AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H118-H125, 2002. First published March 21, 2002; doi:10.1152/ajpheart.00690.2001
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/1/H118    most recent
00690.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matchkov, V. V.
Right arrow Articles by Nilsson, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matchkov, V. V.
Right arrow Articles by Nilsson, H.
Vol. 283, Issue 1, H118-H125, July 2002

Myogenic response of rat femoral small arteries in relation to wall structure and [Ca2+]i

Vladimir V. Matchkov1, Olga S. Tarasova3, Michael J. Mulvany2, and Holger Nilsson1

1 Department of Physiology and 2 Department of Pharmacology, University of Aarhus, DK-8000 Aarhus C, Denmark; and 3 Department of Human and Animal Physiology, Moscow State University, Moscow, RU-119899, Russia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigated the influence of media thickness on myogenic tone and intracellular calcium concentration ([Ca2+]i) in rat skeletal muscle small arteries. A ligature was loosely tied around one external iliac artery of 5-wk-old spontaneously hypertensive rats. At 18 wk of age, femoral artery blood pressure was 102 ± 11 mmHg (n = 15) on the ligated side and 164 ± 6 mmHg (n = 15) on the contralateral side. Small arteries feeding the gracilis muscle had a reduced media cross-sectional area and a reduced media-to-lumen ratio on the ligated side, where also the range of myogenic constriction was shifted to lower pressures. However, when expressed as a function of wall stress, diameter responses were nearly identical. [Ca2+]i was higher in vessels from the ligated hindlimb at pressures above 10 mmHg, but vasoconstriction was not accompanied by changes in [Ca2+]i. Thus the myogenic constriction here seems due primarily to changes in intracellular calcium sensitivity, which are determined mainly by the force per cross-sectional area of the wall and therefore altered by changes in vascular structure.

intracellular calcium concentration; endothelium; fura 2; Ca-channel antagonists


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL KNOWN that vascular smooth muscle contracts in response to increased pressure and dilates when pressure is reduced. These acute changes in vascular diameter are commonly referred to as the myogenic response (19). On the other hand, long-lasting pressure variations affect not only vascular tone but vascular structure as well. Chronic hypertension is associated with an increased wall thickness or an increased wall-to-lumen ratio (8), and the opposite changes are seen after chronic pressure reduction (2, 14). It is likely that such structural changes will interact with the myogenic mechanisms, because wall stress, thought to be a stimulus for the myogenic response (20), is dependent on vessel radius and thickness.

In hypertension, arteries autoregulate at higher pressures than in the normotensive state (8). In accordance with this, the range over which the myogenic response occurs in cerebral (28) or mesenteric (17) arteries of spontaneously hypertensive rats (SHR) is shifted toward higher pressure levels compared with vessels of normotensive Wistar-Kyoto rats. However, in SHR, these changes may be determined not only by vascular structural adaptation to higher pressure but also by a genetically determined propensity toward increased growth of vascular smooth muscle (35). The primary goal of this study was to assess the true effect of local perfusion pressure on vascular structure and myogenic properties. We have therefore undertaken a study of isolated skeletal muscle arteries from SHR using the method of Bund et al. (2). The arterial pressure in one leg was maintained reduced for 3 mo by ligation of the external iliac artery, and the contralateral artery served as a control. With the use of a pressure myograph, we studied the correlation between structure and myogenic responsiveness in these arteries with different structure but from the same genetic and humoral environment.

The cellular mechanisms of the myogenic response include depolarization and subsequent opening of voltage-operated calcium channels (16, 23, 24, 34), but nonelectromechanical coupling mechanisms also appear to be involved (1, 26). It has been shown that pressure-induced activation enhances the calcium sensitivity of the contractile machinery (33). However, the relative contribution of calcium-dependent and -independent mechanisms underlying the myogenic response in vessels with different structure has not been understood. Thus the second goal of this study was to investigate the calcium/tone relationship in vascular smooth muscle in the myogenic response.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals, surgery, and blood pressure measurements. Fifteen male SHR rats aged 4 wk were obtained from the Møllegaard Breeding Centre, housed two per cage, and maintained on tap water and standard laboratory food ad libitum. The systolic blood pressure was measured for each rat at 5 and 18 wk of age by the tail-cuff method.

