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Journal of Physiology (1991), 436, pp. 371-383 With 8 figures Printed in Great Britain
INFLUENCE OF PRESSURE ALTERATIONS ON TONE AND VASOMOTION OF ISOLATED MESENTERIC SMALL ARTERIES OF THE RAT
BY ED VANBAVEL, MAURICE J. M. M. GIEZEMAN, TRUDI MOOIJ AND JOS A. E. SPAAN From the Cardiovascular Research Institute Amsterdam, Department of Medical Physics, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (Received 22 February 1990) SUMMARY
1. Myogenic responses may account for control of organ blood flow. The study of these responses without interference from the organ requires an isolation technique for vessels which contribute significantly to flow resistance. This study reports on experiments on isolated small mesenteric arteries. 2. Distal rat mesenteric arcade arteries and first-order branches (diameter range 145-365 ,um, mean 293 ,um) were manually dissected and cannulated using a doublebarrelled micro-cannula. Luminal cross-sectional area of these vessels was continuously monitored by means of a fluorescence technique. 3. Nine out of eighteen vessels developed basal tone at 80 mmHg distending pressure, resulting in a 45-2 + 5-1 % (mean + S.E.M) decrease of cross-sectional area. Tone was induced in the other vessels by 0-3-1 tim-noradrenaline, resulting in a 59-5 + 741 % decrease in cross-sectional area. 4. In vessels with either spontaneous or induced tone, stepwise changes of pressure resulted in passive effects, followed by myogenic responses. 5. Steady-state pressure-cross-sectional area relations of vessels with basal tone showed a significant negative slope (-0-5% mmHg-'), while pressure-crosssectional area relations of vessels with induced tone were essentially flat between 40 and 120 mmHg. 6. Five vessels with basal tone and eight vessels with induced tone developed vasomotion at 80 mmHg. Frequencies of spontaneous and induced vasomotion were 14 (range 4-31) and 21 (9-25) cycles min-' respectively. Amplitudes were 5 (1-10) and 8 (3-17)% of the passive cross-sectional area. In both groups, frequency was positively, and amplitude negatively correlated with pressure. 7. These data show that myogenic responses are induced by wall stress, rather than by distension of the vascular wall. Basal tone is not a prerequisite for the appearance of myogenic responses.
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E. VANBA VEL AND OTHERS INTRODUCTION
It has been known since the beginning of this century (Bayliss, 1902) that blood vessels react to a raised perfusion pressure by increasing smooth muscle cell tone. This myogenic response may play a role in the autoregulation of flow (Johnson, 1980, 1986) and capillary pressure (Mellander, Oberg & Odelram, 1964; Mellander, Maspers, Bjornberg & Andersson, 1987). Much evidence in favour of a role in autoregulation comes from whole-organ studies. Thus, increasing local pressure by raising venous pressure or transmural pressure induces resistance increase and constriction of observed arterioles (see Johnson 1986, 1989). However, these myogenic responses lead to secondary alterations of local pressures. This stresses the need to actually measure these pressures in the study of myogenic responses, as was done by Davis & Sikes (1989) and Griinde (1989). Further complications in the quantification of myogenic responses in whole organs are flow dependent and metabolic effects, which may become involved when flow is changing due to myogenic constriction. A careful analysis of data is needed to estimate the impact of these effects (Grainde, 1989). Alternatively, one can study myogenic responses in isolated, cannulated vessels. Due to the assumed involvement of myogenic responses in flow control, it would be most relevant to perform these studies on resistance-sized arterial vessels. Up to now, quantification of myogenic responses in isolated resistance vessels has been limited to the brain (Osol & Halpern, 1985), heart (Kuo, Davis & Chilian, 1988) and hamster cheek pouch (Jackson & Duling, 1989). The purpose of this paper is to quantify the myogenic responses in isolated, cannulated mesenteric arteries which are resistance sized, or near to this size. In particular we aimed to establish the relation between basal tone and myogenic responses, and to test whether myogenic responses in the mesenteric vessels are large enough to cause an actual constriction, rather than partial compensation of passive effects, when pressure is raised. Furthermore, the influence of pressure on the vasomotion which was observed in these vessels is documented. METHODS
Isolation procedure Male Wistar rats (350-450 g) were anaesthetized by intraperitoneal injection of pentobarbitone (0-06 mg (g body weight)-'). The abdomen was opened and part of the intestinal tract was removed and placed in cold bicarbonate-buffered Ringer solution (BR, composition in mM: NaCl, 115; KCl, 4-7; KH204, 1-2; MgSO4, 1-2; NaHCO3, 24; CaCl2, 1-25; glucose, 10; solution equilibrated with 95% air, 5% C02; pH adjusted to 7 35). Part of the arterial mesenteric arcade and some first-order branches were cleared from surrounding tissue. This part of the mesenteric arterial tree was removed and placed in the cannulation chamber. Cannulation technique and cross-sectional area measurement Both the cannulation technique and the cross-sectional area measurement have been described in detail elsewhere (VanBavel, Mooij, Giezeman & Spaan, 1990). In short, isolated distal segments of the arterial arcade and first-order branches were cannulated at both ends using micro-cannulae consisting of two concentric pipettes, which are glued together. The end of a vessel was sucked into the inner pipette, and subsequently secured by applying sub-atmospheric pressure to the outer pipette. Cannulated segments were superfused with BR and perfused with BR containing 40 mg 1-l
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FITC-dextran (mnolecular weight 150000). Both perfusate and superfusate were equilibrated with air containing 5 % CO2. The luminal cross-sectional area (CSA) of the cannulated vessel was continuously measured using a fluorescence technique. The cannulated vessel was placed on an inverted microscope (Olympus IA12), which was equipped with a fluorescence attachment. The FITC-dextran present in the lumen of the vessel was excited using a weak light source (wavelength, A = 400-480 nm). The total amount of fluorescence light (A > 515 nm) coming from the lumen was measured with a photomultiplier tube. This amount has been shown to be linearly related to the mean CSA (VanBavel et al. 1990). After cannulation. the temperature was raised to 37 °C and a two-point calibration was performed by comparing fluorescence light with a measurement of the inside contour of the vessel at two widely different vessel sizes, namely at 20 and at 80 mmHg perfusion pressure (maximal dilatation). The CSA measuring technique needed continuous replacement of the luminal solution. Therefore, a small pressure gradient (usually 1 mmHg) was applied between both ends in order to maintain a fluid velocity in the order of 10-100 ,um s-'. This velocity was estimated from the movement of a few red blood cells left in the solution and was much smaller than would be expected under in vivo conditions (Smiesko, Lang & Johnson, 1989).
