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Am J Physiol Heart Circ Physiol 281: H2176-H2183, 2001;
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Vol. 281, Issue 5, H2176-H2183, November 2001

Initial and sustained phases of myogenic response of rat mesenteric small arteries

S. Chlopicki, H. Nilsson, and M. J. Mulvany

Department of Pharmacology, University of Aarhus, 8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A possible role for a metabolite of cytochrome P-450 omega -hydroxylase in the initial and sustained phases of the myogenic response in cannulated rat mesenteric small arteries was studied. With slight preconstriction (norepinephrine and neuropeptide Y), pressure was raised from 60 to 100 mmHg, and both initial (within 2 min) and sustained phases (at 10 min) of the myogenic response were quantified. The myogenic response was fully inhibited by D600 (methoxyverapamil). Ketoconazole and 17-octadecanoic acid did not affect the initial phase but inhibited the sustained phase. In contrast, miconazole did not affect either phase. Charybdotoxin and iberiotoxin potentiated the initial phase but eliminated the sustained phase. Apamin, glibenclamide, 4-aminopyridine, and barium had no effect on either phase. The results demonstrate different mechanisms for the initial and sustained phases of the myogenic response of rat mesenteric small arteries. Only the sustained phase appears mediated through a cytochrome P-450 omega -hydroxylase metabolite and calcium-activated K+ channels. However, both phases of the response are dependent on calcium influx through voltage-dependent calcium channels.

calcium-activated potassium channels; autoregulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CONSTRICTION of a blood vessel in response to an increase in transmural pressure (the myogenic response) has been known for nearly 100 years (1). Nevertheless, whereas it is thought to play an important role in local regulation of capillary pressure and blood flow (8), its exact mechanism remains unknown. Two components of the myogenic response, an initial transient response and a sustained response, have been described in both the intact vascular bed and certain isolated vascular preparations (9, 22). However, so far these phases have not been characterized experimentally using pharmacological tools. In particular, none of the suggested pathways of myogenic response such as stretch-activated ion channels, voltage-dependent calcium channels, or membrane-linked enzymes (phospholipase C and protein kinase C) have proven to be differently involved in initial or sustained phases of myogenic response (4, 32).

In recent years, attention has been focused on a new possible mechanism of the myogenic response. It was found that a cytochrome P-450-dependent arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), is a potent inhibitor of large-conductance, calcium-dependent potassium channels (KCa), causing membrane depolarization and contraction of vascular smooth muscle cells (17). Furthermore, it was demonstrated that inhibitors of cytochrome P-450 (23) and blockade of KCa channels (36) completely blocked pressure-induced vasoconstriction, supporting the involvement of 20-HETE in this response. These studies focused on the tonic component of the myogenic response, and it was therefore of interest to investigate whether the initial part of the response also was affected by interference with the cytochrome P-450 pathway. In the present work, we used cytochrome P-450 inhibitors and KCa channel blockers to examine the role of cytochrome P-450 metabolites in the initial and sustained phases of the myogenic response in isolated, pressurized small arteries from the rat mesentery.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation. Male Wistar rats (200-350 g) were killed by either CO2 or cervical dislocation, the intestines were exposed by a median incision of the abdomen, and one segment of intestine together with mesenteric arteries was quickly excised and placed in a dissection dish containing cold physiological salt solution (PSS; see Drugs and solutions). The tissue was pinned to the bottom of the dish, and a 2- to 3-mm long segment of a second-order branch of the superior mesenteric artery was cleared from surrounding adipose tissue and dissected out under a dissection microscope. After dissection, the artery was transferred to the chamber of a pressure myograph (JP Trading; Aarhus, Denmark) for cannulation. At this stage, the chamber was filled with PSS equilibrated with room air at ambient temperature, and the perfusion line was filled with PSS-albumin (see Drugs and solutions). The chamber contained two glass pipettes (tip outer diameter ~120 µm): one was fixed and the other was mounted on a manipulator for adjusting its position as required. One end of the vessel was cannulated with the inflow pipette and tied securely with 10-0 nylon surgical thread. The inflow pressure was then gently raised from 0 to ~20 mmHg to flush blood out of the vessel lumen. The other end of the vessel was then cannulated with the outflow pipette. After cannulation, the pressure myograph was carefully transferred to the stage of the inverted microscope, the pressure was set to 10 mmHg, and the preparation was heated to 37°C. After equilibration, transmural pressure was elevated to 80 mmHg in 10-mmHg steps. At each step, the axial length was adjusted to eliminate any buckling of the vessel. During that time, the longitudinal force in the vessel was monitored by a force transducer connected to the outlet pipette to avoid loading the vessel longitudinally with >0.5 mN. Higher values of longitudinal force have been found to have a detrimental effect on the viability of the vessel in preliminary experiments. The measured longitudinal extension in 28 vessels was 32 ± 2% relative to unstressed length at 10 mmHg. When pressurized, the vessel was examined under an inverted microscope (Telaval 31, Zeiss) for leaks, which were easily identified by the appearance of albumin in the myograph chamber. In the absence of any leaks, the vessel was allowed to equilibrate for at least 40 min at a transmural pressure of 80 mmHg. During this time, to assist equilibration, a small pressure gradient (1-2 mmHg) was applied to induce flow of ~20 µl/min. Thereafter, experiments were performed without flow.