Protection of the hindlimb vasculature from rising blood pressure was performed as described by Bund et al. (2). At 5 wk of age, rats were anesthetized (75 mg/kg methohexital, Brietal, Lilly), and the external iliac artery was exposed. A stainless steel wire with a diameter of 0.4 mm was placed alongside the artery and tied firmly to the artery by 4-0 braided silk. After the wire was removed, there remained around the artery a fixed-diameter loop of silk, which at this stage did not compress the artery. One-half of the rats were operated on the left side; the others were operated on the right side.

The effect of the ligation was studied at the age of 18 wk by measurement of femoral mean arterial pressure in both ligated and nonligated hindlimbs. Again, the operation was performed under anesthesia (75 mg/kg methohexital), and the femoral artery was cannulated with polyethylene-10 tubing filled with heparinized (20 IU/ml) physiological solution. The pressures were recorded under continued anesthesia using a pressure transducer (Medex Novatrans 2) and a chart recorder (Thermal Arraycorder, WR-7730-4).

Cannulation and mounting of the vessels. After the femoral artery blood pressure was measured, the anaesthetized rats were killed by a blow to the head. The muscular branch of the femoral artery feeding the gracilis muscles in each hind limb was dissected out. Therefore, an experiment in each rat utilized one artery distal to a ligature (from the ligated hindlimb) and one from the control, nonligated hindlimb. The artery was cleaned from extraneous fat and connective tissue. An ~8- to 10-mm segment of the artery was cannulated at either end using glass microcannulas and mounted in a pressure myograph (P100, DMT). All detectable branches were tied off to avoid leakage.

Experiments were performed under no-flow conditions. The myograph was placed on the stage of an inverted microscope (Leica) equipped with a charge-coupled device camera (model XC-75CE). Vessel diameter was measured from video images of the preparation using contrast analysis (VesselView software, DMT).

Measurement of intracellular calcium concentration. The cannulated vessels were loaded with 5 mM fura 2-AM for 2 h at room temperature, followed by a washout period of 45 min, during which the myograph was heated to 37°C. Fura 2-AM dissolved in DMSO was added to the superfusing solution, maintaining the final DMSO concentration below 0.01%. Details of the equipment used (Photon Technology) have been given elsewhere (29). Excitation was achieved using a 75-W xenon light source, vessels were excited alternately at 340 and 380 nm, and emitted light at 515 nm was measured at 10 Hz. All signals were collected and stored digitally using Felix software (version 1.11, Photon Technology).

At the end of each experiment, the minimal and maximal values for the 340-to-380-nm ratio (Rmin and Rmax, respectively) were determined following the procedure described by Jensen et al. (18). Finally, background fluorescence levels after quenching with 20 mM MnCl2 were determined and subtracted from all previously obtained measurements. The intracellular calcium concentration ([Ca2+]i) was calculated from the equation [Ca2+]i = Kd × beta [(R - Rmin)/(Rmax - R)], where beta  is the ratio between maximal and minimal fluorescence at 380 nm and the dissociation constant of fura 2:Ca2+ (Kd) was set to 224 nM (11).

To show that vessel wall movement within the sampling window had no effect on the recorded fura 2 ratio signal, we performed [Ca2+]i measurements in completely relaxed vessels. The vessels (n = 7) were placed in calcium-free solution for 20 min at 80 mmHg, pressure cycled between 10 and 120 mmHg three times, and finally activated in calcium-free high-K+ solution for 5 min to deplete them of intracellular calcium. This procedure was in control experiments found to inhibit the responses of diameter and [Ca2+]i to 10 µM norepinephrine in calcium-free solution, indicating depletion of intracellular stores. Other arteries (n = 3) were additionally treated with ionomycin (40 µM) during the initial equilibration in calcium-free solution to more effectively equilibrate [Ca2+]i and extracellular calcium concentrations. Typical traces for inner diameter and [Ca2+]i in response to the pressure steps in these vessels under calcium-clamped (traces 2 and 3) and control (trace 1) conditions are presented in Fig. 1.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Typical traces of intracellular calcium concentration ([Ca2+]i; A) and internal diameter (B) as a function of time. Trace 1 shows responses in calcium-containing solution, trace 2 shows responses in calcium-free solution after depletion of intracellular calcium (see MATERIALS AND METHODS), and trace 3 shows responses in the presence of ionomycin. The unlabeled trace shows pressure (dotted line, right y-axis).