Drugs FITC-dextran, noradrenaline hydrochloride and acetylcholine were obtained from Sigma. Phentolamine was obtained from Ciba-Geigy. Drugs were dissolved in BR. Stock solutions of drugs were freshly prepared for each experiment and kept on ice. All vasoactive drugs were applied extraluminally and concentrations refer to final bath concentrations.
Protocol After cannulation, the preparation was heated to 37 °C and the CSA calibration performed. The perfusion pressure was subsequently maintained at 80 mmHg for 30-60 min in order to test whether the vessel segment developed basal tone. In cases where basal tone did not develop, noradrenaline was applied extraluminally in a concentration sufficient to evoke a 25-75 % CSA reduction. Functional integrity of the endothelium was checked routinely by applying 025-5 ,umacetylcholine, which is an endothelial-mediated vasodilator of mesenteric arteries (Furchgott, Carvalho, Khan & Matsunaga, 1987). Perfusion pressure was changed step-wise (pressure step completed in about 2 s) from 80 mmHg to either 20, 40, 60, 100 or 120 mmHg, kept at that level until CSA was constant for at least 1 min, and brought back to 80 mmHg. The sequence of pressure steps was randomized. Pressure was changed simultaneously at both cannulated ends, in order to keep flow approximately constant. Each pressure step was performed once or twice for each of eighteen vessels tested. In nine cases the sequence of pressure steps was also performed at maximal dilatation.
Definitions and data analysis The definitions of terms related to myogenic mechanisms are in close accordance with the proposals in the discussion of Johansson (1989). Thus, tone in this paper refers to a sustained CSA reduction due to activation of the contractile apparatus in the smooth muscle cells. Basal tone is tone in the absence of any activation imposed by the investigator. A myogenic response is an alteration of tone imposed by a change in transmural pressure. Vasomotion refers to phasic variations of CSA due to oscillations in contractile activity, and in this paper can be either spontaneous or drug induced. The CSA and left and right cannula pressure signals were filtered at 2 Hz and sampled at 5 Hz. Steady-state CSA was determined by calculating the time average over at least 30 s. Peak values of CSA which occurred just after a change in perfusion pressure were estimated from the mean value over 1-2 s. In cases where vasomotion developed, steady-state means were taken over a number of complete cycles and peak levels as the time average over one cycle. For each pressure step (either from 80 mmHg to a test pressure or the reverse) peak and steady-state CSA values were normalized by dividing them by the steady-state CSA at 80 mmHg. Speed of myogenic responses was quantified as t50 values. The definition of t50 is illustrated in Fig. 1. t50 is the half-time of the total CSA change from the peak value just after a pressure step to the steady-state value. Vasomotion is described by peak-to-peak amplitude and frequency.
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Statistical significance of results was tested using unpaired two-sided t tests, unless indicated otherwise. Results are considered significant at the 5 % level. RESULTS
Passive CSA as a function of perfusion pressure was determined for nine vessels. For vessels which developed basal tone this was done either in the first few minutes
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TimeFig. 1. Schematic representation of the procedure used to calculate t50 values of myogenic responses. t5 was defined as the time elapsed before half of the total response (x in this figure) had occurred.
after heating the preparation to 37 °C (during which period the vessel was still dilated), or after maximal dilatation using an appropriate concentration. of acetylcholine (typically 1 ,UM). As shown in Fig. 2, compliance (the slope of the pressure-CSA relation) decreased strongly with increasing pressure. Eighteen arteries (internal diameter range: 145-365 ,tm at 80 mmHg and maximal dilatation, mean 293 /tm) were dissected, cannulated and pressurized to 80 mmHg. Nine of these vessels developed basal tone, resulting in a mean CSA decrease of 452 + 51 % (mean +s.E.M.). Basal tone at 80 mmHg was relatively constant for several hours of experimentation. In order to test whether endogenous release of noradrenaline plays a role in the development of basal tone, we tried to reverse the tone by adding 50 /SM-phentolamine. This had no effect on the level of tone of the two vessels tested. The nine vessels which did not develop basal tone in the stabilizing period were preconstricted with noradrenaline (0-4-1-2 /SM). This resulted in a mean CSA decrease equal to 59-9 + 71 % of the passive CSA. Constriction was readily and completely reversed after a-blockade (0'6 ,cM-phentolamine, n = 2). In vessels with either basal or induced tone, step-wise changes of distending
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pressure induced passive, elastic effects, followed by myogenic responses. These myogenic responses resulted in CSA alterations which were maintained as long as the pressure level was maintained. Figure 3 illustrates this for a vessel with basal tone. This vessel lost almost all of its tone after reducing perfusion pressure to 20 mmllg. This can be concluded from the peak CSA value after restoration of the pressure to 80 mmHg. This peak value is about equal to the CSA at maximal vasodilatation induced by acetylcholine at that same pressure. Moreover, steady-state CSAs at 20 mmHg with and without acetylcholine are almost the same as well.