The middle of the vessel segment was viewed through the inverted microscope (magnification ×100), and its outer diameter was measured by a videomicroscopic technique. The signal from a video camera attached to the inverted microscope was fed to a frame grabber and then to a dimension-analyzing program (VesselView, JP Trading). Hydrostatic pressures of both inlet and outlet reservoirs were measured by pressure transducers connected to the perfusion line on the inlet and outlet side, respectively. All experiments were performed under no-flow conditions where inlet and outlet pressures were equal. The temperature of the chamber was maintained at 37°C. Outer diameter and inlet and outlet pressures were measured continuously at 3 Hz.

Drugs and solutions. The PSS had the following composition (in mM): 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 25 NaHCO3, 1.18 KH2PO4, 0.027 EDTA, 5.5 glucose, and 2.0 pyruvate. Intraluminal PSS solution contained additionally 1% bovine serum albumin. Solutions were equilibrated with 21% O2-5% CO2 balanced with N2 and had a pH of 7.4-7.5. Drugs were applied extraluminally to the myograph chamber; concentrations are given as final bath concentration. The following drugs were used: neuropeptide Y (NPY), norepinephrine, miconazole (epoxygenase inhibitor), methoxyverapamil (D600, calcium entry blocker); barium chloride (inward rectifier K+ channel blocker), glibenclamide (ATP-dependent K+ channel blocker), apamin (small conductance KCa channel blocker), and 4-aminopyridine (delayed rectifier K+ channel blocker) from Sigma; charybdotoxin (nonspecific KCa channel blocker) and iberiotoxin (large conductance KCa channel blocker) from Latoxan; and 17-octadecanoic acid (17-ODYA) and ketoconazole (structurally dissimilar cytochrome P-450 inhibitors) from BioMol. The concentrations used (see RESULTS) were chosen on the basis found to be effective, with reasonable specificity, as used elsewhere (13, 24, 31, 37).

Experimental protocol. Experiments were performed under no-flow conditions. After equilibration at 80 mmHg for 40 min, the pressure was set to 60 mmHg. The artery was activated with a threshold concentration of NPY (1-3 nM) and then with increasing concentrations of norepinephrine starting from 10 nM until a preconstriction of ~10% of basal lumen diameter was achieved. As also noted elsewhere (16), we found the effects of NPY activation to resist washing out with PSS; therefore, NPY was not included for the second preconstriction (see below).

The rationale for including NPY in the preconstriction solution was that in initial studies, using 17 vessels, we had found an unacceptable degree of variation in the concentration of norepinephrine needed to induce the required 10% level of tone, and the tone was not maintained. Applying norepinephrine on top of NPY appeared to reduce the variance, and the concentration of norepinephrine needed to induce the preconstriction. These indications were later found justified when the parameters from the initial studies were compared with the results of the full study (Fig. 1). Furthermore, the combination of NPY and norepinephrine provided more stable precontractions and a reduction in the amplitude of vasomotion (which made the myogenic response more difficult to analyze), whereas the reproducibility of the myogenic response in this preparation was increased (Table 1).


