a no-flow state with a burette monometer filled with PSS. This pressure was chosen for the microvessel studies to avoid any stretch-dependent effects that begin at higher pressures.5 6 14 15 16 With an inverted microscope (×40 to 200, IMT-2, Olympus) connected to a videocamera, the vessel image was projected onto a television monitor (Panasonic). A videoelectronic dimension analyzer (Living Systems Instrumentation) was used to measure internal lumen diameter and wall thickness. Measurements were recorded with a four-channel Western Graphtec Recorder. After 30 minutes of equilibration in oxygenated PSS at 37°C, baseline diameter (in μm) was recorded. The protocol described above was used except that diameter was recorded. The efficiency of these methods of endothelial denudation for both tissue rings and microvessels has previously been confirmed in our laboratory with the use of agents such as acetylcholine, bradykinin, and sodium nitroprusside.
Human Coronary Artery Studies
Experimental vascular preparations were obtained from patients undergoing heart transplantation due to end-stage heart failure and followed the guidelines of a protocol approved by the Committee for the Protection of Human Subjects at the Beth Israel Hospital and Brigham and Women's Hospital, Boston, Mass. Tissue from patients diagnosed as having coronary artery disease was not used in the present study. Tissue was placed into oxygenated PSS immediately on harvest, and all experiments were completed within 24 hours. Viability was determined through stimulation with elevated potassium PSS. Tissue strip length ranged from 7.5 to 9.0 mm, and weight ranged from 4.4 to 5.4 mg.
For most experiments, PSS was used and contained (in mmol/L) NaCl 120, KCl 5.9, NaHCO3 25, NaH2PO4 1.2, CaCl2 2.5, MgCl2 1.2, and dextrose 11.5. Calcium-free PSS was similar except that no CaCl2 was added and it contained 2 mmol/L EGTA. Elevated potassium PSS was similar except that the KCl concentration was increased to 51 mmol/L and an equimolar amount of NaCl was removed.
Adenosine, staurosporine, forskolin, nitroprusside, hydralazine, and nifedipine were purchased from Sigma Chemical Co. Milrinone was obtained from Sterling Winthrop Laboratories. All other chemicals (reagent grade or better) were obtained from Fisher.
All values were expressed as mean±SEM. The number of different animals from which preparations were taken are indicated. Significance of difference was detected with paired t test or ANOVA as indicated in figure legends. Statistical significance was defined as P<.05.
Demonstration of Intrinsic Tone in Ferret Aorta
A typical experiment illustrating the method used to establish the degree of temperature-dependent intrinsic tone is shown in Fig 1⇓ (top). Maximal force generation was measured through a challenge with elevated potassium PSS, which in this case resulted in a force of 1.0g (Fig 1,⇓ top). To determine the position of the passive force level at Lo, cooling was performed over a 5-minute interval, and adequate time (30 minutes) was allowed to achieve steady state. Rewarming the tissue resulted in a return to baseline level (before cooling). Intrinsic tone is defined as the increase in active tone with rewarming that amounted to 0.52g force (Fig 1,⇓ top). Thus, intrinsic tone, when expressed as a percentage of total vascular tone (1.0g plus 0.52g) in this case was 33%. The mean intrinsic tone seen in our study was 43±2.4%. That this is an active tone has been shown in a previous study9 and was confirmed in the present study by the fact that preincubation with 10 mmol/L NaCN, a concentration that decreased the response to 51 mmol/L KCl by 85±4.3% (n=4), decreased intrinsic tone by 77±3.2% (n=4). Thus, a significant amount of vascular tone could be elicited in a reversible manner with use of the above protocol. Whether this is a phenomenon unique to the ferret aorta or a more fundamental property of vascular tissue was then considered.
Demonstration of Intrinsic Tone in Ferret Conductance and Resistance Vessels
The ferret pulmonary and large epicardial coronary vessels are readily accessible and more likely than the aorta to contribute to physiological and pathophysiological regulation of blood flow. The protocol shown in Fig 1⇑ (top) was used to study the ferret pulmonary artery and LAD. Pulmonary artery intrinsic tone amounted to 42±3.8% of total vascular tone. In the LAD, intrinsic tone was found to be 34±4.4%. These results are summarized in Fig 1⇑ (bottom). Thus, there is a significant potential vascular reserve in three different conductance vessels.
Despite the potential physiological and pathophysiological importance of intrinsic tone in large conductance vessels, it is generally acknowledged that the major determinant of tissue perfusion is the diameter of resistance vessels. Fig 1⇑ (bottom) shows the degree of intrinsic tone in ferret coronary microvessels compared with other vessels. In the coronary microvessels, the degree of intrinsic tone, 41±1.4%, was similar to that seen in conductance vessels, confirming its importance. It is important to recognize that in the microvessels, intrinsic tone is defined by changes in vessel diameter, not by changes in force. The use of diameter as an indirect measure of force is accepted in the study of the microvasculature,5 6 12 14 15 16 although the limitations of extrapolation to force must be realized.