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Figure 4A and B respectively shows the compiled results of vessels with basal and with induced tone. In this figure CSA* denotes the actual CSA, normalized by dividing it by the steady-state CSA at 80 mmHg. As clarified by the inset of Fig. 4A, the triangles refer to peak levels, while squares indicate steady-state levels. Filled 1.6
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Pressure (mmHg) 4. Mean area (CSA*) as a function of perfusion pressure. normalized cross-sectional Fig. A, mean results from nine vessels with basal tone, showing a statistically significant decrease of CSA* between 60 and 100 mmHg. The inset shows a schematic drawing of the normalized CSA and pressure as a function of time. B, mean results from nine vessels in which tone was induced by noradrenaline. *, CSA* calculated from the ratio of steadystate CSA at test pressure and CSA at 80 mmHg prior to changing pressure. [, CSA* calculated from the ratio of steady-state CSA at test pressure and CSA after restoration of perfusion pressure to 80 mmHg. A and A, peak CSA* immediately after changing perfusion pressure to a test pressure and 80 mmHg respectively, due to passive distension or collapse of the vessel. Error bars are S.E.M. (only one side shown).
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symbols indicate pressure steps from 80 mmHg to any test pressure, while open symbols denote the back-going pressure steps. The slight difference in steady-state levels during on-going and back-going steps is due to the separate normalizations. In Fig. 4A a drop in pressure from 80 to 60 mmHg induces a passive collapse (a), followed by a myogenic dilatation (b). Stepping back to 80 mmHg results in a passive
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distension (c), followed by a myogenic constriction (d). The closed triangles form a pressureXCSA* relation at constant smooth muscle cell activity, while the squares indicate the steady-state relation after myogenic alteration of tone. The difference between these two relations indicates the influence of pressure on both basal (Fig. 4A) and induced (Fig. 4B) tone. The slope of the steady-state relation is significantly negative between 60 and 100 mmHg in vessels with basal tone, while it is essentially flat between 40 and 120 mmHg in vessels with induced tone. The length of time needed for 50% of the myogenic response to appear (t50) varied considerably and ranged between 6 s and 5 min (mean: 66 s) in the group of vessels with basal tone, and 11 s to 2 min (mean: 47 s) in the vessels with induced tone. In neither group of vessels was the speed of the myogenic reactions dependent on pressure. Five of the nine vessels which developed basal tone, and eight of the nine vessels with noradrenaline-induced tone, exhibited vasomotion at 80 mmHg. Frequency of spontaneous vasomotion ranged from 4 to 31 cycles min-1 (mean over five vessels: 14 cycles min'l). Amplitude was 1-10% (mean over five vessels: 5%) of the passive CSA. Variations in these parameters during one single experiment were large. Typically two- to threefold changes were found. Frequency of noradrenaline-
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induced vasomotion ranged from 9 to 25 cycles min-' (mean over eight vessels: 21 cycles min-'). Amplitude ranged from 3 to 17 % (mean over eight vessels: 7-5 %). Thus, noradrenaline-induced vasomotion was somewhat faster and with larger amplitude than spontaneous vasomotion. Variation of induced vasomotion during 1.0 c
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Fig. 6. Development of vasomotion as a function of perfusion pressure. The ordinate shows the fraction of observations in which vasomotion which had developed at 80 mmHg was maintained after switching to another pressure. Filled bars, spontaneously active vessels. Hatched bars, noradrenaline-induced vasomotion.
an experiment was small: usually frequency varied by no more than 10% during subsequent periods of perfusion at 80 mmHg. Amplitude varied by a factor of
approximately 1P5. Figure 5 shows examples of vessels with spontaneous (A) and induced (B) vasomotion, subjected to changes in perfusion pressure. As can be seen, both types of vasomotion disappeared at low pressures. When pressure was increased, amplitude of vasomotion decreased and frequency increased. The vasomotion patterns are superposed on myogenic responses. Figure 6 demonstrates the existence of a threshold pressure for both types of vasomotion. As is clear, no vasomotion occurred at 20 mmHg. All vessels exhibiting vasomotion at 80 mmHg did so at 100 mmHg. At 120 mmHg vasomotion appeared to stop in some vessels, although possibly vasomotion may have been overlooked due to the small amplitude at this pressure. In Fig. 7 average amplitude and frequency of induced vasomotion are plotted as functions of pressure. This figure shows that the amplitude of vasomotion decreases strongly when pressure is raised. The vasomotion frequency increases slightly upon raising pressure. The amount of data on spontaneous vasomotion is too small to make a quantification and comparison with induced vasomotion feasible. However, the effect of pressure on vasomotion, when it occurred, was consistent with that observed in vessels with noradrenaline-induced vasomotion: frequency increased, and amplitude decreased with pressure.
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Fig. 7. Effects of pressure on noradrenaline-induced vasomotion. A shows amplitude of vasomotion, normalized to its value at 80 mmHg. B shows frequency of vasomotion, again normalized to the values found at 80 mmHg. Error bars indicate s.E.M. Asterisks indicate values significantly different from the values at 80 mmHg (t test, P < 0-05).