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  		Fig. 1.   Effect of a threshold concentration of neuropeptide Y (NPY, 1-3 nM, see METHODS) on the concentration of norepinephrine (NE) required to constrict mesenteric arteries by 10% of resting lumen diameter. Points show mean for each group (without NPY, n = 17; with NPY, n = 58); vertical bars show SD. Both the means (P < 0.01) and the variances (P < 0.001) were different between the two groups.


                              
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  		Table 1.   Effect of inhibitors on threshold norepinephrine concentrations

When a stable level of preconstriction (10%) had been attained with the norepinephrine-neuropeptide mixture using the above protocol, a myogenic response was evoked by pressure elevation (within 10 s) from 60 to 100 mmHg. The pressure of 100 mmHg was maintained for at least 10 min to allow the vessel to reach steady-state diameter. The pressure was then returned to 60 mmHg, and the tissue was relaxed in PSS. A selected pharmacological inhibitor (see below) was thereafter added to the chamber and incubated for at least 20 min. Subsequently, the vessel was again preconstricted with norepinephrine (but without NPY, see above) to the same level of tone, and when stable the pressure step to 100 mmHg was repeated. In the control experiments, where norepinephrine was given alone for the second preconstriction, the norepinephrine concentration used was the same as for the first preconstriction, because this resulted in approximately the same 10% level of tone. However, after the vessel had been incubated with drugs affecting vascular tone, the concentration of norepinephrine had to be adjusted to obtain the same 10% level of tone. Ketoconazole, 17-ODYA, and D600 required higher concentrations of norepinephrine; charybdotoxin, iberiotoxin, and barium chloride required lower concentrations of norepinephrine (data not shown). To keep a no-flow condition during pressure changes, pressure changes were made simultaneously at both inlet and outlet reservoirs.

At the end of all experiments, a pressure jump from 60 to 100 mmHg was performed in Ca-free PSS to determine the passive pressure-diameter relationship. In some experiments, a pressure step from 60 to 100 mmHg was also performed in nonactivated vessels at the beginning of the experiments. All experiments were completed within 3 h.

Data analysis. A vessel was only accepted for experiment if 1) it was not leaky, 2) not >200 nM norepinephrine [added on the top of the threshold concentration of NPY (1-3 nM)] was needed to preconstrict the vessel by 10%, and 3) this preconstriction was uniform along the whole vessel length. Any artery that did not fulfil these criteria was discarded from the data analysis. Of 120 vessels mounted, 62 were rejected. Interestingly, all vessels that fulfilled criteria 1-3 showed myogenic responsiveness, that is, the vessel diameter returned to its initial value after a pressure step from 60 to 100 mmHg.

The myogenic response was quantified (Fig. 2) with the use of three parameters. The first was initial myogenic response (I) equal to DI - DC, where DI is the minimum diameter during the first 2 min after the pressure step and DC is the diameter of the vessel at the pressure of 60 mmHg before the pressure step. Second, sustained myogenic response (S) was equal to DS - DC, where DS is the diameter of the vessel 10 min after pressure elevation. The third was the rate of myogenic response (R), defined as the reciprocal of the time (Tr) that elapsed from the pressure jump until the vessel, at a pressure of 100 mmHg, regained the same diameter as it had before the pressure elevation.


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  		Fig. 2.   Myogenic lumen diameter response to change in transmural pressure from 60 to 100 mmHg. I, initial myogenic response, i.e., maximum contraction from diameter at 60 mmHg during first 2 min; S, sustained myogenic response, i.e., contraction at 10 min; Tr, myogenic response time, i.e., time after raising pressure to achieving initial diameter. Rate of myogenic response defined as R = 1/Tr. See METHODS for further details.

