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Am J Physiol Regul Integr Comp Physiol 282: R969-R978, 2002; doi:10.1152/ajpregu.00405.2001
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Vol. 282, Issue 4, R969-R978, April 2002

Multiple dilator pathways in skeletal muscle contraction-induced arteriolar dilations

Coral L. Murrant1 and Ingrid H. Sarelius2

1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 1J6; and 2 Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether nitric oxide (NO), adenosine (Ado) receptors, or ATP-sensitive potassium (KATP) channels play a role in arteriolar dilations induced by muscle contraction, we used a cremaster preparation in anesthetesized hamsters in which we stimulated four to five muscle fibers lying perpendicular to a transverse arteriole (maximal diameter ~35-65 µm). The diameter of the arteriole at the site of overlap of the stimulated muscle fibers (the local site) and at a remote site ~1,000 µm upstream (the upstream site) was measured before, during, and after muscle contraction. Two minutes of 4-Hz muscle stimulation (5-15 V, 0.4 ms) produced local and upstream dilations of 19 ± 1 and 10 ± 1 µm, respectively. Nomega -nitro-L-arginine (10-4 M; NO synthase inhibitor), xanthine amine congener (XAC; 10-6 M; Ado A1, A2A, and A2B receptor antagonist), or glibenclamide (Glib; 10-5 M; KATP channel inhibitor) superfused over the preparation attenuated the local dilation (by 29.7 ± 12.7, 61.8 ± 9.0, and 51.9 ± 14.9%, respectively), but only XAC and Glib attenuated the upstream dilation (by 68.9 ± 6.8 and 89.1 ± 6.4%, respectively). Furthermore, only Glib, when applied to the upstream site directly, attenuated the upstream dilation (48.1 ± 9.1%). Neither XAC nor Glib applied directly to the arteriole between the local and the upstream sites had an effect on the magnitude of the upstream dilation. We conclude that NO, Ado receptors, and KATP channels are involved in the local dilation initiated by contracting muscle and that both KATP channels and Ado receptor stimulation, but not NO, play a role in the manifestation of the dilation at the upstream site.

microvasculature; adenosine; nitric oxide; adenosine 5'-triphosphate-sensitive potassium channels; metabolic control of blood flow


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN SKELETAL MUSCLE, CHANGES in metabolic rate lead to closely related changes in blood flow (2, 4, 6, 16, 24, 26, 33). This very close coupling between muscle metabolism and tissue perfusion involves the coordination and integration of multiple mechanisms of blood flow control, including direct local dilation of the terminal arteriolar microvasculature in response to contraction of adjacent muscle fibers (14, 28). This dilator response involves at least two components: 1) a local dilation that occurs at the site where the active muscle fibers and the arteriole are directly associated, at the site of contracting muscle fiber-arteriole overlap (11, 14, 25, 28), and 2) a dilation that is initiated at the site of contracting muscle fiber-arteriole overlap and is transmitted along the arteriolar wall to sites remote from the contracting muscle fibers (28). The local dilation induced by muscle contraction at 4 Hz is approximately twice the magnitude of the dilation that is transmitted along the blood vessel wall. A characteristic of this remote response is that it does not decay along the length of the vessel (28), suggesting that the mechanism of transmission of the dilation along the vessel wall may include a regenerative signaling component. From these observations, we have hypothesized that muscle contraction-induced arteriolar dilations are the product of at least two dilator pathways, one that produces a dilation only locally and another dilation that is produced locally and transmitted to remote arteriolar sites.

It is generally accepted that multiple dilator pathways contribute to the local dilations associated with active hyperemia and that the relative contribution of different dilators will depend on characteristics of the activity of the contracting muscle fibers, such as the rate or duration of muscle contraction, and the muscle fiber type. For example, the majority of evidence supports a role for adenosine (Ado) in functional hyperemia (10, 19, 30-32, 39), although its contribution varies with muscle fiber type (7, 31, 32, 39) and the magnitude of its contribution is dependent on stimulation and exercise parameters (21, 32) as well as contraction duration (30, 31, 39). A role for nitric oxide (NO) during changes in perfusion in contracting skeletal muscle has also been identified (e.g., Refs. 1, 13, 15, 17, 23), although not consistently (e.g., Refs. 20, 29, 34), a difference that may be linked to contraction frequency (1). Furthermore, although it is generally accepted that multiple dilator pathways are used to produce the dilations that occur during muscle contraction, the extent to which these pathways are activated simultaneously (vs., for example, having different local mediators predominating under different contraction conditions) is not established. In addition, our finding that local muscle contraction induces both local and remote dilations (28) raises the question of whether these responses reflect activation of different local metabolic dilator pathways and led directly to our hypothesis.