DISCUSSION
Preparation The current experiments show myogenic responses in isolated small arteries. These responses can be large enough to cause an actual constriction with increasing pressure, and thus may possibly be involved in the autoregulation of flow. Furthermore, myogenic alterations of both basal and drug-induced tone were found. The vessels in this study were distal parts of the arterial arcade and first-order branches. Pressure distal to the mesenteric arteries, at the entrance of the intestine,
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is about 50 % of aortic pressure (see Lundgren, 1984). Occlusion of an arcading vessel leads to a significant drop of flow velocity in distally located non-arcading branches, despite the availability of alternative pathways (Smiesko, Lang & Johnson, 1989). These findings suggest that the vessels we studied are resistance sized. Tone in such arteries may be dependent on local oxygen tension (Jackson & Duling, 1983), and the oxygen tension of 150 mmHg may seem unphysiologically high. However, Lang & Johnson (1988) could not demonstrate effects of oxygen tension changes between 4 and 134 mmHg on in situ cat mesenteric arterioles. Furthermore, these authors report oxygen tension in the abdominal cavity to be 67 mmHg, showing that hypoxia is not a normal condition for these vessels. Only 50 % of the vessels we tested developed basal tone at 80 mmHg. Furthermore the time course of myogenic responses, as well as the spontaneous vasomotion parameters, varied considerably. One can only speculate about the cause of this heterogeneity: acetylcholine caused dilatation, indicating the functioning of the endothelium (Furchgott et al. 1987). However, this does not exclude the possibility that partial damage was present in some vessels. Alternatively, the amount of lengthening of the vessels might be important (Cox, 1983). We found the in situ length of mesenteric vessels hard to estimate, if at all constant. After cannulation, all vessels were lengthened until they were straight at 80 mmHg perfusion pressure. Despite this, some heterogeneity might have been present in the ratio of in vitro and in vivo length of the segments. Few studies are available on myogenic responses in cannulated resistance vessel segments. Osol & Halpern (1985) found myogenic responses in cannulated cerebral blood vessels (about 200,um) with basal tone. Responses in vessels from normotensive rats were somewhat larger than the initial passive distension, while in vessels from spontaneous hypertensive rats the responses just compensated these initial effects. Kuo et al. (1988) observed overcompensating responses in cannulated epicardial arterioles, while the steady-state pressure-diameter relation of endocardial vessels was flat. Jackson and Duling found overcompensating responses of hamster cheek pouch arterioles (80,um) with basal tone, but not with potassium- or phenylephrineinduced tone. These latter findings closely resemble the current results. Vasomotion is commonly observed in vivo in arterioles (Meyer, Lindbom & Intaglietta, 1987; Hundley, Renaldo, Levasseur & Kontos. 1988; Oude Vrielink, Slaaf, Tangelder, Weijmer-VanVelzen & Reneman, 1990). No earlier reports have been made of the in vitro spontaneous vasomotion in the mesenteric vessels studied by us. Mulvany, Nilsson & Flatman (1982) describe noradrenaline-induced oscillations in tension and membrane potential of isometrically mounted mesenteric vessels, which resemble the induced vasomotion patterns we found. In vitro spontaneous vasomotion has been observed in cerebral arteries (inside diameter about 200 ,tm) of spontaneously hypertensive rats (Osol & Halpern, 1988). The frequency and amplitude of vasomotion in that study respectively increased and decreased with increasing pressure, as was the case in both groups in our study. The same relation between pressure and vasomotion parameters has also been observed in in vivo studies of the mesenteric microcirculation (Burrows & Johnson, 1981).
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Mechanismn of myogenic vasoconstriction The stimulus for myogenic responses may be either related to stretch of the vessel wall or tension in the wall (Johansson & Mellander, 1975; Johansson, 1989). However, in our experiments the myogenic responses in the vessels with basal tone 1.0
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Fig. 8. Active wall tension as a function of total tension, calculated for vessels with basal (A) and noradrenaline-induced (0) tone. Both axes have been normalized to the tension at 80 mmHg and maximal dilatation. The dashed line represents the identity relation.
cannot be induced solely by stretch of the vessel wall, since in this case the response would eliminate its cause and steady constrictions upon elevating pressure could not have been observed. Alternatively, total circumferential wall tension (approximately being equal to the product of pressure and inner radius) can remain elevated upon a pressure increase, despite a constriction of the vessel which overcompensates the initial distension. Johnson (1980, 1986) points out that in this case wall tension becomes the controlled variable. Total tension in our data can be calculated from the pressure-CSA* relation (*) in Fig. 4. The part of this tension carried by the passive elements follows from the pressure-CSA* relation at maximal dilatation (Fig. 2), and the remaining part is active tension. Figure 8 plots active tension as a function of total tension for both groups of vessels. According to the above hypothesis, this plot represents the stimulus-response relationship for myogenic responses. As can be seen, the relationships for both groups of vessels differ in the level of tone, as evidenced by the shift between the curves, but hardly at all in the myogenic responsiveness, indicated by the slope of the curves. Yet the last difference is of importance: at points where the slope of the curve exceeds one, an increase in total tension due to pressure elevation leads to a more than proportional increment in active tension, and thus to a decrement in passive tension and therefore in radius. Such a range was found in the vessels with basal tone, but not with noradrenalineinduced tone. The cellular mechanism by which wall tension could be sensed remains to be established. Possibly activation leads to a heterogenous load on the smooth muscle
382 E. VANBAVEL AND OTHERS 382 cell membrane. Evidence for this comes from studies of isolated smooth muscle cells (VanDijk & Laird, 1984), the membrane of which shows evaginations at activation, but not at relaxation. Stretch-dependent calcium channels (Laher & Bevan, 1989) in these parts of the membrane could act as tension sensors. Financial support from The Netherlands Heart Foundation is gratefully acknowledged. authors would like to thank Rob Tigchelaar for the construction of the micro-cannulae, Professor M. I. M. Noble for previewing the manuscript.