All results were expressed as means ± SE unless otherwise stated. Differences between means were analyzed with Student's paired t-test and considered statistically significant if P < 0.05. Difference in variances for two samples were evaluated with F-test and considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basic characteristics of myogenic response. The basal diameter of the entire group of 58 mesenteric small arteries at a transmural pressure of 60 mmHg was 342 ± 5 µm. No vessel developed spontaneous myogenic tone. Thus, in the absence of activation with norepinephrine or NPY, the response of vessels to an increase in pressure to 100 mmHg was only passive distension, the final diameter at 10 min being close to that obtained in Ca2+-free PSS solution (Fig. 3). However, when arteries were preconstricted by 10.3 ± 0.6% of resting lumen diameter with a threshold concentration of NPY (2 ± 0.2 nM, range 1-3 nM) along with norepinephrine (77 ± 4 nM, range 30-200 nM), rapid elevation of transmural pressure from 60 to 100 mmHg resulted in a myogenic response (n = 58). An example of the changes of lumen diameter in response to a pressure jump from 60 to 100 mmHg is shown in Fig. 2, where the initial passive distention is followed by a myogenic contraction that has been divided into the indicated two phases. The constrictor response of the vessel to the pressure jump varied considerably between preparations, both in terms of the initial (range = +5 µm to -35 µm) and sustained (range -1 µm to -45 µm) myogenic responses, as well as in the myogenic response time (range 6-213 s). However, the myogenic response within the same preparation was highly reproducible (for two repeated pressure steps, myogenic responses were the following: initial, -10.4 ± 2.9 µm and 12.4 ± 2.2 µm; sustained, -12.1 ± 2.6 µm and 13.5 ± 2.3 µm; n = 7). Thus each vessel was used as its own control.


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  		Fig. 3.   Pressure-diameter relations of isolated, pressurized mesenteric arteries in physiological salt solution (normal PSS, open diamonds, n = 25) and in Ca2+-free PSS (open circles, n = 58) and in vessels subjected to a 10 ± 0.6% preconstriction with combination of NPY (2 ± 0.2 nM) and norepinephrine (77 ± 4 nM) (closed symbols) when pressure was raised from 60 to 100 mmHg. At 100 mmHg, closed circles show I phase and closed square shows S phase, as defined in Fig. 2. Values are means ± SE.

Spontaneous vasomotion was not observed in any vessel, but in 8 of 58 investigated vessels, vasomotion of low amplitude (<4% of resting lumen diameter) was observed upon addition of norepinephrine.

Effects of cytochrome P-450 inhibitors on myogenic response. Figure 4 illustrates the effects of three different inhibitors of cytochrome P-450-dependent enzymes on the myogenic response. In the presence of 17-ODYA (20 µM, Fig. 4, A and B) pressure elevation induced an initial myogenic response not different from control. However, after the initial contraction the vessel gradually lost the tone induced by the pressure jump. Pretreatment of the vessel with another cytochrome P-450 inhibitor, ketoconazole (100 µM), had similar effects. The initial response to a pressure step from 60 to 100 mmHg was unaffected, whereas the sustained phase was inhibited (Fig. 4, C and D). In contrast, the epoxygenase inhibitor miconazole (1 µM) did not significantly affect either phase of the myogenic response (Fig. 4, E and F).


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  		Fig. 4.   Effect of cytochrome P-450 inhibitors on myogenic response evoked by pressure step from 60 to 100 mmHg in isolated mesenteric small arteries. Left: typical traces showing lumen diameter response to pressure step from 60 to 100 mmHg in absence and presence of 20 µM 17-ODYA (A), 100 µM ketoconazole (C), and 1 µM miconazole (E). Right (B, D, F): corresponding bar graphs depicting effects of cytochrome P-450 inhibitors on I and S myogenic response. Columns represent mean; bars show SE; n = 5-8. *Significant (P < 0.001) difference between test from the corresponding control value in the same vessel.

Effects of D600 on the myogenic response. D600 (0.2 µM) inhibited the contractile response of the vessel to potassium chloride by 83 ± 2%, n = 5. In the presence of this concentration of D600, pressure elevation resulted in only a passive distension of the vessel, which was not followed by a myogenic contraction (Fig. 5, A and B)


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  		Fig. 5.   Effects of methoxyverapamil (D600) on myogenic response evoked by pressure step from 60 to 100 mmHg in isolated small mesenteric arteries. A: typical trace showing complete inhibition of lumen diameter myogenic response to pressure step from 60 to 100 mmHg by D600 (0.2 µM). B: corresponding bar graph summarizing inhibitory effects of D600 on I and S myogenic response (n = 4). Columns represent mean; vertical bars show SE; n = 4. *Significant (P < 0.01) difference between test value from the corresponding control value in the same vessel.