We therefore sought to test whether multiple dilator pathways act simultaneously to produce the local arteriolar dilation associated with muscle activity and whether all dilators that act locally are responsible for the production of the associated remote dilations. Because both Ado and NO have been identified as playing a role in active hyperemia in hamster cremaster muscle, we explored their contributions to these dilations by using an Ado receptor antagonist [xanthine amine congener (XAC), an A1, A2A, and A2B membrane receptor antagonist] and a NO synthase inhibitor [Nomega -nitro-L-arginine (L-NNA)]. The actions of Ado receptor activation (8, 10, 18, 22) and NO dilator capacity (27, 42) have been linked to ATP-sensitive potassium (KATP) channel function. At the microvascular level, KATP channels are functional in arterioles at rest (18), and both Ado and KATP channels have been identified as important in arteriolar dilations during muscle contraction (32, 36). Thus we also used a KATP channel inhibitor (glibenclamide) to explore a role for KATP channels in the manifestation of the local and remote dilations produced by 2 min of 4-Hz muscle contraction.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and Muscle Fiber Stimulation

Adult male Golden hamsters (100-130 g) were anesthetized with pentobarbital sodium (70 mg/kg ip) and tracheotomized. Catheters were placed in the left femoral artery and left femoral vein to monitor mean arterial pressure and for supplemental pentobarbital administration, respectively. Supplemental pentobarbital was given as needed during surgery and constantly infused (10 mg/ml saline, 0.56 ml/h) throughout the experimental protocol. Hamster esophageal temperature was maintained at 37°C via convective heat. The right cremaster was prepared for in situ microscopy as previously described (3, 28, 37). Briefly, the cremaster was isolated, cut longitudinally, separated from the testis and epididymis, and gently spread over a semicircular Lucite platform. The edges of the tissue were secured with insect pins to maintain tension but not stretch the muscle. Once exposed, the cremaster muscle was constantly superfused with a bicarbonate-buffered salt solution containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 30 NaHCO3, equilibrated with gas containing 5% CO2-95% N2 (pH 7.35-7.45). Cremaster muscle temperature was maintained at 34.0°C by heating the superfusion solution. After the surgery, all preparations were allowed to stabilize for 45-60 min before data collection.

The cremaster microvasculature was visualized by transillumination with a xenon lamp and with a Leitz Laborlux (or an Olympus BX50WI) microscope using a ×25 long-working-distance objective (numerical aperture 0.22). The microscope image was displayed via charge-coupled device camera (model MTI CCD72S, DAGE) on a Sony monitor and recorded with a 3/4-in. videotape recorder (model VO-9600, Sony). Final magnification of the site was ×1,420. Diameter measurements were reproducible to ±0.3 µm, which is ~1-2% of the expected diameter.

We observed transverse arterioles (Fig. 1) with maximal diameter between 35 and 65 µm. Transverse arterioles were identified as previously described (41). Briefly, capillaries were initially identified and traced back to their inflow arteriole. The inflow arteriole was traced back to its vessel of origin, and this arteriole (the branch) was in turn traced back to its arteriole of origin. If more than two branches arose from this vessel, it was considered a transverse arteriole [transverse arterioles typically have 3-8 branches (5)]. Our only selection criterion required that muscle fibers associated with the transverse arteriole run approximately perpendicular to the vessel. This architecture is common and can be found in all areas of the tissue preparation.


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  		Fig. 1.   Schematic (not drawn to scale) of the microvascular experimental site in the cremaster muscle, consisting of a transverse arteriole [third-order arteriole; from Wiedeman (43)] originating from a feed arteriole and diverging into many branch arterioles. Located on the schematic are the local site where the arteriole overlaps the contracting muscle fibers and the remote upstream site ~1,000 µm upstream of the contracting muscle fibers. Arrowhead A represents the site of micropipette placement for blocking upstream dilations directly at the upstream site (pipette A). Arrowhead B represents the site of micropipette placement for blocking the transmission pathway for the upstream dilation (pipette B).

