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Am J Physiol Heart Circ Physiol 278: H1916-H1923, 2000;
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Vol. 278, Issue 6, H1916-H1923, June 2000

Remote arteriolar dilations in response to muscle contraction under capillaries

Kenneth D. Cohen, Bradley R. Berg, and Ingrid H. Sarelius

Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In hamster cremaster muscle, it has been shown previously that contraction of skeletal muscle fibers underlying small groups of capillaries (modules) induces dilations that are proportional to metabolic rate in the two arteriolar generations upstream of the stimulated capillaries (Berg BR, Cohen KD, and Sarelius IH. Am J Physiol Heart Circ Physiol 272: H2693-H2700, 1997). These remote dilations were hypothesized to be transmitted via gap junctions and not perivascular nerves. In the present study, halothane (0.07%) blocked dilation in the module inflow arteriole, and dilation in the second arteriolar generation upstream, the branch arteriole, was blocked by both 600 mosM sucrose and halothane but not tetrodotoxin (2 µM). Dilations in both arterioles were not blocked by the gap junction uncoupler 18-beta -glycyrrhetinic acid (40 µM), and 80 mM KCl did not block dilation of the module inflow arteriole. These data implicate a gap junctional-mediated pathway insensitive to 18-beta -glycyrrhetinic acid in dilating the two arterioles upstream of the capillary module during "remote" muscle contraction. Dilation in the branch arteriole, but not the module inflow arteriole, was attenuated by 100 µM Nomega -nitro-L-arginine. Thus selective contraction of muscle fibers underneath capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. The observed insensitivities to 18-beta -glycyrrhetinic acid, to KCl, and to Nomega -nitro-L-arginine suggest, however, that there are multiple signaling pathways by which remote dilations can be initiated in these microvessels.

microcirculation; cell signaling; conducted dilation; gap junctions


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH TISSUE BLOOD FLOW and capillary function are closely matched to metabolism in skeletal muscle. Increases in metabolism (e.g., in exercise) result in capillary recruitment, and it is established that control of capillary recruitment resides in arterioles upstream of capillaries (22). It is also established that changes in functional capillary density (capillary recruitment) can occur separately from changes in arteriolar conductance (14), with capillary recruitment occurring early in exercise, followed by continuing increases in blood flow through the muscle. This implies that the signaling mechanisms governing capillary recruitment may be distinct from those mechanisms that are responsible for dilating arterioles during increases in muscle metabolism.

In skeletal muscle, capillary networks are composed of capillary groups known as modules (3). Capillary networks are also identified as the recruitable unit in cremaster muscle (22). In a previous study (2), we showed that contraction of small groups of muscle fibers underneath capillary modules resulted in increased blood flow through that individual capillary module. The increased blood flow was a consequence of vasodilations occurring in arterioles upstream of the capillary module. Preliminary observations in that study suggested that dilations in the first arteriolar generation upstream from the capillary module (the module inflow arteriole) might be due to a signal conducted along the blood vessel wall via gap junction communication, i.e., a conducted vasodilation. This would constitute a mechanism whereby a signal arising at the capillary level results in dilation of the arterioles controlling capillary recruitment, i.e., vasodilator signals of capillary origin were transmitted to a remote arteriolar site upstream, resulting in increased perfusion of the downstream capillaries. The present study was undertaken to characterize these dilations and determine specifically whether a conducted vasodilation is indeed the mechanism underlying the remote dilation in the two arteriolar generations upstream from capillaries.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and Muscle Fiber Stimulation

Adult male golden hamsters (HSD Han:Aura; ~100-150 g) were anesthetized with pentobarbital sodium (70 mg/kg ip) with supplemental anesthesia maintained through a left femoral venous catheter. Body temperature was kept at 37 ± 0.5°C by radiant heating, and in most animals blood pressure was monitored through a left femoral arterial catheter. The right cremaster muscle was prepared and viewed as described elsewhere (1, 22). Briefly, the cremaster was cut longitudinally, separated from the testis, and gently spread over a semicircular Lucite platform. The muscle was secured with insect pins while taking care not to unduly stretch the muscle. The muscle was continuously superfused with warmed (34 ± 0.5°C) bicarbonate-buffered physiological salt solution containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 30 NaHCO3, and equilibrated with 5% CO2-95% N2 to maintain pH at 7.35-7.45.