Effect of potassium channel blockers on the myogenic response. After pretreatment with charybdotoxin (100 nM), a pressure jump tended to induce greater initial constriction, although not statistically different from the control responses. This response, however, was not sustained in that the sustained myogenic response was lost (Fig. 6, A and B). Also, another inhibitor of large-conductance potassium channels, iberiotoxin (100 nM), affected the diameter response to pressure elevation in a similar way (Fig. 6, C and D). None of the following potassium channel blockers used in the study significantly influenced the myogenic response (Fig. 7): apamin (100 nM), glibenclamide (1 µM), barium chloride (100 µM), and 4-aminopyridine (500 µM).


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  		Fig. 6.   Effect of large-conductance potassium channel inhibitors on the myogenic response evoked by a pressure step from 60 to 100 mmHg in isolated mesenteric small arteries. Left: representative traces showing vessel diameter response to pressure step from 60 to 100 mmHg in absence and presence of 100 nM charybdotoxin (A) and 100 nM iberiotoxin (C). Right (B and D): corresponding bar graphs summarizing effects of large-conductance potassium channel inhibitors on I and S myogenic responses. Columns represent mean; bars show SE; n = 7. *Significant (P < 0.01) difference between test value from the corresponding control value in the same vessel.



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  		Fig. 7.   Effects of inhibitors of various potassium channels on I and S myogenic responses. A: 100 nM apamin (n = 3); B: 1 µM glibenclamide (n = 3); C: 100 µM barium chloride (n = 4); D: 0.5 mM 4-aminopyrydine (n = 4). Columns represent mean; bars show SE. There were no significant differences (P > 0.05) from the corresponding test and control values in the same vessels.

Effects of the inhibitors used in the study on the rate of myogenic response. In control conditions, the second myogenic response was slightly more rapid (rate of second myogenic response was 130 ± 14% of the first). Of all drugs used in the study, only blockers of large-conductance potassium channels substantially affected the rate of myogenic response (Fig. 8). Thus in the presence of iberiotoxin (100 nM) or charybdotoxin (100 nM), the rate of myogenic response increased up to 483% and 672% of control values, respectively (n = 7, P < 0.05).


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  		Fig. 8.   Effect of various inhibitors used in the study on the rate of myogenic response evoked by a pressure step from 60 to 100 mmHg in isolated mesenteric small arteries. Data are presented as a percentage of control response. Columns represent mean; vertical bars show SE; n = 3-8 experiments. Note that the rate of the myogenic response was affected only after pretreatment with charybdotoxin (100 nM, n = 7) or iberiotoxin (100 nM, n = 7). *Significant (P < 0.05) difference between test value (filled bars) and the corresponding control value (open bar) in the same vessel.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments have defined an initial and a sustained phase of the myogenic response of rat mesenteric small arteries. Using pharmacological tools, we demonstrated for the first time that the sustained phase, but not the initial phase, was eliminated by cytochrome P-450 inhibitors (17-ODYA and ketoconazole) and by large-conductance KCa channel inhibitors (charybdotoxin and iberiotoxin). Neither the epoxygenase inhibitor miconazole nor inhibitors of other types of potassium channels (glibenclamide, apamin, 4-aminopyridine, or barium chloride) influenced either phase of the myogenic response. However, both phases were abolished by an inhibitor of L-type calcium channels (D600).

Preconstriction. The rat mesenteric small arteries used in this investigation (diameter 200-400 µm) contribute substantially to the vascular resistance and play an important role in the regulation of blood flow (7). In contrast to more distal arteries (33), these densely innervated arteries (6) have little myogenic tone under resting conditions in vitro (37). However, moderate activation of the smooth muscle has previously been shown to allow myogenic responses (35). We therefore used a combination of the neural cotransmitters NPY and norepinephrine to induce a defined level of tone. The more consistent responses obtained by inclusion of NPY could relate to suppression of cAMP activity caused by beta -adrenergic activation (27).