The and
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Journal of Physiology (1991), 436, pp. 371-383 With 8 figures Printed in Great Britain
INFLUENCE OF PRESSURE ALTERATIONS ON TONE AND VASOMOTION OF ISOLATED MESENTERIC SMALL ARTERIES OF THE RAT
BY ED VANBAVEL, MAURICE J. M. M. GIEZEMAN, TRUDI MOOIJ AND JOS A. E. SPAAN From the Cardiovascular Research Institute Amsterdam, Department of Medical Physics, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (Received 22 February 1990) SUMMARY
1. Myogenic responses may account for control of organ blood flow. The study of these responses without interference from the organ requires an isolation technique for vessels which contribute significantly to flow resistance. This study reports on experiments on isolated small mesenteric arteries. 2. Distal rat mesenteric arcade arteries and first-order branches (diameter range 145-365 ,um, mean 293 ,um) were manually dissected and cannulated using a doublebarrelled micro-cannula. Luminal cross-sectional area of these vessels was continuously monitored by means of a fluorescence technique. 3. Nine out of eighteen vessels developed basal tone at 80 mmHg distending pressure, resulting in a 45-2 + 5-1 % (mean + S.E.M) decrease of cross-sectional area. Tone was induced in the other vessels by 0-3-1 tim-noradrenaline, resulting in a 59-5 + 741 % decrease in cross-sectional area. 4. In vessels with either spontaneous or induced tone, stepwise changes of pressure resulted in passive effects, followed by myogenic responses. 5. Steady-state pressure-cross-sectional area relations of vessels with basal tone showed a significant negative slope (-0-5% mmHg-'), while pressure-crosssectional area relations of vessels with induced tone were essentially flat between 40 and 120 mmHg. 6. Five vessels with basal tone and eight vessels with induced tone developed vasomotion at 80 mmHg. Frequencies of spontaneous and induced vasomotion were 14 (range 4-31) and 21 (9-25) cycles min-' respectively. Amplitudes were 5 (1-10) and 8 (3-17)% of the passive cross-sectional area. In both groups, frequency was positively, and amplitude negatively correlated with pressure. 7. These data show that myogenic responses are induced by wall stress, rather than by distension of the vascular wall. Basal tone is not a prerequisite for the appearance of myogenic responses.
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E. VANBA VEL AND OTHERS INTRODUCTION
It has been known since the beginning of this century (Bayliss, 1902) that blood vessels react to a raised perfusion pressure by increasing smooth muscle cell tone. This myogenic response may play a role in the autoregulation of flow (Johnson, 1980, 1986) and capillary pressure (Mellander, Oberg & Odelram, 1964; Mellander, Maspers, Bjornberg & Andersson, 1987). Much evidence in favour of a role in autoregulation comes from whole-organ studies. Thus, increasing local pressure by raising venous pressure or transmural pressure induces resistance increase and constriction of observed arterioles (see Johnson 1986, 1989). However, these myogenic responses lead to secondary alterations of local pressures. This stresses the need to actually measure these pressures in the study of myogenic responses, as was done by Davis & Sikes (1989) and Griinde (1989). Further complications in the quantification of myogenic responses in whole organs are flow dependent and metabolic effects, which may become involved when flow is changing due to myogenic constriction. A careful analysis of data is needed to estimate the impact of these effects (Grainde, 1989). Alternatively, one can study myogenic responses in isolated, cannulated vessels. Due to the assumed involvement of myogenic responses in flow control, it would be most relevant to perform these studies on resistance-sized arterial vessels. Up to now, quantification of myogenic responses in isolated resistance vessels has been limited to the brain (Osol & Halpern, 1985), heart (Kuo, Davis & Chilian, 1988) and hamster cheek pouch (Jackson & Duling, 1989). The purpose of this paper is to quantify the myogenic responses in isolated, cannulated mesenteric arteries which are resistance sized, or near to this size. In particular we aimed to establish the relation between basal tone and myogenic responses, and to test whether myogenic responses in the mesenteric vessels are large enough to cause an actual constriction, rather than partial compensation of passive effects, when pressure is raised. Furthermore, the influence of pressure on the vasomotion which was observed in these vessels is documented. METHODS
Isolation procedure Male Wistar rats (350-450 g) were anaesthetized by intraperitoneal injection of pentobarbitone (0-06 mg (g body weight)-'). The abdomen was opened and part of the intestinal tract was removed and placed in cold bicarbonate-buffered Ringer solution (BR, composition in mM: NaCl, 115; KCl, 4-7; KH204, 1-2; MgSO4, 1-2; NaHCO3, 24; CaCl2, 1-25; glucose, 10; solution equilibrated with 95% air, 5% C02; pH adjusted to 7 35). Part of the arterial mesenteric arcade and some first-order branches were cleared from surrounding tissue. This part of the mesenteric arterial tree was removed and placed in the cannulation chamber. Cannulation technique and cross-sectional area measurement Both the cannulation technique and the cross-sectional area measurement have been described in detail elsewhere (VanBavel, Mooij, Giezeman & Spaan, 1990). In short, isolated distal segments of the arterial arcade and first-order branches were cannulated at both ends using micro-cannulae consisting of two concentric pipettes, which are glued together. The end of a vessel was sucked into the inner pipette, and subsequently secured by applying sub-atmospheric pressure to the outer pipette. Cannulated segments were superfused with BR and perfused with BR containing 40 mg 1-l
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FITC-dextran (mnolecular weight 150000). Both perfusate and superfusate were equilibrated with air containing 5 % CO2. The luminal cross-sectional area (CSA) of the cannulated vessel was continuously measured using a fluorescence technique. The cannulated vessel was placed on an inverted microscope (Olympus IA12), which was equipped with a fluorescence attachment. The FITC-dextran present in the lumen of the vessel was excited using a weak light source (wavelength, A = 400-480 nm). The total amount of fluorescence light (A > 515 nm) coming from the lumen was measured with a photomultiplier tube. This amount has been shown to be linearly related to the mean CSA (VanBavel et al. 1990). After cannulation. the temperature was raised to 37 °C and a two-point calibration was performed by comparing fluorescence light with a measurement of the inside contour of the vessel at two widely different vessel sizes, namely at 20 and at 80 mmHg perfusion pressure (maximal dilatation). The CSA measuring technique needed continuous replacement of the luminal solution. Therefore, a small pressure gradient (usually 1 mmHg) was applied between both ends in order to maintain a fluid velocity in the order of 10-100 ,um s-'. This velocity was estimated from the movement of a few red blood cells left in the solution and was much smaller than would be expected under in vivo conditions (Smiesko, Lang & Johnson, 1989).