Experimental protocols. After fura 2 loading, when the arteries had equilibrated at 37°C under a pressure of 50 mmHg, pressure was cycled three times between 10 and 120 mmHg to decrease mechanical hysteresis (28). The cycling was performed in large steps (10 right-arrow 30 right-arrow 60 right-arrow 80 right-arrow 120 mmHg) of 5-min duration. After the third cycle, the vessel was left at 10 mmHg and 20 min later was subjected to test series of pressure steps from 10 to 120 mmHg. Each step was accomplished in ~1 s with 5 min between consecutive steps. Throughout this series, lumen diameter, [Ca2+]i, and pressure were continuously recorded. Finally, the vessel was relaxed in calcium-free solution for 15 min and subjected to another series of pressure steps (from 10 to 120 mmHg), during which the passive pressure-diameter relation was recorded.

Effect of endothelium removal. Because other studies have demonstrated that vasoconstrictor factors from the endothelium can have a decisive influence on myogenic properties of blood vessels in SHR (7, 32), we examined the role of the endothelium in the small arteries examined here. In a separate series of experiments, the dependence of diameter and [Ca2+]i on pressure was determined in intact arteries as described above. Subsequently, the endothelium was removed with the use of a rat's whisker, and the relation was determined again. The absence of endothelium was verified by testing with 10 µM acetylcholine. As seen in Fig. 2, the endothelium had little influence on the myogenic response of these arteries, whether in terms of the mechanical response or the response of [Ca2+]i to pressure elevation. All other experiments were therefore performed on intact arteries.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of transmural pressure on internal diameter (A) and [Ca2+]i (B) of small arteries from 18-wk-old spontaneously hypertensive rat (SHR) hindlimbs before (squares) and after endothelium removal (circles). Open symbols, measurements in calcium-free solution; solid symbols, measurements in calcium-containing solution. Curves before and after endothelium removal were compared by fitting fourth-order polynomials and comparing the coefficients; in all comparisons, differences were not statistically significant.

Drugs and solutions. Experiments were performed using a solution of the following composition (in mM): 140.0 NaCl, 1.17 MgSO4, 1.6 CaCl2, 4.6 KCl, 10.0 D-glucose, 0.026 EDTA, 1.18 KH2PO4, and 5.0 HEPES. Calcium-free solution had the same composition but with 0.1 mM EGTA and no CaCl2. The pH of these solutions was titrated to 7.4 using 1 M NaOH. High-K+ solution composition was as the calcium-free solution but with NaCl replaced by KCl.

For calibration of the fluorescence signals, the following solution was used (in mM): 125 KCl, 1.17 MgCl2, 5.5 D-glucose, and 5.0 HEPES. To determine Rmin, 40 µM ionomycin and 2 mM EGTA was added; for determining Rmax, 5.0 mM CaCl2 was added on top. The pH of the calibration solutions was titrated to 7.45 using 1 M KOH. All solutions were oxygenated with 100% O2.

Fura 2-AM was obtained from Molecular Probes. All other drugs were obtained from Sigma.

Histological examination. After the myograph experiments, the arteries were fixed with 4% formaldehyde, dehydrated in an alcohol series in the myograph bath, embedded in paraffin, sectioned, and stained with Giemsa stain. The stained sections had clearly distinguishable smooth muscle cells (Fig. 3). From these sections, the inner and outer circumference of the media was measured (Optimas version 5.1, Media Cybernetics), and from this the inner and outer diameters (ID and OD, respectively) were calculated to determine the media cross-sectional area as pi  × (OD2 - ID2)/4 and the media-to-lumen ratio as (OD - ID)/ID.


View larger version (76K):
[in this window]
[in a new window]
  		Fig. 3.   Photomicrographs of Giemsa-stained cross sections of small arteries from ligated (A) and nonligated hindlimbs (B).

Data analysis. Passive and active pressure-diameter curves were obtained by recording pressure and lumen diameter during pressure steps series in calcium-free and calcium-containing solutions, respectively.

The degree of spontaneous tone at each level of pressure was quantified as [(PD - AD)/PD], where AD is the active lumen diameter (in calcium-containing solution) and PD is the passive lumen diameter (in calcium-free solution) as measured in the myograph by the videocamera. Wall stress was calculated as [P × (ID + OD)]/[2 × (OD - ID)], where P is the transmural pressure and ID and OD are the inner and outer diameters as measured by the videocamera.

All data are presented as means ± SE. Where curves were compared, these were analyzed by fitting a third- or fourth-order polynomial and analyzing between the coefficients. Other data were analyzed with unpaired Student's t-test. In all cases, P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Between the ages of 5 and 18 wk, the rats showed a significant blood pressure rise: at 5 wk, the systolic blood pressure measured in the tail artery was 122 ± 2 mmHg, and at 18 wk, it was 187 ± 6 mmHg.