After surgery was completed, the preparation was allowed to stabilize for 30-60 min. The cremaster muscle microcirculation was visualized using a Leitz Laborlux microscope with a Leitz ×25 long working distance objective. The microscope image was displayed via an RCA video camera or a CCD camera (Dage MTI CCD72S) on a Hitachi monitor. To assess preparation viability a brisk dilatory response to 100 µM topical adenosine and constriction to transient superfusate equilibration with 10% oxygen were verified in three randomly selected arterioles before the start of data collection. If preparations did not meet the above criteria, they were discarded.

The relevant anatomy for these protocols is schematized in Fig. 1. We chose module inflow and branch arterioles using previously described criteria (2, 22). Briefly, a transverse arteriole arising from a feed arteriole in a clear central region of the tissue was selected, and a module in a capillary network arising from the transverse arteriole was selected for the study. Capillary modules were identified by the network anatomy and the inability of these vessels to respond to topical adenosine application and increased superfusate oxygen. Capillary modules for study were also selected according to the orientation of the muscle fibers relative to the vasculature (generally perpendicular to the module inflow arteriole, see Fig. 1) and the ability to clearly visualize all relevant parts of the vasculature. Baseline flow or the number of capillaries in the module were never used as selection criteria. Many suitable sites are seen in each cremaster preparation.


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  		Fig. 1.   Schematic of experimental site. A transverse arteriole gives rise to a branch arteriole. Branch arterioles, from which capillary networks arise, give rise to daughter arterioles termed module inflow arterioles, which in turn feed groups of capillaries (modules). Number of modules in a capillary network is usually 5-8 (see Ref. 3). For clarity, only two modules are shown in this schematic. A, transmission pathway for module inflow arteriole. B, transmission pathway for branch arteriole.

To stimulate muscle fibers underlying capillary modules to contract, boroscillate glass tubes (OD/ID, 1.5/1.2) were pulled and beveled to make stimulating microelectrodes with tip diameters of ~2-3 µm and filled with 3 M NaCl. Alternatively, microelectrodes consisting of a 25-µm platinum/iridium (90:10%) wire connected by soldering to the silver wire in a microelectrode holder (World Precision Instruments) were used. The wires were enclosed in a glass pipette, and the tip was insulted with silicone (Dow Corning) so that ~3 µm of the platinum wire was exposed. Square-wave pulses of 0.4 ms and 5-30 V were used to stimulate small bundles of muscle fibers underlying specific capillary modules for 2 min at each of 2, 4, and 8 Hz. A recovery period of 3 min was allowed before proceeding to the next highest frequency. Microelectrodes were placed >= 1,000 µm away from the module and network of interest to minimize any possible direct electrical effects of electrode activity on the vasculature.

Experimental Protocols

Upstream arteriolar dilations in response to stimulation of muscle fibers underlying capillary modules. In all experiments, diameters were measured in module inflow and branch arterioles (i.e., in the two arteriolar generations upstream of the selected capillary module) at rest and in response to muscle contraction underlying the capillary module (Fig. 1). Module inflow and branch arteriole observation sites were always >= 500 µm away from the capillary module and contracting muscle fibers; we refer to this as "remote" muscle contraction.

To characterize the dilation of the module inflow arteriole during remote muscle contraction, test substances were directed, via micropipette (~10 µm tip diameter), to flow across the signal transmission pathway between the contracting muscle fibers and the observation site. Module inflow arteriolar diameters were measured near their origin from the parent branch arteriole. In separate sets of experiments, to characterize the dilations of branch arterioles during remote muscle contraction, test substances were applied via micropipette to the module inflow/branch arteriolar junction (i.e., across the transmission pathway). Branch arterioles were observed near their origin from transverse arterioles. Vessels were exposed to micropipette contents for 10-20 min (depending on the test substance) before the muscle contraction protocol was repeated. Ejection of micropipette contents in all experiments was achieved by raising the pressure in a water manometer system connected to the pipette (9, 16). Micropipette exposure sites during remote muscle contraction were always >= 200 µm downstream of the observation sites. Fluorescein isothiocyanate dextran (100 µM) was added to the micropipette contents, and brief epifluoresence was used to verify that the pipette contents were flowing only over the required region of the microvasculature and not flowing over the stimulated muscle fibers or observation sites. The micropipette was then turned off, the tissue was allowed to recover from test substance exposure for 20-30 min, and the muscle contraction protocol was repeated (recovery).