Demonstration of Intrinsic Tone in Human Coronary Arteries
Further investigations were undertaken to determine whether these results could be extrapolated to human vessels. Fig 1⇑ (bottom) shows that temperature-dependent intrinsic tone in human epicardial coronary vessels (right coronary artery and LAD) was 35±0.5% of total vascular tone, a value similar to that in ferret vessels. Despite the limited number of preparations available, these results strongly suggest the presence of an important potential component of vascular reserve in human coronary arteries.
Dependence on Extracellular Calcium
Calcium is known to play a central role in the development and maintenance of vascular tone. Therefore, we were interested in determining the calcium dependence of intrinsic tone in the vessels used in the present study. The effect of the calcium channel blocker nifedipine on intrinsic tone in the ferret aorta, pulmonary artery, and coronary microvessels was studied first. As seen in Fig 2⇓ (top), the effect of nifedipine (1×10−5 mol/L) on intrinsic tone in ferret aorta, pulmonary artery, and microvessels was minimal and did not achieve statistical significance.
To test whether intrinsic tone and myogenic tone represent distinct processes and because of the differential selectivity of various calcium channel blockers, the effects of calcium-free PSS plus EGTA on intrinsic tone were investigated. As seen in Fig 2⇑ (bottom), calcium-free solution had no effect on the intrinsic tone in ferret aorta and pulmonary artery. A small but statistically insignificant effect was seen in the ferret coronary microvessels (40 mmHg). Thus, our results clearly demonstrate that temperature-dependent intrinsic tone is not dependent on extracellular calcium.
We sought to explain the disparity regarding calcium dependency in myogenic and intrinsic tone. Kuo et al15 suggested that pig epicardial arterioles developed statistically significant myogenic activity at intraluminal pressure of >100 cm H2O (73 mm Hg). Similarly, Osol et al16 reported the appearance of myogenic tone between 50 and 75 mm Hg. Our experiments were conducted at 40 mm Hg; at this pressure, there was no significant effect of calcium-free solutions (Fig 2,⇑ bottom). However, when vessel pressure was increased incrementally to 120 mm Hg, myogenic activity was noted, and the addition of calcium-free solution did decrease tone (Fig 2,⇑ bottom). Thus, the intrinsic tone component of basal tone is not dependent on extracellular calcium, whereas the myogenic component of basal tone is partially calcium dependent.
Concentration-Response Relationship for Various Vasodilatory Agents in Ferret Aorta
The question arises as to the best pharmacological approach with which to exploit the potential vascular reserve reflected in intrinsic tone. For this reason, the effects of a number of vasodilatory agents were determined. Fig 3⇓ depicts the concentration-response relationship for these agents in ferret aorta between 1×10−9 and 1×10−4 mol/L. Agents that increase cAMP levels (eg, milrinone, forskolin) or decrease protein kinase C activity (eg, staurosporine) were effective in decreasing intrinsic tone by ≈30%. Interestingly, sodium nitroprusside, hydralazine, nifedipine, and adenosine caused no statistically significant decrease in intrinsic tone. As can be noted in Fig 3,⇓ most agents that were effective in decreasing intrinsic tone had a near-maximal effect at 1×10−5 mol/L concentration. Forskolin and milrinone continued to show increasing effectiveness at higher concentrations, but these were not used to avoid nonspecific effects. A summary of the percent decrease in intrinsic tone at this concentration (1×10−5 mol/L) for all agents tested in ferret aorta is shown in Fig 4⇓ (top).
Effect of Vasodilatory Agents on Intrinsic Tone in Ferret Pulmonary Artery and Coronary Microvessels
Whether the results noted above for the aorta can be extended to the pulmonary artery and resistance vessels (coronary microvessels) was also determined. As was seen in the aorta, both ferret pulmonary artery and microvessel showed (Fig 4,⇑ middle and bottom) decreases in intrinsic tone in response to agents that elevate cAMP levels (eg, forskolin, milrinone) or decrease protein kinase C activity (eg, staurosporine). A slightly greater effect of forskolin and milrinone was seen in microvessels than in other vascular beds studied, with a 45% to 50% decrease in intrinsic tone. Sodium nitroprusside, adenosine, and hydralazine did not demonstrate a significant effect on intrinsic tone in the pulmonary artery or coronary microvessels.
Our results suggest several important conclusions: (1) intrinsic tone accounts for ≈40% of total tone in several ferret vascular beds, (2) intrinsic tone can be demonstrated in both conductance and resistance vessels, (3) intrinsic tone appears to be present in human coronary arteries, (4) intrinsic tone is not significantly dependent on extracellular calcium, (5) agents that increase cAMP levels or those that inhibit protein kinase C activity are partially effective (30%) in decreasing intrinsic tone, and (6) nitrates, calcium channel blockers, adenosine, and hydralazine are not effective in decreasing intrinsic tone. We believe that intrinsic tone represents a potentially significant component of vascular tone that has not been previously recognized. Pathophysiological alteration of this vascular reserve may be of great importance in conditions under which reduction in vascular resistance may be of significant use (eg, hypertension, coronary artery disease, peripheral vascular disease). Thus, defining the mechanisms responsible for intrinsic tone is of significant importance.