Drugs FITC-dextran, noradrenaline hydrochloride and acetylcholine were obtained from Sigma. Phentolamine was obtained from Ciba-Geigy. Drugs were dissolved in BR. Stock solutions of drugs were freshly prepared for each experiment and kept on ice. All vasoactive drugs were applied extraluminally and concentrations refer to final bath concentrations.
Protocol After cannulation, the preparation was heated to 37 °C and the CSA calibration performed. The perfusion pressure was subsequently maintained at 80 mmHg for 30-60 min in order to test whether the vessel segment developed basal tone. In cases where basal tone did not develop, noradrenaline was applied extraluminally in a concentration sufficient to evoke a 25-75 % CSA reduction. Functional integrity of the endothelium was checked routinely by applying 025-5 ,umacetylcholine, which is an endothelial-mediated vasodilator of mesenteric arteries (Furchgott, Carvalho, Khan & Matsunaga, 1987). Perfusion pressure was changed step-wise (pressure step completed in about 2 s) from 80 mmHg to either 20, 40, 60, 100 or 120 mmHg, kept at that level until CSA was constant for at least 1 min, and brought back to 80 mmHg. The sequence of pressure steps was randomized. Pressure was changed simultaneously at both cannulated ends, in order to keep flow approximately constant. Each pressure step was performed once or twice for each of eighteen vessels tested. In nine cases the sequence of pressure steps was also performed at maximal dilatation.
Definitions and data analysis The definitions of terms related to myogenic mechanisms are in close accordance with the proposals in the discussion of Johansson (1989). Thus, tone in this paper refers to a sustained CSA reduction due to activation of the contractile apparatus in the smooth muscle cells. Basal tone is tone in the absence of any activation imposed by the investigator. A myogenic response is an alteration of tone imposed by a change in transmural pressure. Vasomotion refers to phasic variations of CSA due to oscillations in contractile activity, and in this paper can be either spontaneous or drug induced. The CSA and left and right cannula pressure signals were filtered at 2 Hz and sampled at 5 Hz. Steady-state CSA was determined by calculating the time average over at least 30 s. Peak values of CSA which occurred just after a change in perfusion pressure were estimated from the mean value over 1-2 s. In cases where vasomotion developed, steady-state means were taken over a number of complete cycles and peak levels as the time average over one cycle. For each pressure step (either from 80 mmHg to a test pressure or the reverse) peak and steady-state CSA values were normalized by dividing them by the steady-state CSA at 80 mmHg. Speed of myogenic responses was quantified as t50 values. The definition of t50 is illustrated in Fig. 1. t50 is the half-time of the total CSA change from the peak value just after a pressure step to the steady-state value. Vasomotion is described by peak-to-peak amplitude and frequency.
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Statistical significance of results was tested using unpaired two-sided t tests, unless indicated otherwise. Results are considered significant at the 5 % level. RESULTS
Passive CSA as a function of perfusion pressure was determined for nine vessels. For vessels which developed basal tone this was done either in the first few minutes
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after heating the preparation to 37 °C (during which period the vessel was still dilated), or after maximal dilatation using an appropriate concentration. of acetylcholine (typically 1 ,UM). As shown in Fig. 2, compliance (the slope of the pressure-CSA relation) decreased strongly with increasing pressure. Eighteen arteries (internal diameter range: 145-365 ,tm at 80 mmHg and maximal dilatation, mean 293 /tm) were dissected, cannulated and pressurized to 80 mmHg. Nine of these vessels developed basal tone, resulting in a mean CSA decrease of 452 + 51 % (mean +s.E.M.). Basal tone at 80 mmHg was relatively constant for several hours of experimentation. In order to test whether endogenous release of noradrenaline plays a role in the development of basal tone, we tried to reverse the tone by adding 50 /SM-phentolamine. This had no effect on the level of tone of the two vessels tested. The nine vessels which did not develop basal tone in the stabilizing period were preconstricted with noradrenaline (0-4-1-2 /SM). This resulted in a mean CSA decrease equal to 59-9 + 71 % of the passive CSA. Constriction was readily and completely reversed after a-blockade (0'6 ,cM-phentolamine, n = 2). In vessels with either basal or induced tone, step-wise changes of distending
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pressure induced passive, elastic effects, followed by myogenic responses. These myogenic responses resulted in CSA alterations which were maintained as long as the pressure level was maintained. Figure 3 illustrates this for a vessel with basal tone. This vessel lost almost all of its tone after reducing perfusion pressure to 20 mmllg. This can be concluded from the peak CSA value after restoration of the pressure to 80 mmHg. This peak value is about equal to the CSA at maximal vasodilatation induced by acetylcholine at that same pressure. Moreover, steady-state CSAs at 20 mmHg with and without acetylcholine are almost the same as well.