Characterization of upstream arteriolar dilations. To determine whether the dilations in module inflow and branch arterioles during remote muscle contraction were due to a conducted dilation involving gap junctions, we used separate sets of animals and micropipette exposure to one of three nonspecific gap junction uncouplers. We established previously that sucrose in superfusate (600 mosM) blocks dilation in the module inflow arteriole during remote muscle contraction (2). With this in mind, in separate sets of experiments, we applied either halothane (0.07%) or 18-beta -glycyrrhetinic acid (beta -GA, 40 µM; Refs. 5 and 27) via a micropipette to block module inflow arteriole dilations to remote muscle contraction. Furthermore, in separate sets of experiments, we used halothane, hypertonic sucrose, or beta -GA to attempt to block dilations in the branch arterioles during remote muscle contraction. The response of the module inflow arteriole was unaffected by the application of gap junction uncouplers to the branch transmission pathway (data not shown). Because beta -GA did not block upstream dilations (see RESULTS), we verified that beta -GA was reaching the tissue by initiating a conducted dilation using ACh (100 µM) applied to module capillaries while observing the diameter of the module inflow arteriole before and during beta -GA application to the transmission pathway. In some experiments, micropipette application of hypertonic sucrose and halothane produced a small dilation that spread ~200 µm beyond the site of application. We have shown elsewhere (9) that local vasodilation does not prevent transmission of a conducted response: in the present study we used 10-5 M sodium nitroprusside-induced local vasodilation (data not shown) to confirm that the upstream response was not affected by local dilator effects of the applied drugs.

In an earlier study (2), we found no role for perivascular nerve communication in dilation of the module inflow arteriole during remote muscle contraction. To test for involvement of a perivascular neural pathway in mediating the upstream dilations in the branch arteriole, we applied the sodium channel antagonist tetrodotoxin (TTX, 2 µM) via a micropipette to the transmission pathway for this vessel during remote muscle contraction.

We and others (7, 9) have demonstrated an involvement of nitric oxide (NO) in conducted responses in small arterioles. We have also shown that, during remote muscle contraction, wall shear stress increases in module inflow arterioles but remains at baseline values in branch arterioles (2), suggesting that there could be differing involvement of flow-sensitive mechanisms in the branch verses the module inflow arteriole. Therefore, in this set of experiments, we tested whether NO synthase (NOS) inhibition affected the dilations of module inflow and branch arterioles during remote muscle contraction using micropipette application of the NOS antagonist Nomega -nitro-L-arginine (L-NNA, 100 µM) to the transmission pathways of either arteriole.

Segal and Duling (18) found that a depolarizing solution of KCl could significantly attenuate a conducted dilation initiated by ACh, suggesting that a conducted hyperpolarization is a component of this conducted dilation. We sought to determine whether a component of the dilation in the module inflow arteriole was due to conduction of a hyperpolarization along the vessel wall. We applied a depolarizing solution of 80 mM KCl via a micropipette to the module inflow arteriole transmission pathway while observing diameter changes in this arteriole during remote muscle contraction.

Materials

Sucrose solution consisted of control superfusate plus the required amount of sucrose to reach 600 mosM. Halothane and ACh solutions were made up fresh daily in control superfusate. High KCl solution was made by substituting KCl for NaCl in control superfusate. L-NNA and adenosine were made as 10-2 M stock solutions in distilled water and diluted to final concentrations in control superfusate. beta -GA was made daily in control superfusate using 0.04% DMSO as a solvent. We have determined (data not shown) along with others (12) that this concentration of DMSO has no effect on vessel tone and reactivity to agonists or muscle stimulation. DMSO was purchased from J. T. Baker (Phillipsburg, NJ), and halothane was purchased from Ayerst (Philadelphia, PA). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Data Analysis and Statistics