Muscle fiber bundles (4-5 fibers) were stimulated directly by using a platinum wire microelectrode (tip diameter ~25 µm) placed onto muscle fibers running approximately perpendicular to the arteriole. The microelectrode was positioned at least 1,000 µm away from the chosen site of the arteriole-stimulated muscle fiber intersection. The ground electrode was placed in the superfusate around the outer rim of the tissue support pedestal. We stimulated muscle fibers via a square-wave pulse of 0.4 ms, 4-10 V at 4 Hz (model S48 stimulator, Grass, Quincy, MA). The overall state of vascular responsiveness in each preparation was assessed. Only the data collected on preparations that clearly displayed arteriolar constriction to 10% O2 and dilation to 10-4 M Ado were kept for further analysis (~4% of all preparations were discarded). After each protocol, maximal arteriolar diameters were recorded after at least 2 min of superfusion of the preparation with 10-4 M Ado. Sodium nitroprusside (10-3 M) was used to determine maximal arteriolar diameter in experiments involving XAC and glibenclamide superfusion.

Experimental Protocols

We used high magnification to optimize the resolution for diameter measurement; thus the local site (defined as the site where the arteriole crossed the contracting muscle fibers; Fig. 1) and the remote upstream site (a site 1,183 ± 70 µm upstream from the contracting muscle fibers, along the same arteriole, Fig. 1) could not be observed simultaneously but had to be observed in sequence. We have established previously that the sequence in which the sites are studied does not influence the observed responses (28). After the 45- to 60-min stabilization period, arteriolar diameter at the local site was continuously recorded for 1 min before muscle stimulation, during 2 min of muscle contraction, and for 2 min of recovery after stimulation (control local data). After 2 min, this protocol was repeated while the upstream site was observed (control upstream data).

Role for NO, Ado receptors, and KATP channels in the local dilation to muscle contraction. To investigate whether NO, Ado receptors, or KATP channels were involved in the local dilations induced by muscle contraction, we recorded the arteriolar diameter at the local site and at the upstream site, before, during and after 2 min of 4-Hz muscle contraction as described above (control data). We then applied either 10-4 M L-NNA (n = 9), 10-6 M XAC (n = 6), or 10-5 M glibenclamide (n = 8) over the whole preparation by adding each agent to the superfusate. These antagonist concentrations were selected on the basis of their ability to attenuate dilations elicited by supramaximal concentrations of the appropriate agonist (9; unpublished data). After 10 min of XAC or L-NNA application or 30 min of glibenclamide application, local and upstream diameter measurements were repeated during 2 min of 4-Hz muscle contraction. The superfusate was then switched back to control superfusate solution, and, after 20 min, washout diameters at the local and upstream sites were again recorded during 2 min of 4-Hz muscle stimulation (recovery measurements). Recovery data were collected to confirm that there was no time-dependent loss of dilator capacity and are not included in any analysis.

Role for Ado receptors and KATP channels in the manifestation of the dilation at the remote upstream site. Because L-NNA in the superfusate had no effect on the remote upstream response (see RESULTS), a role for NO in producing remote dilations was not tested further. To test whether Ado receptors or KATP channels were involved in the manifestation of the upstream response, after the collection of control data micropipettes were filled with either 2 × 10-6 M XAC (n = 7) or 2 × 10-5 M glibenclamide (n = 7). Antagonist concentrations in the micropipette were doubled to allow for possible dilution of pipette contents in the superfuste (12). The micropipette tip was placed at the vessel wall at the upstream observation site (pipette A, Fig. 1). These agents were applied over a very small region (~200 µm) of the arteriole via micropipette as previously described (12, 38). Briefly, micropipettes (tip diameter ~10 µm) were placed with the tip as close to the vessel wall as possible without touching the cremaster preparation itself. The observed arterioles were located either on the surface of the preparation or below from one to four muscle fibers. The micropipette was attached to a water manometer that, once pressurized, caused flow from the micropipette (12, 38). FITC-dextran (100 µM) was added to each micropipette solution so that brief epifluorescence could be used to verify flow from the micropipette and the flow direction of the micropipette contents. Care was taken to ensure that micropipette contents flowed approximately perpendicular to the arteriole under observation and that no other part of the arteriole or the contracting muscle fiber bundle were exposed to the micropipette contents. Care was also taken to ensure that any tissue movement that occurred during muscle stimulation did not impede flow out of the micropipette. After either 10 min of XAC application or 30 min of glibenclamide application to the upstream site, local and upstream diameter measurements were repeated during 4-Hz muscle contraction. Pipette flow was then stopped, and, after 20 min, recovery measurements were taken.