At 18 wk, femoral arterial pressure distal to the ligature was ~60% of that in the control leg (Table 1). Histological examination revealed differences in structure of the small arteries taken from ligated and control hindlimbs (Fig. 3). Distal to a ligation, the arteries had a lower media cross-sectional area and a lower media-to-radius ratio than arteries from control hindlimbs (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Blood pressure and morphological characteristics of small arteries from ligated and nonligated hindlimbs

Passive and active pressure-diameter relationships. In calcium-free solution, arteries were fully relaxed as indicated by the absence of a dilator response to a supramaximal dose of sodium nitroprusside (3 mM). Over the entire pressure range, the passive diameter of the arteries from ligated hindlimbs was significantly greater than that of the control arteries (Fig. 4A).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of transmural pressure on internal diameter (A), tone (B), and [Ca2+]i (C) of small arteries from ligated (circles) and nonligated hindlimbs (squares). Data were replotted in D to show the relation between tone and [Ca2+]i. Open symbols, relations in calcium-free solution; solid symbols, relations in calcium-containing solution. Curves were fit by second-order (calcium-free solution) or fourth-order (calcium-containing solution) polynomials. * P < 0.05, ** P < 0.01, and *** P < 0.001, significant differences in coefficients between curves.

In calcium-containing solution, the arteries developed spontaneous tone, as evidenced by smaller diameters than in calcium-free conditions (Fig. 4A). The magnitude of this basal tone was pressure dependent. Tone increased with increasing pressure, reached a maximum, and finally declined again (Fig. 4B). The maximal levels of achieved tone were not significantly different between the two groups of arteries. However, the pressure-tone relation for arteries from ligated hindlimbs was shifted toward lower pressures compared with the control arteries (Fig. 4B). Vessels from ligated hindlimbs reached maximal spontaneous tone at 70 mmHg, whereas in control arteries maximal tone was at 100 mmHg.

Effect of intraluminal pressure on [Ca2+]i. In the presence of extracellular calcium, [Ca2+]i increased with increasing pressure between 10 and 30 mmHg. This initial increment was followed by a plateau up to ~70 mmHg, after which [Ca2+]i increased again (Fig. 4C). The initial level of [Ca2+]i at the pressure of 10 mmHg was similar in vessels from ligated and nonligated hindlimbs. Both groups of vessels showed the initial increase in [Ca2+]i, but this rise was more pronounced in vessels from ligated hindlimbs, resulting in higher [Ca2+]i at the plateau (Fig. 4C). The final rise of pressure was also more marked in vessels from ligated hindlimbs (Fig. 4C).

Diameter-[Ca2+]i relations. The data shown in Fig. 4, B and C, are replotted in Fig. 4D, which shows calculated tone in relation to [Ca2+]i. Increasing pressure from 10 mmHg caused a rise in [Ca2+]i as described above with little change in tone. Subsequently, tone increased but calcium remained quite constant (the vertical part of the curves in Fig. 4D). Both groups of vessels reached similar maximal tone levels, but calcium was throughout higher in vessels from ligated hindlimbs. After the peak, tone declined again in the face of a further [Ca2+]i increase.

Relations with wall stress. Wall stress, calculated from transmural pressure and internal and external diameters as described in MATERIALS AND METHODS, is a measure of the force per cross-sectional area of the vessel wall and thus of the load on individual muscle cells. As shown in Fig. 5A, the vasoconstriction in response to pressure elevation occurred at similar values of wall stress in the two groups, although the pressure ranges over which this occurred were offset by ~30 mmHg (Fig. 4A). In contrast, the relations between wall stress and [Ca2+]i differed between the groups (Fig. 5B) at all levels of wall stress. [Ca2+]i was higher in vessels from ligated hindlimbs, and the changes in [Ca2+]i levels did not occur at similar wall stresses.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 5.   Influence of wall stress on internal diameter (A) and [Ca2+]i (B) for small arteries from ligated (circles) and nonligated hindlimbs (squares). Curves were fit by fourth-order polynomials. * P < 0.05, significant difference in coefficients between curves.

Effect of D-600. Exposure to the calcium entry blocker D-600 (0.1 µM) fully inhibited spontaneous tone. There were no differences between diameters of arteries in calcium-free solution and in the presence of D-600. Under these conditions, a pressure step resulted in passive increase of diameter at all pressures tested (Fig. 6A).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of transmural pressure on internal diameter (A) and [Ca2+]i (B) of small arteries from ligated (circles) and nonligated hindlimbs (squares). In A, symbols with Xs through them show measurements in calcium-free solution and solid symbols show measurements calcium-containing solution with 0.1 µM D-600. In B, open symbols show measurements in calcium-containing solution and solid symbols show measurements in calcium-containing solution with 0.1 µM D-600. Curves were fit by second-order (in A) or fourth-order (in B) polynomials. ** P < 0.01, significant differences in coefficients between curves.