Resting arteriolar diameters were measured as the average diameter in the 15-30 s before each bout of muscle fiber stimulation (baseline diameter), and responses to muscle contraction were measured in the first 10 s after the cessation of stimulation at each frequency. In some experiments time course data were taken at 10-s intervals throughout 2 min of 4-Hz muscle stimulation where possible, i.e., when tissue movement during muscle contraction did not obscure focus of the observed arteriolar site. All data are reported as means ± SE and expressed as a percentage of baseline diameter or as absolute values (in µm). Maximal arteriolar diameter was measured at the end of each experiment by exposing the tissue to 100 µM adenosine in the superfusate for 5 min. One observation was made in each animal. Group means were compared using a one-way or repeated measures ANOVA or paired Student's t-test as appropriate (21). If the ANOVA showed a significant effect among stimulation frequencies or between conditions, group means were compared using a post hoc Newman-Keuls multiple comparison test, with all differences being significant at P <=  0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dilation of Upstream Arterioles in Response to Remote Muscle Contraction

Mean resting and maximal diameters of module inflow arterioles were 8.4 ± 0.7 and 21.1 ± 1.1 µm, respectively. For branch arterioles, mean resting diameter was 15.0 ± 1.0 µm, and maximal diameter was 29.4 ± 1.2 µm. In pooled data from all experiments, remote stimulation of muscle fibers at 2, 4, and 8 Hz induced a dilation of 131.9 ± 4.7, 165.2 ± 6.3, and 194.6 ± 8.2% baseline, respectively, in module inflow arterioles (n = 34) under control conditions. Branch arterioles dilated to 115.7 ± 2.1, 131.9 ± 3.4, and 145.6 ± 5.3% baseline diameter at 2, 4, and 8 Hz of remote muscle contraction, respectively (n = 25) under control conditions. All increases are significant. Figure 2 shows the time course of diameter changes in module inflow and branch arterioles during remote muscle contraction at 4 Hz. Both module inflow and branch arterioles exhibited dilations that reached a steady-state plateau at ~60 s.


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  		Fig. 2.   Time course of diameter changes in module inflow arterioles (n = 17) and branch arterioles (n = 12) during 4-Hz remote muscle contraction. All increases are significant except module inflow arteriole dilation at 10 s.

Dilation of Upstream Arterioles in Response to Remote Muscle Contraction During Application of Gap Junction Inhibitors

Our previous observation that hypertonic sucrose abolishes dilation in the module inflow arteriole during remote muscle contraction (2) suggested that this response might be transmitted via gap junctions. In the present study we tested other gap junction inhibitors, halothane and beta -GA. Dilations in the module inflow arteriole at 2, 4, and 8 Hz were completely abolished by application of halothane (Table 1), supporting a role for gap junctions in transmission of this response. Interestingly, however, beta -GA did not block dilations in the module inflow arteriole (Table 1). We tested whether beta -GA had the capacity to block a conducted response in the module inflow arteriole that was initiated by micropipette application of ACh to module capillaries. beta -GA was effective in significantly attenuating a conducted dilation to ACh (144.2 ± 7.8% baseline diameter dilation under control conditions compared with 107.4 ± 8.7% dilation in the presence of beta -GA; n = 7, P <=  0.05).

                              
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  		Table 1.   Diameter changes of module inflow arterioles with application of halothane and 18-beta -glycyrrhetinic acid during remote muscle stimulation

Figure 3, A and B, shows that hypertonic sucrose and halothane also significantly inhibited branch arteriolar dilations during remote contraction of skeletal muscle underneath capillary modules. Control dilations to 112.4 ± 4.1, 124.3+7.4, and 137.7 ± 7.4% baseline diameter at 2, 4, and 8 Hz, respectively, were completely abolished by micropipette application of hypertonic sucrose, and control dilations to 115.5 ± 4.2, 129.9 ± 2.9, and 146.7 ± 8.0% baseline diameter at 2, 4, and 8 Hz were significantly attenuated by halothane. However, as also seen in the module inflow arteriole, beta -GA did not attenuate the contraction-induced dilation in the branch arteriole (Fig. 3C).


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  		Fig. 3.   Responses of branch arterioles after 2 min of remote muscle contraction under control conditions, in presence of hypertonic sucrose (A, n = 5), halothane (B, n = 7), 18-beta -glycyrrhetinic (beta -GA; C, n = 4), or tetrodotoxin (TTX; D, n = 7), and after recovery. Bars indicate means ± SE. *Significant difference from control at that same frequency.

Dilation of Branch Arterioles in Response to Remote Muscle Contraction During Application of TTX

In an earlier study (2), we showed that micropipette application of TTX to the transmission pathway did not affect dilation in the module inflow arteriole, suggesting that the dilation in this vessel is independent of a neural mechanism. To address if perivascular nerves play a role in dilating the branch arteriole during remote muscle contraction, we applied TTX to the transmission pathway and observed the branch arteriolar response. Figure 3D shows that TTX failed to attenuate this dilation. Effectiveness of TTX was verified as described previously (2).