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Figure 4A and B respectively shows the compiled results of vessels with basal and with induced tone. In this figure CSA* denotes the actual CSA, normalized by dividing it by the steady-state CSA at 80 mmHg. As clarified by the inset of Fig. 4A, the triangles refer to peak levels, while squares indicate steady-state levels. Filled 1.6
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Pressure (mmHg) 4. Mean area (CSA*) as a function of perfusion pressure. normalized cross-sectional Fig. A, mean results from nine vessels with basal tone, showing a statistically significant decrease of CSA* between 60 and 100 mmHg. The inset shows a schematic drawing of the normalized CSA and pressure as a function of time. B, mean results from nine vessels in which tone was induced by noradrenaline. *, CSA* calculated from the ratio of steadystate CSA at test pressure and CSA at 80 mmHg prior to changing pressure. [, CSA* calculated from the ratio of steady-state CSA at test pressure and CSA after restoration of perfusion pressure to 80 mmHg. A and A, peak CSA* immediately after changing perfusion pressure to a test pressure and 80 mmHg respectively, due to passive distension or collapse of the vessel. Error bars are S.E.M. (only one side shown).
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symbols indicate pressure steps from 80 mmHg to any test pressure, while open symbols denote the back-going pressure steps. The slight difference in steady-state levels during on-going and back-going steps is due to the separate normalizations. In Fig. 4A a drop in pressure from 80 to 60 mmHg induces a passive collapse (a), followed by a myogenic dilatation (b). Stepping back to 80 mmHg results in a passive
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distension (c), followed by a myogenic constriction (d). The closed triangles form a pressureXCSA* relation at constant smooth muscle cell activity, while the squares indicate the steady-state relation after myogenic alteration of tone. The difference between these two relations indicates the influence of pressure on both basal (Fig. 4A) and induced (Fig. 4B) tone. The slope of the steady-state relation is significantly negative between 60 and 100 mmHg in vessels with basal tone, while it is essentially flat between 40 and 120 mmHg in vessels with induced tone. The length of time needed for 50% of the myogenic response to appear (t50) varied considerably and ranged between 6 s and 5 min (mean: 66 s) in the group of vessels with basal tone, and 11 s to 2 min (mean: 47 s) in the vessels with induced tone. In neither group of vessels was the speed of the myogenic reactions dependent on pressure. Five of the nine vessels which developed basal tone, and eight of the nine vessels with noradrenaline-induced tone, exhibited vasomotion at 80 mmHg. Frequency of spontaneous vasomotion ranged from 4 to 31 cycles min-1 (mean over five vessels: 14 cycles min'l). Amplitude was 1-10% (mean over five vessels: 5%) of the passive CSA. Variations in these parameters during one single experiment were large. Typically two- to threefold changes were found. Frequency of noradrenaline-
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induced vasomotion ranged from 9 to 25 cycles min-' (mean over eight vessels: 21 cycles min-'). Amplitude ranged from 3 to 17 % (mean over eight vessels: 7-5 %). Thus, noradrenaline-induced vasomotion was somewhat faster and with larger amplitude than spontaneous vasomotion. Variation of induced vasomotion during 1.0 c
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Fig. 6. Development of vasomotion as a function of perfusion pressure. The ordinate shows the fraction of observations in which vasomotion which had developed at 80 mmHg was maintained after switching to another pressure. Filled bars, spontaneously active vessels. Hatched bars, noradrenaline-induced vasomotion.
an experiment was small: usually frequency varied by no more than 10% during subsequent periods of perfusion at 80 mmHg. Amplitude varied by a factor of
approximately 1P5. Figure 5 shows examples of vessels with spontaneous (A) and induced (B) vasomotion, subjected to changes in perfusion pressure. As can be seen, both types of vasomotion disappeared at low pressures. When pressure was increased, amplitude of vasomotion decreased and frequency increased. The vasomotion patterns are superposed on myogenic responses. Figure 6 demonstrates the existence of a threshold pressure for both types of vasomotion. As is clear, no vasomotion occurred at 20 mmHg. All vessels exhibiting vasomotion at 80 mmHg did so at 100 mmHg. At 120 mmHg vasomotion appeared to stop in some vessels, although possibly vasomotion may have been overlooked due to the small amplitude at this pressure. In Fig. 7 average amplitude and frequency of induced vasomotion are plotted as functions of pressure. This figure shows that the amplitude of vasomotion decreases strongly when pressure is raised. The vasomotion frequency increases slightly upon raising pressure. The amount of data on spontaneous vasomotion is too small to make a quantification and comparison with induced vasomotion feasible. However, the effect of pressure on vasomotion, when it occurred, was consistent with that observed in vessels with noradrenaline-induced vasomotion: frequency increased, and amplitude decreased with pressure.
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Fig. 7. Effects of pressure on noradrenaline-induced vasomotion. A shows amplitude of vasomotion, normalized to its value at 80 mmHg. B shows frequency of vasomotion, again normalized to the values found at 80 mmHg. Error bars indicate s.E.M. Asterisks indicate values significantly different from the values at 80 mmHg (t test, P < 0-05).