Exposure to D-600 also lowered intracellular calcium levels and much reduced the changes in [Ca2+]i with pressure (Fig. 6B). However, pressures above 60 mmHg still induced a small elevation of [Ca2+]i. This was in contrast to vessels that had been depleted of intracellular calcium, in which [Ca2+]i was constant (Fig. 1; see MATERIALS AND METHODS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main results of the present study are as follows: 1) both arterial wall structure and myogenic properties are influenced by long-term changes in transmural pressure; 2) wall stress is a major determinant of the myogenic response; 3) whereas changes in wall structure correlate with changes in the myogenic response, [Ca2+]i levels do not; and 4) the myogenic response is to a large extent dependent on pressure-dependent modulation of intracellular calcium sensitivity.

Effect of ligation. In this study, we followed the ligation procedure initially described by Bund et al. (2). In that study, the media-to-lumen ratios of the arteries from hypertensive rats was reduced by ligation to become similar to those of normotensive rats. This is in good agreement with several other studies where blood pressure was reduced in hypertensive rats either by drug treatment (3, 10, 27) or by ligation (9). In the present study, we observed a similar structural change after ligation, as did Bund et al. (2). Because in this model both arteries are obtained from the same animal, genetic and humoral causes are excluded, and thus the observed changes in structure and function can be attributed only to the effects of the ligation. A potential problem with this technique is that the ligature may affect the intramural sympathetic nerves of the external iliac artery and consequently the sympathetic innervation in more distal parts of the vascular tree. However, it is difficult to sympathetically denervate distal blood vessels by proximal periarterial denervation (21, 22), which makes an effect on intramural nerves in the investigated arteries less likely as a major cause for the observed effects, although we cannot exclude that it may contribute. The ligature will not only influence transmural pressure but also flow, which is an important determinant of vascular structure. However, reduced blood flow results in luminal reduction [inward remodeling (30)] and is thus unlikely to explain the observed increase in lumen diameter after ligation in the present study. We therefore believe the observed changes in the arteries from the ligated side to be caused primarily by the reduced blood pressure.

Relation between vascular structure and myogenic response. Several studies comparing hypertensive and normotensive rats have demonstrated that the myogenic range is shifted towards higher pressures in hypertensive rats [cerebral arteries (28); mesenteric arteries (17); skeletal muscle arterioles (6)]. While this difference may be attributed to hypertension, genetic strain differences cannot be excluded. However, in the present study, genetic factors can be excluded because both hypertensive and normotensive arteries were taken from the same animal.

If the myogenic response is activated by an increase in wall stress (20, 31), it follows that in thick-walled arteries the myogenic response would require higher transmural pressure to develop than in vessels with a thinner wall, at least if the elastic modulus is similar in the thick and the thin wall. This could explain the resetting of the myogenic response in hypertensive arteries with increased wall-to-lumen ratio. The observed relation between diameter and wall stress is consistent with this hypothesis. To be quite correct, this relation ought to be based on active, not total, wall stress. However, the passive wall stress at the diameters where myogenic tone was observed is very small and not possible to determine with any accuracy, and the error from using total wall stress is minimal. The relation between diameter and wall stress was similar for vessels from the ligated and nonligated side over the pressure range of the myogenic response. This means that the physical load on the smooth muscle cells appears to be the primary determinant of its degree of contraction.

Thus protection of vessels from hypertensive pressure results in a shift of the pressure-tone curve to lower pressures without a change in the maximal tone developed at the "optimal" pressure level. The studies have shown that pressure elevation is accompanied by a shift of the myogenic range towards higher pressures. The present study shows that the converse is also true and suggests that a regression of structural changes may be of central importance for achieving normal vascular function in the treatment of hypertension. If the myogenic range would not follow the long-term average pressure level in this way, pressure reduction would increase the risk for increased variability in perfusion through organs normally highly dependent on flow autoregulation, such as the brain and kidney, with organ malfunction or damage as the ultimate consequence.