Addition of the various inhibitors (Table 1) changed the level of tone and therefore made it necessary to adjust the concentration of the agonists. It is conceivable that the altered agonist concentration might affect the myogenic response independently of any effect of the agonist (28). However, it has been demonstrated that myogenic responses depend more on the level of agonist-induced tone than on agonist concentration (34). For this reason, we chose to carefully adjust the level of preconstriction to a constant level to allow us to determine the direct effect of the inhibitors on the myogenic response.

Arachidonic acid metabolism. Arachidonic acid metabolites from the cytochrome P-450 pathway have been suggested to play an important role in the myogenic response (15, 17). Thus inhibition of cytochrome P-450 enzymes abolished pressure-induced constriction (23), and the metabolite 20-HETE, a major product of cytochrome P-450 4A omega -hydroxylase in smooth muscle cells and an endogenous inhibitor of KCa channels (15, 17, 37), constricts arterioles in the nanomolar range (21). Indeed, the cytochrome P-450 4A protein has been detected in vascular smooth muscle (16), and formation of 20-HETE was demonstrated in small resistance vessels (21) but not in large conduit vessels such as the aorta (30) lacking myogenic response. It was thus proposed that 20-HETE by its action on KCa channels would play a major role in pressure-induced constriction (15, 17), although the pathways of 20-HETE generation in vascular smooth muscle in response to pressure elevation remain unclear.

Whereas direct detection of arachidonic acid metabolites (e.g., 20-HETE) is notoriously difficult in smooth muscle cells, there is evidence for a reduction of 20-HETE production by the same inhibitors as used in this study (15, 39). Moreover, in patch-clamp studies of smooth muscle cells isolated from cat cerebral arteries, administration of 17-ODYA (20 µM) increased K+ channel activity, an effect that was reversed by 20-HETE (38). Our pharmacological analysis of myogenic response is thus consistent with a cytochrome P-450 metabolite such as 20-HETE being a mediator necessary for maintenance of the myogenic response.

Two phases in the myogenic response. The concept of two distinct components (transient and tonic, respectively) in the myogenic response was suggested previously in studies in vitro on portal vein strips (22), on isolated arterioles from hamster cheek pouch (5), as well as in vivo on whole skeletal muscle vascular beds (2, 9-11). In the present study, two components were not distinguishable initially, but could be clearly demonstrated after pharmacological intervention, suggesting that also here different mechanisms are involved. Whether the two phases observed here relate to the two phases described previously remains to be determined.

Initial phase. The present study shows that the initial phase was dependent on influx of calcium through L-type calcium channels, being inhibited by D600. This is consistent with the prevailing hypothesis that myogenic contraction is initiated by smooth muscle cell depolarization (14, 36), possibly through stretch-activated channels (4, 27), which then regulates Ca2+ entry through voltage-gated calcium channels (18, 20, 29, 37). The rise in intracellular Ca2+ concentration ([Ca2+]i) will itself promote contraction, but it may also activate phospholipase A2 or phospholipase C, causing release of arachidonic acid and possibly elevating the concentration of 20-HETE.

Both the increase in [Ca2+]i and vascular smooth muscle depolarization will activate KCa channels, and it was proposed that activation of these channels could act as a negative feed-back mechanism to limit voltage-gated Ca2+ entry (38). Our observation that the rate of the myogenic response was enhanced by KCa channel blockers supports a counterregulatory role for such channels in the initial phase of the response.

In some preparations (3, 19, 25) the role of calcium has been questioned. Furthermore, there is also evidence for direct modulation of L-type calcium channels by stretch (26) and for pressure-induced calcium sensitization (35). However, for the present preparation, the evidence suggests that depolarization-induced calcium influx is essential for the initial phase of the myogenic response.