Dilation of Upstream Arterioles in Response to Remote Muscle Contraction During Application of L-NNA

Figure 4A shows that L-NNA applied via micropipette to the transmission pathway did not block dilation in the module inflow arteriole compared with control dilations. In contrast, Fig. 4B shows that local application of L-NNA via micropipette to the module inflow/branch arteriolar junction (the transmission pathway for branch dilation) attenuated branch arteriolar dilations, an overall significant reduction of 46.8 ± 18.3%.


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  		Fig. 4.   Responses of module inflow (A, n = 8) or branch arterioles (B, n = 7) after 2 min of muscle contraction under control conditions, in presence of Nomega -nitro-L-arginine (L-NNA), and after recovery. Bars indicate means ± SE. *Significant difference from control at that same frequency.

Dilation of Module Inflow Arterioles in Response to Remote Muscle Contraction During Application of KCl

We sought to determine whether a conducted hyperpolarization was a component of the dilation seen in the module inflow arteriole using KCl applied via a micropipette as described in METHODS. As can be seen in Fig. 5, control dilations to 133.9 ± 13.4, 174.2 ± 21.0, and 193.0 ± 25.6% baseline diameter at 2, 4, and 8 Hz were not significantly different during KCl application to the transmission pathway (129.0 ± 8.3, 157.4 ± 19.1, and 227.3 ± 46.7% baseline at 2, 4, and 8 Hz).


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  		Fig. 5.   Responses of module inflow arterioles (n = 5) after 2 min of muscle contraction under control conditions, in presence of 80 mM KCl, and after recovery. There are no significant differences between conditions at any frequency.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The findings in this study suggest that both module inflow and branch arterioles dilate during remote contraction of muscle fibers underlying capillary modules through a signal likely transmitted via gap junctions. We conclude that signal transmission involves gap junctions because the dilations are inhibited by the nonspecific gap junction uncouplers hypertonic sucrose and halothane and are unaffected by neural blockade. However, the conducted signal is more complex than a simple electrotonic spread of membrane potential change such as has been identified for conducted responses initiated by ACh. This is suggested by 1) the observation that beta -GA, unlike hypertonic sucrose and halothane, does not block dilations in either arteriole; 2) by the observation that KCl does not affect the response in the module inflow arteriole; and 3) by the different responses of module inflow arterioles compared with branch arterioles during L-NNA application to the transmission pathway.

Application of the gap junction uncouplers 600 mosM sucrose or halothane abolished dilation in the module inflow and branch arterioles during remote muscle contraction. However, when we tested the effects of the gap junction uncoupler beta -GA on both arterioles, it failed to block remote dilations. We verified that beta -GA was reaching the tissue by showing that it attenuated a conducted dilation in the module inflow arteriole during ACh application to capillaries. One interpretation of these observations is that muscle contraction and ACh utilize different signaling pathways for conducted arteriolar dilations. Signaling pathways for conducted responses are also known to differ from tissue to tissue (20). Gap junction inhibitors may have differential effects on different connexins (6, 10), and gap junctions are known to exhibit various conductance states (23). This could allow selective movement of specific signaling molecules while preventing movement of others. To date, the gap junction proteins connexins 37, 40, and 43 have been identified in blood vessels (13, 28). Thus beta -GA may block a specific gap junction pathway (e.g., connexin 43) that is important in conducting dilator signals to ACh application but that is not involved in conduction of signals that are initiated by muscle contraction. An alternative interpretation is that different signaling molecules could be traversing the gap junctions and that beta -GA could be targeting the action of one such molecule. Either way, our data support the conclusion that there are multiple gap junction-dependent transmission pathways in these microvessels. This conclusion is dependent on the assumption that the primary effect of each of the three blockers used in these experiments was to modulate gap junction transmission. Because each of these agents exhibits other nonspecific effects in addition to uncoupling gap junctions, we cannot eliminate the possibility that non-gap junctional-mediated signaling contributed to the responses we observed.