Inhibitors of protein kinase C were able to produce a 25% to 30% decrease in intrinsic tone, leading us to conclude that a protein kinase C-dependent signal transduction pathway plays at least a partial role in the development of intrinsic tone. This is not surprising given the growing evidence supporting an important role for protein kinase C in the regulation of vascular smooth muscle tone.4 16 17 18 19 20 Recently, we observed that Ca+-independent isoenzymes of protein kinase C may also be involved in the regulation of smooth muscle contraction.16 17 19 The apparent calcium independence of intrinsic tone suggests the involvement of a calcium-independent isoform of protein kinase C. Identification of specific isoenzyme(s) that may be involved in intrinsic tone may allow for greater manipulation with more specific inhibitors than staurosporine. The concentration of staurosporine used in this study was relatively high, and thus effects on other protein kinases21 cannot be excluded.
Agents that elevate levels of cAMP also decrease intrinsic tone by ≈30%. Effects on the contractile apparatus itself, in addition to effects on calcium homeostasis, have been implicated in the ability of cAMP to mediate relaxation.18 Which of these mechanisms is responsible for decreasing intrinsic tone is unclear from our results and will require further investigation. Ideally, agents with more targeted actions, perhaps at one of the effector sites of the actions of cAMP, will allow greater exploitation of intrinsic tone.22
Adenosine was ineffective in reducing intrinsic tone in both conductance and resistance vessels. The latter result is somewhat surprising given that adenosine is known for its vasodilatory effects in resistance vessels. It should be noted, however, that the well-recognized vasodilatory action of adenosine is generally observed in conditions under which the tissue is preconstricted.23 24 In the present study, we examined the effect of this agent on basal tone in the resting state. Also, it is unlikely that the degradation of adenosine plays a significant role as the agent is applied directly to smooth muscle cells and previous studies that involved the same methodology in preconstricted coronary microvessels have shown significant vasorelaxation.23
The lack of dependency of intrinsic tone on extracellular calcium in conductance and resistance beds is an important observation. It is clear that calcium is a major determinant of vascular tone. There is extensive evidence that myogenic tone is dependent on extracellular calcium20 25 in resistance arteries. Although our results appear to conflict with previous data, we believe that there is a simple explanation that points to the general concept that intrinsic tone represents a process that is distinct from myogenic tone. As stated earlier, several investigators have demonstrated the development of myogenic tone at intraluminal pressures of >50 to 75 mm Hg.15 16 Our studies demonstrating calcium independence of intrinsic tone in resistance vessels used intraluminal pressures well below this (40 mm Hg). In fact, at higher pressures at which a myogenic response was generated, a dependency on extracellular calcium was also seen in the present study.
Based on our results, we propose a model that may help define and clarify the confusing terminology in the literature (Fig 5⇓). We define resting tone as consisting of basal active tone and passive tone. Basal active tone is comprised of two components: myogenic tone and intrinsic tone. Myogenic tone is stretch, calcium, and protein kinase C dependent.16 Our results suggest that intrinsic tone is universally calcium independent. Intrinsic tone is sensitive to cAMP levels and protein kinase C inhibition and can be elicited through rewarming cooled tissue, even under conditions in which myogenic responses are not expected. The dotted line in Fig 5⇓ between intrinsic tone and myogenic tone reflects the fact that various vascular beds may have different proportions of these two variables comprising basal tone. Tone above resting tone is regulated by neural, humoral, and metabolic factors (agonist-induced tone). Intrinsic tone does not appear to require an exogenous initiating factor but is regulated, as shown by this study.
In summary, our results show that intrinsic tone represents an important degree of vascular reserve that is partially sensitive to cAMP levels and protein kinase C inhibition but not extracellular calcium. Other agents with known vasodilatory effects that occur through alternative mechanisms were ineffective in reducing intrinsic tone. Thus, inhibition of the currently understood pathways leading to smooth muscle contraction leave ≈70% of intrinsic tone unaffected, suggesting the presence of new mechanisms to be defined.
This work was supported by PHS grants HL-31704 and HL-42293 (Dr Morgan) and NIH HL-46716 and by American Heart Association, Massachusetts Affiliate, Grant-in-Aid (Dr Sellke). We are indebted to Christopher Parker and Christa Lajoie for their technical assistance and to Debora Fischer for help in preparation of the manuscript.
- Received October 30, 1995.
- Revision received February 13, 1996.
- Accepted March 6, 1996.
- Copyright © 1996 by American Heart Association
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