Determination of intracellular calcium. Even though the fura 2 ratio is quite immune to changes in the amount of dye observed, such as may occur by tissue moving into or out of the field of view, we performed initial experiments to determine the impact of such movements in practice. As described in MATERIALS AND METHODS and Fig. 1, if [Ca2+]i was maintained at a low, constant level by depleting the tissue of calcium with or without the use of ionomycin, changes in vessel diameter by nearly a factor of 2 had virtually no influence on the ratio and thus on the estimated calcium concentration. Furthermore, the pattern of changes in [Ca2+]i observed in the study was different from the pattern of changes in diameter, again indicating little influence of tissue movement upon the measurements. Therefore, unless pressurization or vasoconstriction in some way change the affinity of the dye for calcium, we believe our measurements indeed reflect actual changes in [Ca2+]i.

Intracellular calcium levels and myogenic response. In contrast to the mechanical response, intracellular calcium levels changed in a similar fashion over the same pressure range in both groups of arteries. Upon pressurization of the artery, [Ca2+]i increased slightly and then levelled off or even tended to fall slightly upon continued pressure elevation. The initial increase of ~50-100 nM is similar to that seen by McCarron et al. (25) in rat cerebral arteries, who found calcium to increase by ~100 nM during myogenic constriction over the pressure range of 30-70 mmHg.

In arteries from the ligated side, calcium levels were throughout higher than on the control side. While this could be thought to correlate with the thinner wall experiencing a higher load at any given pressure, and thus indicate that the calcium level is influenced by wall stress, even when taken in relation to wall stress, the calcium levels were higher throughout in the arteries from the ligated side. It is thus difficult to explain the regulation of [Ca2+]i in the two types of vessels by the physical forces on the smooth muscle cells.

A striking result was that pressure-induced vasoconstriction occurred with minimal changes in [Ca2+]i. As Fig. 5 demonstrates, virtually all vasoconstriction occurred at constant [Ca2+]i, which started to increase only when the vessel began to dilate in response to overly high pressure. At pressures above 30 mmHg, [Ca2+]i remained nearly constant up to at least 70-90 mmHg, whereas myogenic constriction started at 30 or 70 mmHg in low- and high-pressure arteries, respectively. Thus [Ca2+]i does not directly determine the myogenic response, but the latter is rather regulated by a mechanism distinct from that determining [Ca2+]i. It is evident that pressure-induced vasoconstriction is nearly exclusively regulated by changes in intracellular calcium sensitivity in this artery. While this is in contrast to the myogenic constriction of cerebral arteries (25), which could not be attributed to changes in calcium sensitivity, modulation of calcium sensitivity seems to contribute to the myogenic response in a number of arteries (4, 13, 33).

Over the pressure range where myogenic constriction was observed, calcium elevation could be prevented by blocking calcium entry, which suggests it was mediated mainly by calcium influx through voltage-operated channels. Calcium entry blockade also inhibited contraction, indicating that the calcium elevation was necessary for contraction. This is consistent with observations in several other blood vessels (16, 23, 24, 34): that myogenic tone is susceptible to calcium antagonists, although some arteries may generate tone by a mechanisms insensitive to such drugs (12, 15, 26). The finding that myogenic tone was inhibited by the calcium channel antagonist D-600, even though it was dependent on changes in calcium sensitivity, indicates that the pressure-induced enhancement of sensitivity was not large enough to activate contraction at low levels of calcium but that a suprabasal [Ca2+]i was a prerequisite.

At high pressures, calcium began to rise again, a rise that was not eliminated by blocking calcium entry. This calcium elevation seemed to coincide with the pressure-induced forced dilatation of the arteries and started at lower pressures in arteries from the ligated side. Even in the presence of calcium entry blockers, a rise in [Ca2+]i was seen in this pressure range, suggesting it was not mediated by calcium influx through L-type calcium channels. The mechanism behind this calcium rise [e.g., intracellular calcium release or stretch-activated channels (5)] was not investigated further.

In conclusion, this study gives support to the hypothesis that vascular structure influences the myogenic response, probably by altering the wall stress at a given pressure level. In the arteries studied, elevated [Ca2+]i is required for the myogenic response to occur, but the response itself is determined by changes in intracellular calcium sensitivity.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Danish Medical Research Council, the University of Aarhus, and the Russian Foundation for Basic Research.


    FOOTNOTES

Address for reprint requests and other correspondence: V. V. Matchkov, Dept. of Physiology, Univ. of Aarhus, Universitetsparken 160, DK-8000 Aarhus C, Denmark (E-mail: vvm{at}fi.au.dk).

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.