To test whether Ado and KATP channels were part of a common signaling pathway, we first compared the ability of Ado and KATP channel stimulation in inducing an upstream response by directly micropipette applying either Ado [10-4 M (n = 3) and 10-5 M (n = 10) to achieve submaximal dilations; results from the 2 groups were pooled] or 10-4 M pinacidil (n = 5) at a site equivalent to the local site in Fig. 1. Arteriolar diameter at the local site was continuously recorded 1 min before and during 2 min of micropipette application of either Ado or pinacidil and for 2 min of recovery after drug removal. In separate sets of experiments, we measured peak local arteriolar dilations to 2 min of micropipette application of either 10-4 M Ado (n = 9) or 10-5 M Ado (n = 7) before and after 30-min superfusion of 10-5 M glibenclamide. To ensure that glibenclamide was sufficient to block KATP channels, we measured the magnitude of the local dilation produced by 10-4 M pinacidil (n = 5) and then remeasured peak local dilation after 30 min of 10-5 M glibenclamide superfusion.

We tested whether Ado receptors or KATP channel activity was involved in transmission of the dilator signal along the vessel wall. After the collection of control data, either 2 × 10-6 M XAC (n = 7) or 2 × 10-5 M glibenclamide (n = 7) were applied via a micropipette to the arteriole in the signal transmission pathway between the local and the upstream sites, and ~500 µm from either the local or the remote upstream site (pipette B; Fig. 1). After either 10 min of micropipette application of XAC (n = 7) or 30 min of glibenclamide (n = 8) between the local and the upstream sites, local and upstream diameter measurements were repeated as described above during 4-Hz muscle contraction. Pipette flow was then stopped, and, after 20 min, diameters at the local and upstream sites were again recorded during 4-Hz muscle stimulation (recovery measurements). Our laboratory has shown elsewhere (12, 28) that use of this approach to locally apply blockers to the vessel wall is effective in blocking transmission of the remote dilation that is initiated by acetylcholine.

To ensure that the function of the skeletal muscle was not altered by superfusion of L-NNA, glibenlamide (and DMSO), or XAC, we isolated paired strips of hamster cremaster muscle, secured at each end with suture silk, and hung them vertically in a tissue organ bath between a fixed point and a force transducer. The muscles were immersed in superfusion solution and stimulated to contract for 2 min at 4 Hz (same stimulation parameters as in situ preparation). One muscle strip was then exposed to either 10-6 M XAC (n = 4) or 10-4 M L-NNA (n = 4) for 10 min while the other strip served as a time control. After 10 min, the muscle strips were stimulated again for 2 min at 4 Hz. In a like manner, pairs of muscle strips were also exposed to either 10-5 M glibenclamide (n = 4) or DMSO (control; n = 4) for 30 min and then stimulated to contract for 2 min at 4 Hz.

Materials

All reagents were obtained from Sigma Chemical (St. Louis, MO). Stock solutions of glibenclamide were made in 100% DMSO and further diluted in superfusate to final working concentrations of 10-5 M or 2 × 10-5 M (final DMSO concentrations 0.04 and 0.08%, respectively, to avoid direct effects of the solvent). Stock solutions of pinacidil were made in 100% DMSO and further diluted in superfusate to final working concentrations of 10-4 M and a final DMSO concentration of 0.4%. This concentration of DMSO alone had no effect on arterioles over the 2-min application period. All other reagents were dissolved in superfusate.

Data Analysis and Statistics

Only one arteriole per preparation was used to collect data. All experiments were videotaped and analyzed off-line. Arteriolar lengths and diameters were measured using video calipers generated by a modified video analyzer (model 321, Colorado Video), calibrated using a videotaped stage micrometer. All data are reported as means ± SE. Time course data were analyzed with a repeated-measures ANOVA. When the ANOVA identified significant differences, means between conditions were further analyzed by using Bonferroni post hoc analysis. A paired Student's t-test was used to compare all other control and experimental group means (40). Differences were considered significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Resting and maximal diameters of the vessels used in this study are summarized in Table 1. Neither L-NNA, XAC, nor glibenclamide in the superfusate altered resting arteriolar tone (Table 1). We also verified that the force of contraction and the rate of fatigue of isolated cremaster muscle strips in vitro were not altered by incubation with either 10-4 M L-NNA, 10-6 M XAC, or 10-5 M glibenclamide. When normalized to a percentage of the initial force developed, force developed by control muscles after 2 min of stimulation (197.6 ± 11.9%) was not significantly different from XAC (181 ± 14.1%), L-NNA (194.9 ± 11.9%), or glibenclamide (199.7 ± 18.4%) at the same time point. Therefore, there was no direct effect of these antagonists and inhibitors on skeletal muscle in experiments in which they were added to the superfusate.