DISCUSSION
Preparation The current experiments show myogenic responses in isolated small arteries. These responses can be large enough to cause an actual constriction with increasing pressure, and thus may possibly be involved in the autoregulation of flow. Furthermore, myogenic alterations of both basal and drug-induced tone were found. The vessels in this study were distal parts of the arterial arcade and first-order branches. Pressure distal to the mesenteric arteries, at the entrance of the intestine,
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is about 50 % of aortic pressure (see Lundgren, 1984). Occlusion of an arcading vessel leads to a significant drop of flow velocity in distally located non-arcading branches, despite the availability of alternative pathways (Smiesko, Lang & Johnson, 1989). These findings suggest that the vessels we studied are resistance sized. Tone in such arteries may be dependent on local oxygen tension (Jackson & Duling, 1983), and the oxygen tension of 150 mmHg may seem unphysiologically high. However, Lang & Johnson (1988) could not demonstrate effects of oxygen tension changes between 4 and 134 mmHg on in situ cat mesenteric arterioles. Furthermore, these authors report oxygen tension in the abdominal cavity to be 67 mmHg, showing that hypoxia is not a normal condition for these vessels. Only 50 % of the vessels we tested developed basal tone at 80 mmHg. Furthermore the time course of myogenic responses, as well as the spontaneous vasomotion parameters, varied considerably. One can only speculate about the cause of this heterogeneity: acetylcholine caused dilatation, indicating the functioning of the endothelium (Furchgott et al. 1987). However, this does not exclude the possibility that partial damage was present in some vessels. Alternatively, the amount of lengthening of the vessels might be important (Cox, 1983). We found the in situ length of mesenteric vessels hard to estimate, if at all constant. After cannulation, all vessels were lengthened until they were straight at 80 mmHg perfusion pressure. Despite this, some heterogeneity might have been present in the ratio of in vitro and in vivo length of the segments. Few studies are available on myogenic responses in cannulated resistance vessel segments. Osol & Halpern (1985) found myogenic responses in cannulated cerebral blood vessels (about 200,um) with basal tone. Responses in vessels from normotensive rats were somewhat larger than the initial passive distension, while in vessels from spontaneous hypertensive rats the responses just compensated these initial effects. Kuo et al. (1988) observed overcompensating responses in cannulated epicardial arterioles, while the steady-state pressure-diameter relation of endocardial vessels was flat. Jackson and Duling found overcompensating responses of hamster cheek pouch arterioles (80,um) with basal tone, but not with potassium- or phenylephrineinduced tone. These latter findings closely resemble the current results. Vasomotion is commonly observed in vivo in arterioles (Meyer, Lindbom & Intaglietta, 1987; Hundley, Renaldo, Levasseur & Kontos. 1988; Oude Vrielink, Slaaf, Tangelder, Weijmer-VanVelzen & Reneman, 1990). No earlier reports have been made of the in vitro spontaneous vasomotion in the mesenteric vessels studied by us. Mulvany, Nilsson & Flatman (1982) describe noradrenaline-induced oscillations in tension and membrane potential of isometrically mounted mesenteric vessels, which resemble the induced vasomotion patterns we found. In vitro spontaneous vasomotion has been observed in cerebral arteries (inside diameter about 200 ,tm) of spontaneously hypertensive rats (Osol & Halpern, 1988). The frequency and amplitude of vasomotion in that study respectively increased and decreased with increasing pressure, as was the case in both groups in our study. The same relation between pressure and vasomotion parameters has also been observed in in vivo studies of the mesenteric microcirculation (Burrows & Johnson, 1981).
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Mechanismn of myogenic vasoconstriction The stimulus for myogenic responses may be either related to stretch of the vessel wall or tension in the wall (Johansson & Mellander, 1975; Johansson, 1989). However, in our experiments the myogenic responses in the vessels with basal tone 1.0
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Fig. 8. Active wall tension as a function of total tension, calculated for vessels with basal (A) and noradrenaline-induced (0) tone. Both axes have been normalized to the tension at 80 mmHg and maximal dilatation. The dashed line represents the identity relation.
cannot be induced solely by stretch of the vessel wall, since in this case the response would eliminate its cause and steady constrictions upon elevating pressure could not have been observed. Alternatively, total circumferential wall tension (approximately being equal to the product of pressure and inner radius) can remain elevated upon a pressure increase, despite a constriction of the vessel which overcompensates the initial distension. Johnson (1980, 1986) points out that in this case wall tension becomes the controlled variable. Total tension in our data can be calculated from the pressure-CSA* relation (*) in Fig. 4. The part of this tension carried by the passive elements follows from the pressure-CSA* relation at maximal dilatation (Fig. 2), and the remaining part is active tension. Figure 8 plots active tension as a function of total tension for both groups of vessels. According to the above hypothesis, this plot represents the stimulus-response relationship for myogenic responses. As can be seen, the relationships for both groups of vessels differ in the level of tone, as evidenced by the shift between the curves, but hardly at all in the myogenic responsiveness, indicated by the slope of the curves. Yet the last difference is of importance: at points where the slope of the curve exceeds one, an increase in total tension due to pressure elevation leads to a more than proportional increment in active tension, and thus to a decrement in passive tension and therefore in radius. Such a range was found in the vessels with basal tone, but not with noradrenalineinduced tone. The cellular mechanism by which wall tension could be sensed remains to be established. Possibly activation leads to a heterogenous load on the smooth muscle
382 E. VANBAVEL AND OTHERS 382 cell membrane. Evidence for this comes from studies of isolated smooth muscle cells (VanDijk & Laird, 1984), the membrane of which shows evaginations at activation, but not at relaxation. Stretch-dependent calcium channels (Laher & Bevan, 1989) in these parts of the membrane could act as tension sensors. Financial support from The Netherlands Heart Foundation is gratefully acknowledged. authors would like to thank Rob Tigchelaar for the construction of the micro-cannulae, Professor M. I. M. Noble for previewing the manuscript.
The and
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