First published March 21, 2002;10.1152/ajpheart.00690.2001

Received 2 August 2001; accepted in final form 14 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bakker, EN, Kerkhof CJ, and Sipkema P. Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle. Cardiovasc Res 41: 229-236, 1999[Abstract/Free Full Text].

2.   Bund, SJ, West KP, and Heagerty AM. Effects of protection from pressure on resistance artery morphology and reactivity in spontaneously hypertensive and Wistar-Kyoto rats. Circ Res 68: 1230-1240, 1991[Abstract/Free Full Text].

3.   Christensen, KL, Jespersen LT, and Mulvany MJ. Development of blood pressure in spontaneously hypertensive rats after withdrawal of long-term treatment related to vascular structure. J Hypertens 7: 83-90, 1989[ISI][Medline].

4.   D'Angelo, G, Davis MJ, and Meininger GA. Calcium and mechanotransduction of the myogenic response. Am J Physiol Heart Circ Physiol 273: H175-H182, 1997[Abstract/Free Full Text].

5.   Davis, MJ, Meininger GA, and Zawieja DC. Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 263: H1292-H1299, 1992[Abstract/Free Full Text].

6.   Falcone, JC, Granger HJ, and Meininger GA. Enhanced myogenic activation in skeletal muscle arterioles from spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 265: H1847-H1855, 1993[Abstract/Free Full Text].

7.   Falcone, JC, and Meininger GA. Endothelin mediates a component of the enhanced myogenic responsiveness of arterioles from hypertensive rats. Microcirculation 6: 305-313, 1999[ISI][Medline].

8.   Folkow, B. Physiological aspects of primary hypertension. Physiol Rev 62: 347-504, 1982[Free Full Text].

9.   Folkow, B, Gurevich M, Hallbäck M, Lundgren Y, and Weiss L. The hemodynamic consequences of regional hypotension in spontaneously hypertensive and normotensive rats. Acta Physiol Scand 83: 532-541, 1971[ISI][Medline].

10.   Folkow, B, Hallbäck M, Lundgren Y, and Weiss L. The effect of intense treatment with hypotensive drugs on structural design of the resistance vessels in spontaneously hypertensive rats. Acta Physiol Scand 83: 280-282, 1971[ISI][Medline].

11.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract/Free Full Text].

12.   Hill, MA, and Meininger GA. Calcium entry and myogenic phenomena in skeletal muscle arterioles. Am J Physiol Heart Circ Physiol 267: H1085-H1092, 1994[Abstract/Free Full Text].

13.   Hill, MA, Zou H, Davis MJ, Potocnik SJ, and Price S. Transient increases in diameter and [Ca2+]i are not obligatory for myogenic constriction. Am J Physiol Heart Circ Physiol 278: H345-H352, 2000[Abstract/Free Full Text].

14.  Hughes JM and Bund SJ. Influence of Experimental Reduction of Arterial Media:Lumen Ratio on the Myogenic Response. Proc 7th Intl Symp: Resistance Arteries, Muskoka Sands, ON, Canada, 2001, p. 56.

15.   Hwa, JJ, and Bevan JA. A nimodipine-resistant Ca2+ pathway is involved in myogenic tone in a resistance artery. Am J Physiol Heart Circ Physiol 251: H182-H189, 1986[Abstract/Free Full Text].

16.   Hynes, MR, and Duling BR. Ca2+ sensitivity of isolated arterioles from the hamster cheek pouch. Am J Physiol Heart Circ Physiol 260: H355-H361, 1991[Abstract/Free Full Text].

17.   Izzard, AS, Bund SJ, and Heagerty AM. Myogenic tone in mesenteric arteries from spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 270: H1-H6, 1996[Abstract/Free Full Text].

18.   Jensen, PE, Mulvany MJ, and Aalkjær C. Endogenous and exogenous agonist-induced changes in the coupling between [Ca2+]i and force in rat resistance arteries. Pflügers Arch 420: 536-543, 1992[ISI][Medline].

19.   Johansson, B. Myogenic tone and reactivity: definitions based on muscle physiology. J Hypertens Suppl 7: S5-S8, 1989[Medline].

20.   Johnson, PC. The myogenic response. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Soc, 1980, sect. 2, vol. II, chapt. 15, p. 409-442.

21.   Kaarela, OI. Perivascular sympathectomy of the metacarpal artery of the rabbit paw fails to remove distal adrenergic innervation. Scand J Plast Reconstr Surg Hand Surg 25: 121-124, 1991[ISI][Medline].