                              
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  		Table 1.   Resting and maximal diameters of arterioles used in experiments investigating processes involved in the local and remote dilations to local 4-Hz muscle contraction

NO Is Implicated in the Local, but Not the Remote, Dilation to Muscle Contraction

Two minutes of 4-Hz muscle stimulation under the arteriole induced a large dilation locally and a dilation of approximately one-half of this magnitude upstream (controls, Fig. 2), consistent with previous work (28). L-NNA in the superfusate attenuated the local muscle contraction-induced dilation by 29.7 ± 12.7% while leaving the remote dilation unaffected (Fig. 2), suggesting that a component of the local dilation was dependent on NO synthase activity but that the remote dilation was via a NO-independent mechanism.


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  		Fig. 2.   Peak local and upstream dilations resulting from local 4-Hz muscle contraction under control conditions, with Nomega -nitro-L-arginine (L-NNA) in the superfusate, and during recovery from drug application (n = 9). Values are means ± SE. + Significant difference from local control, P < 0.05.

Ado Receptors Are Implicated in the Local Dilation and Also in the Initiation of the Remote Dilation

Both the local and remote dilations were attenuated in the presence of XAC in the superfusate (Fig. 3A) by 61.8 ± 9.0 and 68.9 ± 6.8%, respectively. Note that, in the presence of XAC, both the local and upstream dilations were attenuated proportionally, with the upstream dilation remaining at approximately one-half of the local dilation. This indicates that Ado mediates a component of the local response and, furthermore, suggests that the manifestation of the remote response may be dependent on this Ado-dependent component of the local dilation.


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  		Fig. 3.   Peak local and upstream dilations resulting from 4-Hz muscle contraction under control conditions, with xanthine amine congener (XAC) either in the superfusate (A; n = 6) or micropipette applied directly at the upstream site (B; n = 7) (pipette A, Fig. 1), and during recovery from drug application. Values are means ± SE. + Significant difference from local control, P < 0.05. * Significant difference from upstream control, P < 0.05.

It is also possible that Ado receptors were involved in the manifestation of the remote dilation directly and that this, too, was blocked by XAC in the superfusate. To test this possibility, we used a micropipette to apply XAC directly to the remote site during muscle contraction at the local site. Application of XAC directly to the remote site did not change the magnitude of the upstream dilation (Fig. 3B), indicating that the remote vasodilation was not directly mediated by Ado receptor activation. Application of XAC via micropipette to the arteriole midway between the local and remote sites confirmed that Ado receptors are also not involved in the transmission of the remote response, because the remote response during control conditions (12 ± 2 µm) was not different from that seen with XAC applied to the transmission pathway (11 ± 2 µm). It has been verified that XAC applied locally to the arteriole blocks ~70% of the local vasodilation produced by application of 10-4 M Ado (9).

KATP Channel Activity Is Implicated in the Local Response to Muscle Contraction and Also in the Remote Upstream Vasodilation

Glibenclamide in the superfusate attenuated contraction-induced dilations at both the local and the upstream sites by 51.9 ± 14.9 and 98.1 ± 6.4%, respectively. We confirmed that DMSO vehicle alone did not alter the metabolic dilation (n = 4); the dilation before DMSO in the superfusate (15 ± 4 µm) was not different from after 30 min of DMSO exposure (14 ± 4 µm). With glibenclamide present, the local and remote dilations were not attenuated proportionally, the upstream dilation being almost completely abolished (Fig. 4A). This suggests that KATP channel activity is involved in the local dilation and that it is also necessary for manifestation of the remote dilation upstream. This conclusion is further supported by the finding that direct application of glibenclamide via micropipette to the remote upstream site was able to attenuate the upstream dilation (Fig. 4B); in contrast, micropipete application of glibenclamide to the arteriole midway between the local and upstream sites did not affect the magnitude of the muscle contraction-induced upstream dilation (10 ± 2 µm in controls vs. 13 ± 3 µm with glibenclamide). Thus, although KATP channel activity is necessary for the production of both the local and remote dilations, our data suggest that it is probably not required for transmission of the dilator signal along the arteriolar wall.


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  		Fig. 4.   Peak local and upstream dilations resulting from 4-Hz muscle contraction under control conditions, with glibenclamide either in the superfusate (A; n = 8) or micropipette applied across the arteriole directly at the upstream site (B; n = 7) (pipette A, Fig. 1), and during recovery from drug application. Values are means ± SE. + Significant difference from local control, P < 0.05. * Significant difference from upstream control, P < 0.05.