Sustained phase. As indicated above, both the increase in [Ca2+]i and vascular smooth muscle depolarization will activate KCa channels, which would then repolarize the smooth muscle cells and thus limit the myogenic response. It is thus possible that the sustained phase is due to prevention of this KCa channel activation, for which, as indicated above, 20-HETE could be acting as the inhibitor. Our data extend previous studies and suggest that 20-HETE may be specifically involved in the sustained but not in the initial phase of the myogenic response, because only this phase was inhibited by inhibitors of cytochrome P-450 activity. Thus this phase was inhibited by 17-ODYA or ketoconazole at concentrations where they have been shown to substantially inhibit omega -hydroxylase, 20-HETE formation, and epoxygenase activity in vascular smooth muscle (39), whereas miconazole at 1 µM a selective inhibitor of epoxygenase activity (39), was not effective. Furthermore, under conditions where KCa channels were inhibited (using charybdotoxin of iberiotoxin), no sustained phase was seen, consistent with Wesselman et al. (36), who found that charybdotoxin prevents sustained pressure-induced depolarization. The present evidence thus supports the suggestion (15, 17) that the sustained phase of the myogenic response is dependent on omega -hydroxylase activity and 20-HETE-mediated inhibition of KCa channels, but further work is needed to confirm this.

In summary, we have shown that the initial and sustained phases of the myogenic response of rat mesenteric small arteries are mediated through separate mechanisms. Whereas both phases required calcium influx through voltage-dependent calcium channels, the sustained phase specifically depended on a cytochrome P-450 metabolite, possibly 20-HETE.


    ACKNOWLEDGEMENTS

This project was supported by the Aarhus University Research Foundation, the Danish Medical Research Council, and the Danish Heart Foundation.


    FOOTNOTES

Current address for S. Chlopicki: Dept. of Pharmacology, Medical College, Jagiellonian University, Grzegórzecka 16, 31-531 Krakow, Poland (E-mail: mfschlop{at}cyf-kr.edu).

Address for reprint requests and other correspondence: M. J. Mulvany, Dept. of Pharmacology, Aarhus Univ., Univ. Park 240, 8000 Aarhus C, Denmark (E-mail: mm{at}farm.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.

Received 25 October 2000; accepted in final form 18 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bayliss, WM. On the local reactions of the arterial wall to changes in internal pressure. J Physiol (Lond) 28: 220-231, 1902.

2.   Borgström, P, Grände PO, and Lindbom L. Responses of single arterioles in vivo in cat skeletal muscle to change in arterial pressure applied at different rates. Acta Physiol Scand 113: 207-212, 1981[ISI][Medline].

3.   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].

4.   Davis, MJ, and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387-423, 1999[Abstract/Free Full Text].

5.   Davis, MJ, and Sikes PJ. Myogenic responses of isolated arterioles: test for a rate-sensitive mechanism. Am J Physiol Heart Circ Physiol 259: H1890-H1900, 1990[Abstract/Free Full Text].

6.   Donoso, MV, Brown N, Carrasco C, Cortes V, Fournier A, and Huidobro-Toro JP. Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y-Y1 receptors. J Neurochem 69: 1048-1059, 1997[ISI][Medline].

7.   Fenger-Gron, J, Mulvany MJ, and Christensen KL. Intestinal blood flow is controlled by both feed arteries and microcirculatory resistance vessels in freely moving rats. J Physiol (Lond) 498: 215-224, 1997[ISI][Medline].

8.   Folkow, B, and Neil E. The principles of vascular control. In: Circulation, edited by Folkow B, and Neil E.. London: Oxford University Press, 1971, p. 285-306.

9.   Grände, PO. Dynamic and static components in the myogenic control of vascular tone in cat skeletal muscle. Acta Physiol Scand Suppl 476: 1-44, 1979[Medline].

10.   Grände, PO, Lundvall J, and Mellander S. Evidence for a rate-sensitive regulatory mechanism in myogenic microvascular control. Acta Physiol Scand 99: 432-447, 1977[ISI][Medline].

11.   Grände, PO, and Mellander S. Characteristics of static and dynamic regulatory mechanisms in myogenic microvascular control. Acta Physiol Scand 102: 231-245, 1978[ISI][Medline].

12.   Gustafsson, H, and Nilsson H. Endothelium-independent potentiation by neuropeptide Y of vasoconstrictor responses in isolated arteries from rat and rabbit. Acta Physiol Scand 138: 503-507, 1990[ISI][Medline].

13.   Gustafsson, H, and Nilsson H. Rhythmic contractions in isolated small arteries of rat: role of K+ channels and the Na+,K+-pump. Acta Physiol Scand 150: 161-170, 1993.