22.   Kaarela, OI, Raatikainen TK, Junila JA, and Waris TH. Distal adrenergic innervation remains normal after perivascular sympathectomy. Microsurgery 15: 52-54, 1994[ISI][Medline].

23.   Karibe, A, Watanabe J, Horiguchi S, Takeuchi M, Suzuki S, Funakoshi M, Katoh H, Keitoku M, Satoh S, and Shirato K. Role of cytosolic Ca2+ and protein kinase C in developing myogenic contraction in isolated rat small arteries. Am J Physiol Heart Circ Physiol 272: H1165-H1172, 1997[Abstract/Free Full Text].

24.   Laher, I, and Bevan JA. Stretch of vascular smooth muscle activates tone and 45Ca2+ influx. J Hypertens Suppl 7: S17-S20, 1989[Medline].

25.   McCarron, JG, Crichton CA, Langton PD, MacKenzie A, and Smith GL. Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries. J Physiol 498: 371-379, 1997[ISI][Medline].

26.   Meininger, GA, and Davis MJ. Cellular mechanisms involved in the vascular myogenic response. Am J Physiol Heart Circ Physiol 263: H647-H659, 1992[Abstract/Free Full Text].

27.   Nyborg, NC, and Mulvany MJ. Lack of effect of anti-hypertensive treatment with felodipine on cardiovascular structure of young spontaneously hypertensive rats. Cardiovasc Res 19: 528-536, 1985[ISI][Medline].

28.   Osol, G, and Halpern W. Myogenic properties of cerebral blood vessels from normotensive and hypertensive rats. Am J Physiol Heart Circ Physiol 249: H914-H921, 1985[Abstract/Free Full Text].

29.   Peng, H, Jensen PE, Nilsson H, and Aalkjær C. Effect of acidosis on [Ca2+]i in rat cerebral arteries: is there a role for membrane potential? Am J Physiol Heart Circ Physiol 274: H655-H662, 1998[Abstract/Free Full Text].

30.   Pourageaud, F, and De Mey JG. Structural properties of rat mesenteric small arteries after 4-wk exposure to elevated or reduced blood flow. Am J Physiol Heart Circ Physiol 273: H1699-H1706, 1997[Abstract/Free Full Text].

31.   Schubert, R, and Mulvany MJ. The myogenic response: established facts and attractive hypotheses. Clin Sci 96: 313-326, 1999[Medline].

32.   Ungvari, Z, and Koller A. Endothelin and prostaglandin H2/thromboxane A2 enhance myogenic constriction in hypertension by increasing Ca2+ sensitivity of arteriolar smooth muscle. Hypertension 36: 856-861, 2000[Abstract/Free Full Text].

33.   VanBavel, E, Wesselman JP, and Spaan JA. Myogenic activation and calcium sensitivity of cannulated rat mesenteric small arteries. Circ Res 82: 210-220, 1998[Abstract/Free Full Text].

34.   Wesselman, JP, VanBavel E, Pfaffendorf M, and Spaan JA. Voltage-operated calcium channels are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries. J Vasc Res 33: 32-41, 1996[ISI][Medline].

35.   Yamori, Y, Igawa T, Kanbe T, Kihara M, Nara Y, and Horie R. Mechanisms of structural vascular changes in genetic hypertension: analyses on cultured vascular smooth muscle cells from spontaneously hypertensive rats. Clin Sci 61, Suppl7: 121s-123s, 1981.

Am J Physiol Heart Circ Physiol 283(1):H118-H125
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
V. V. Matchkov, A. Rahman, L. M. Bakker, T. M. Griffith, H. Nilsson, and C. Aalkjaer
Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries
Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H357 - H367.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
W. D. Strain, N. Chaturvedi, C. J. Bulpitt, C. Rajkumar, and A. C. Shore
Albumin Excretion Rate and Cardiovascular Risk: Could the Association Be Explained by Early Microvascular Dysfunction?
Diabetes, June 1, 2005; 54(6): 1816 - 1822.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
C. Dubroca, D. You, B. I. Levy, L. Loufrani, and D. Henrion
Involvement of RhoA/Rho Kinase Pathway in Myogenic Tone in the Rabbit Facial Vein
Hypertension, May 1, 2005; 45(5): 974 - 979.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. F. Brekke, N. I. Gokina, and G. Osol
Vascular smooth muscle cell stress as a determinant of cerebral artery myogenic tone
Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2210 - H2216.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/1/H118    most recent
00690.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matchkov, V. V.
Right arrow Articles by Nilsson, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matchkov, V. V.
Right arrow Articles by Nilsson, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online