Both Ado Receptors and KATP Channels Are Implicated in Production of Local and Upstream Dilations

We have shown previously that local micropipette application of the NO donor sodium nitroprusside is able to produce a local, but not a remote, dilation in these arterioles (28). We used micropipette application to the local site to verify that both Ado and the KATP channel opener pinacidil were able to produce both local and upstream dilations (Fig. 5). Ado (Fig. 5A) significantly increased arteriolar diameter, both locally and at the upstream site, from resting levels of 16 ± 1.0 and 19 ± 1 µm, respectively, to 28 ± 2 µm and 22 ± 1 µm, respectively (maximal diameters 43 ± 2 and 47 ± 23 µm, respectively). Pinacidil (Fig. 5B) was also able to significantly increase arteriolar diameter both locally and at the upstream site, from resting levels of 17 ± 1 µm locally and 18 ± 2 µm upstream to 30 ± 5 and 23 ± 2 µm, respectively (maximal diameter 37 ± 4 and 41 ± 3 µm, respectively). We explored this further by testing whether a locally produced dilation to Ado could be blocked by glibenclamide. Micropipette application of 10-4 M Ado resulted in a dilation of 80.3 ± 2.7% of maximal diameter (Fig. 6A) that could not be attenuated by incubation with 10-5 M glibenclamide. Similarly, 10-5 M Ado resulted in a dilation of 4 ± 1 µm (12.6 ± 3.6% of maximal diameter) that was not significantly attenuated in the presence of glibenclamide (4 ± 1 µm). Glibenclamide (10-5 M) could, however, significantly attenuate the dilation to pinacidil (Fig. 6B). The observation that a local dilation to Ado could not be attenuated by glibenclamide at a concentration known to significantly attenuate both a pinacidil-induced local dilation (Fig. 6B) and the contraction-induced response (Fig. 3A) suggests that, in this system, Ado is not acting via KATP channel activity.


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  		Fig. 5.   Time course of the changes in diameter of the local site and the remote upstream site during 2-min application of either adenosine (A; n = 13) or pinacidil (B; n = 5) at the local site. Distance between the local and upstream site for adenosine and pinacidil experiments was 789 ± 105 and 1,244 ± 48 µm, respectively. Values are means ± SE. * Significant change in vessel diameter from 0, P < 0.05.



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  		Fig. 6.   Peak local arteriolar dilations induced by adenosine (A) and pinacidil (B) in the absence and the presence of glibenclamide. Resting diameters for arterioles exposed to adenosine and pinacidil were 17 ± 1 and 17 ± 2 µm, respectively. Resting diameters for the adenosine and pinacidil groups in the presence of glibenclamide were 16 ± 2 and 13 ± 3 µm, respectively. Maximal diameters for the adenosine and pinacidil groups were 44 ± 4 and 37 ± 4 µm, respectively. Values are means ± SE. * Significant difference from the control pinacidil dilation in the absence of glibenclamide, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that, in the terminal microvasculature, the arteriolar dilation produced by muscle contraction is dependent on the simultaneous production of multiple dilator signals acting in concert and that not all of these necessarily induce a remote dilation upstream. We show that NO, Ado receptor activation, and KATP channel activity all contribute to the local dilation, and we provide evidence (discussed below) suggesting that neither NO nor Ado acts via a KATP channel-dependent pathway. We also show that the remote upstream dilation is initiated by mechanisms that are dependent on Ado and KATP channel activity (although likely acting via separate pathways) and is dependent on KATP channels, but not Ado, for its manifestation at the remote upstream site directly.

Role of NO

We have demonstrated that NO made a small, but significant, contribution to the arteriolar dilation induced by muscle contraction at the local site and, clearly, has no role in the transmission (the route by which the dilation travels from local to upstream sites) or the manifestation of the upstream dilation directly because L-NNA in the superfusate did not affect the upstream response. We conclude that NO was not acting locally via an action on KATP channels because we have shown elsewhere for these arterioles (28) that NO applied locally does not produce a remote dilation, whereas we show here (Fig. 5) that locally applied pinacidil produces both a local and an upstream response. Furthermore, our results indicate that, whereas glibenclamide attenuates both the local and upstream dilations to muscle contraction, L-NNA attenuates only the local response, leaving the remote dilation unaffected. Together, these findings argue that the NO-dependent component of the local dilation must be acting via a KATP channel-independent pathway. Our finding that NO is involved, although not prominently, in metabolic vasodilation in these small arterioles is consistent with the observation of Hester and colleagues (15), who found that contraction of the entire cremaster muscle produced dilation in large, but not small, arterioles. The arteriolar observation site under those contraction conditions would reflect a combination of both local dilation and dilation transmitted from remote sites. In contrast, the arteriolar dilation due to muscle fiber bundle stimulation in our study is strictly local, and, as we have shown previously (28), there is no contribution by dilations from remote sources. Thus it is possible that the component of the dilations resulting from whole muscle contraction that are dependent on NO is negligible, and only when the dilation to muscle contraction is split into various dilator components can a small effect of NO be observed. Parenthetically, we note that NO-dependent arteriolar dilations have been observed in mouse cremaster muscle during field stimulation (23).