14.   Harder, DR. Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ Res 55: 197-202, 1984[Abstract/Free Full Text].

15.   Harder, DR, Campbell WB, and Roman RJ. Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone. J Vasc Res 32: 79-92, 1995[ISI][Medline].

16.   Harder, DR, Gebremedhin D, Narayanan J, Jefcoat C, Falck J, Campbell WB, and Roman R. Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol Heart Circ Physiol 266: H2098-H2107, 1994[Abstract/Free Full Text].

17.   Harder, DR, Lange AR, Gebremedhin D, Birks EK, and Roman RJ. Cytochrome P450 metabolites of arachidonic acid as intracellular signaling molecules in vascular tissue. J Vasc Res 34: 237-243, 1997[ISI][Medline].

18.   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].

19.   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].

20.   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].

21.   Imig, JD, Zou AP, Stec DE, Harder DR, Falck JR, and Roman R. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. Am J Physiol Regulatory Integrative Comp Physiol 270: R217-R227, 1996[Abstract/Free Full Text].

22.   Johansson, B, and Mellander S. Static and dynamic components in the vascular myogenic response to passive changes in length as revealed by electrical and mechanical recordings from the rat portal vein. Circ Res 36: 76-83, 1975[Abstract/Free Full Text].

23.   Kauser, K, Clark JE, Masters BS, Ortiz de Montellano PR, Ma YH, Harder DR, and Roman RJ. Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries. Circ Res 68: 1154-1163, 1991[Abstract/Free Full Text].

24.   Knot, HJ, and Nelson MT. Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries. Am J Physiol Heart Circ Physiol 269: H348-H355, 1995[Abstract/Free Full Text].

25.   Laher, I, van Breemen C, and Bevan JA. Stretch-dependent calcium uptake associated with myogenic tone in rabbit facial vein. Circ Res 63: 669-672, 1988[Abstract/Free Full Text].

26.   Langton, PD. Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive. J Physiol (Lond) 471: 1-11, 1993[Abstract/Free Full Text].

27.   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].

28.   Meininger, GA, and Faber JE. Adrenergic facilitation of myogenic response in skeletal muscle arterioles. Am J Physiol Heart Circ Physiol 260: H1424-H1432, 1991[Abstract/Free Full Text].

29.   Nordlander, M, and Thalén P. Effects of felodipine on local and neurogenic control of vascular resistance. J Cardiovasc Pharmacol 10, Suppl 1: S100-106, 1987.

30.   Pfister, SL, Falck JR, and Campbell WB. Enhanced synthesis of epoxyeicosatrienoic acids by cholesterol-fed rabbit aorta. Am J Physiol Heart Circ Physiol 261: H843-H852, 1991[Abstract/Free Full Text].

31.   Quayle, JM, McCarron JG, Brayden JE, and Nelson MT. Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries. Am J Physiol Cell Physiol 265: C1363-C1370, 1993[Abstract/Free Full Text].

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

33.   Sun, D, Messina EJ, Kaley G, and Koller A. Characteristics and origin of myogenic response in isolated mesenteric arterioles. Am J Physiol Heart Circ Physiol 263: H1486-H1491, 1992[Abstract/Free Full Text].

34.   VanBavel, E, and Mulvany MJ. Role of wall tension in the vasoconstrictor response of cannulated rat mesenteric small arteries. J Physiol (Lond) 477: 103-115, 1994[ISI][Medline].

35.   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].

36.   Wesselman, JP, Schubert R, VanBavel ED, Nilsson H, and Mulvany MJ. KCa channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries. Am J Physiol Heart Circ Physiol 272: H2241-H2249, 1997[Abstract/Free Full Text].

37.   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].

38.   Zou, AP, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR, and Roman RJ. 20-HETE is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles. Am J Physiol Regulatory Integrative Comp Physiol 270: R228-R237, 1996[Abstract/Free Full Text].

39.   Zou, AP, Ma YH, Sui ZH, Ortiz de Montellano PR, Clark J, Masters BS, and Roman RJ. Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome P450 fatty acid omega-hydroxylase on renal function in rats. J Pharmacol Exp Ther 268: 474-481, 1994[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(5):H2176-H2183
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