Role of Ado Receptors

Ado made a significant contribution to the local dilation observed in our study. The actions of Ado have been linked to KATP channel activation in skeletal muscle (8, 10) as well as in other tissues (18, 22). However, we were unable to attenuate Ado-induced dilations with the concentration of glibenclamide that significantly attenuated both a pinacidil-induced local dilation (Fig. 6B) and the contraction-induced response (Fig. 3A). Thus we conclude that, in these vessels, Ado is unlikely to be acting via KATP channel activation to cause the local contraction-induced dilation.

We show that the upstream dilation to muscle contraction was also related to Ado receptor activation at the local site. If Ado were initiating solely a local dilation, we would expect inhibition by XAC to resemble that of L-NNA, attenuating only the local dilation and leaving the upstream dilation intact. However, XAC in the superfusate not only inhibited the local dilation but also attenuated the upstream response, whereas L-NNA did not. We know from previous work (28) that the upstream dilation is not simply a consequence of any dilation at the local site because many agents can produce local dilations of similar magnitude (e.g., acetylcholine, sodium nitroprusside, Ado, etc.) but result in very different upstream responses. Thus upstream dilations are not simply initiated by "environmental" factors resulting from the local dilation (i.e., local change in endothelial cell stretch, wall shear stress, pressure, etc.). It is also clear that the Ado-induced upstream dilation is unlikely to be part of the generalized regional dilations that have been described as a "network response" (35) because we have shown previously that muscle contraction 1,000 µm away from the upstream site, but not directly associated with the selected arteriole, does not cause dilation at the upstream site (28); thus no generalized dilations are being initiated. The present study shows, however, that the contraction-induced upstream dilation is dependent in some as yet unidentified way on the dilation initiated by Ado locally. This relationship (between the Ado-dependent component of the local contraction-induced dilation and the remote upstream response) is complex, as indicated by the apparently contrasting findings for the effect of XAC on the response to locally applied Ado vs. its effect on the contraction-induced responses. Blocking the Ado response to muscle contraction by using XAC produced a decrease in the local and upstream dilations of ~50% each. However, we also found that micropipette application of Ado directly over the arteriole produced a large dilation locally and only a very small upstream dilation. Given the inhibition of the upstream response that we observed with XAC during muscle contraction, we expected a greater dilation upstream when we directly applied 10-4 M Ado to the arteriole. It is unlikely that XAC is nonspecifically blocking some other process involved in the manifestation of the upstream response because XAC applied at midvessel or directly to the upstream site did not affect the upstream response. More likely, we infer that the signaling actions of Ado from muscle contraction are not the same as the actions of Ado applied directly from a micropipette. Our data indicate that there are multiple dilatory signals initiated by contracting muscle, and hence, if many dilatory signaling pathways are upregulated at once, the signaling mechanisms that produce dilation from Ado in this complex environment may be different from the dilator signaling initiated solely by locally applied Ado. Further experiments will be required to address this possibility.

Role of KATP Channels

KATP channels appear to play a major role in both the local and the remote dilations initiated by muscle contraction. KATP channel activation made a significant contribution to the local dilation observed in our study. As discussed above, we conclude that neither NO nor Ado receptor activation stimulates the opening of KATP channels in this vascular bed, which implies that another, as yet unidentified mediator must be responsible for the activation of KATP channels in this system.

We conclude that the upstream dilation was dependent on KATP channel function at the upstream site because glibenclamide, directly micropipette applied to the upstream site, was able to significantly attenuate the upstream dilation. Interestingly, glibenclamide applied to the transmission pathway (between the local and the upstream sites) did not affect the magnitude of the upstream dilation, implying that the mechanisms by which the dilation is traveling from one location to the other does not involve KATP channels, whereas these channels are involved in the actual manifestation of both the local and remote dilations. Restated, our data indicate that, when the transmitted signal reaches the upstream site, it initiates a signaling cascade involving KATP channels that results in dilation at the upstream site, whereas the process of transmission of the vasodilatory signal itself along the vessel wall may be independent of KATP channel activation.

The mechanisms through which contraction-induced dilator signals are transferred along the arteriolar wall remain unclear. Previous experiments (28) have shown that the gap junction uncouplers 18beta -glycerrhitinic acid and halothane do not inhibit conduction of the muscle contraction-induced dilation from the local to the upstream site, although both of these gap junction uncouplers are able to inhibit acetylcholine-induced conducted responses in the same preparation. This indicates that the mechanism of conduction of muscle contraction-induced dilations to remote sites along the arteriole differs significantly from the mechanisms that have been identified elsewhere for acetylcholine-induced conducted dilator signals. The present study suggests that neither NO, Ado receptor activation, nor KATP channels are involved in the transmission of the dilation to distant sites along the arteriole, although we show that KATP channels are involved in the manifestation of the dilation once it reaches its destination. Direct application of pinacidil was able to produce both a local and a remote dilation (Fig. 5B), indicating that KATP channel stimulation alone can promote a dilation upstream. Our data suggest that it is indeed likely that KATP channels are involved in initiation of the dilator signal that is transmitted along the vessel wall. We were unable to test this concept further because contraction-induced tissue movement at the site of initiation of this signal (i.e., at the local site) precluded use of a micropipette to locally apply glibenclamide to this region during muscle contraction.

Origins of the Dilator Signals and Role in Muscle Blood Flow

The specific source of the NO and the location of the Ado receptors and KATP channels involved in the dilations induced by muscle contraction could not be determined from this study because, during the studies involving muscle contraction (as noted above), we could not apply pharmacological agents to the local site with a micropipette because of the fact that muscle movement during contraction at this site often inhibited the flow of pipette contents and frequently broke the micropipettes. Therefore, all cell types (skeletal muscle cells, endothelial cells, and vascular smooth muscle cells) were exposed to superfused substances, and we could not determine the cellular sources of NO or the cellular sites of Ado receptors or KATP channels. We have, however, clearly identified a role for KATP channels on either vascular smooth muscle cells or endothelial cells in the contraction-induced dilation at the remote upstream site. Inhibition of the upstream contraction-induced dilation during upstream micropipette application of glibenclamide directly to the arteriole indicates that the cells of the arteriolar wall contain functional KATP channels that are an important step in the upstream dilator process.

In summary, we have demonstrated that multiple signaling pathways are involved in the terminal arteriolar dilations associated with muscle contraction. At the local site (the site of contracting muscle fiber-arteriole overlap), muscle contraction produces an arteriolar dilation that is dependent on NO, Ado receptors, and KATP channels. Neither NO or Ado receptors act through KATP channels to produce dilation, indicating that there is at least one more vasodilatory product produced by muscle contraction that dilates arterioles through the stimulation of KATP channels. This local muscle contraction also initiates a signal that is transmitted upstream to cause remote parts of the arteriole to dilate. We were not able to identify a role for NO, Ado, or KATP channels in transmission of this upstream signal along the vessel wall; however, the manifestation of the upstream dilation is directly dependent on KATP channels and indirectly dependent on Ado receptor activation at the local site.

Perspectives

In any muscle, the wide dispersion of the skeletal muscle fibers that comprise an individual motor unit means that a single arteriole may overlie fibers of different metabolic activity and will therefore receive a wide range of local signals as motor units are recruited. In this context, transmission of a local dilation along an arteriole to remote regions of the vessel enables the vessel to dilate as a single coordinated unit, despite the variations in metabolic demand likely to be occurring along its length. Therefore, we speculate that, during submaximal contractions (few motor units recruited), the contribution of Ado and KATP channel-dependent signaling pathways to alteration of tissue perfusion is much greater, because of their ability to induce upstream dilations, than that of NO, which produces only a very localized response. With increased motor unit recruitment, the contribution of NO may be greater because a larger number of muscle fibers spanning the length of the arteriole would contribute to the contraction and arteriolar dilations over a greater arteriolar length would result. Thus the contribution of NO to the redistribution of blood flow and tissue perfusion may be dependent on the recruitment pattern of motor units. The Ado and KATP channel-dependent signaling pathways in the terminal microvasculature appear likely to be much more effective at redistributing blood flow and tissue perfusion at any stimulation frequency because they not only produce a very local dilation but are also involved in producing the dilations transmitted to upstream sites, the dilations required to spread the response to larger regions. Thus, with the ability to spread the dilator response, contraction of a single motor unit will have the capacity to influence regional blood flow.


    ACKNOWLEDGEMENTS

We thank Patricia A. Titus for skilled technical assistance and Tasmia Duza and Joel Wojciechowski for helpful discussion and thorough review of the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-56574 and American Heart Association Fellowship 9820063T.

Address for reprint requests and other correspondence: I. H. Sarelius, Dept. Pharmacology and Physiology, Univ. of Rochester, Box 711, Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: ingrid_sarelius{at}urmc.rochester.edu).

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.

10.1152/ajpregu.00405.2001

Received 16 July 2001; accepted in final form 30 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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