The branch arteriole is the parent vessel of the module inflow arteriole, but despite the fact that they are directly connected, our data suggest that these two vessels respond differently during remote muscle contraction. Blockade of NO production by micropipette application of L-NNA did not block dilation in the module inflow arteriole but attenuated dilations in the branch arteriole (Fig. 4), implying that the vasodilator signal in the branch includes an NO-dependent component. We note that in earlier work (2) in which we measured wall shear stress at the end of 2 min of remote muscle contraction, wall shear stress increased in the module inflow arteriole with increases in contraction frequency but remained unchanged from baseline in the branch. Maintenance of unchanged wall shear stress during vasodilation has been identified as a characteristic of endothelium-mediated flow-dependent arteriolar responses (15). The observation that wall shear stress remained unchanged in the branch suggests, therefore, that the branch response might be sensitive to flow and by implication suggests that it might include an NO-dependent component in its response. Thus we speculate that there may be a component of the transmission pathway signal in the branch arteriole that is NO dependent; involvement of local paracrine signaling in a gap junctional-mediated pathway has been proposed in other cell systems (4).

Previous studies (18, 24-26) have indicated that conducted responses characteristically include membrane potential changes; in those studies short pulses (<= 5 s) of agonist were used to initiate the conducted response. A conducted dilation in response to a short duration of ACh applied via a micropipette can be attenuated by micropipette application of KCl to the transmission pathway (18), suggesting involvement of conducted hyperpolarization. In our study, the remote response that we report follows 2 min of muscle contraction. Our finding that this response is not blocked by application of KCl to the transmission pathway suggests that membrane potential changes are not a primary characteristic of this response and again supports the idea that the transmission pathway along the vessel wall during remote muscle contraction is different from that initiated by ACh. An early membrane potential-dependent component in our study has not been ruled out, because vessel movement during muscle stimulation complicates diameter measurements at early time points in our stimulation periods.

In many studies, micropipette application of different agonists has been used to initiate conducted responses in arterioles (8, 17-19, 24-26). Conducted dilations in the present study are in response to remote muscle contraction underneath groups of capillaries. Muscle contraction initiates these dilations at the capillaries, where smooth muscle is absent, thus it appears likely that endothelial cells are the primary pathway for transmission of the signal. We speculate that the dilator signal(s) initiated in the capillary endothelium by remote muscle contraction is most likely transmitted upstream along the vessel wall to the module inflow and branch arterioles via the endothelial cell layer, because this cell layer is thought to be better coupled morphologically compared with vascular smooth muscle in arterioles (11, 13).

During remote muscle contraction, the module inflow arteriole dilated to ~200% of baseline diameter at 8 Hz. This is proportionately larger than dilations seen in response to conducted responses initiated by agonists in arterioles (17-19, 24-26). There are at least two possibilities that might account for the greater magnitude of this response. First, as discussed earlier, it could reflect differences in the signaling mechanism underlying transmission of an ACh-induced remote dilation compared with that induced by muscle contraction. A second possibility is that the response reflects the sum of many downstream signals arising from each of the module capillaries. It is known that conducted responses from two arterioles can be summed in a common upstream vessel (19). We established that conducted signals initiated at capillaries can also be summed, because application of ACh to increasing numbers of capillaries in a module via micropipette produces a proportionate increase in dilation of the module inflow arteriole (data not shown). Thus the module inflow arteriole does indeed have the capacity to produce a greater dilation when a greater fraction of the downstream capillaries are stimulated; determination of whether this mechanism underlies the responses we describe is beyond the scope of the present work.

Recruitment of blood flow, initiated at the capillaries by increased muscle metabolism, may be a physiologically relevant mechanism by which blood flow is matched to metabolic demand. Contraction of muscle fibers underlying capillary modules dilates the two upstream arteriolar generations; it is known that capillary recruitment and capillary blood flow distribution are regulated by these arterioles (22). These conducted dilations affect only the arterioles upstream from the specific area of increased metabolism, i.e., contraction of muscle fibers underlying a specific capillary module does not cause dilations of module inflow or branch arterioles not associated with the capillary module overlying the active muscle fibers (2). Thus the upstream dilator response is directly coupled to changed muscle metabolism underlying specific modules, and the local nature of this response allows flow to be directed solely to the region of increased metabolic activity.

In summary, we show that selective contraction of small groups of muscle fibers underneath specified groups of capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. This conducted dilation shares many common characteristics with conducted responses produced by agonist application but with some exceptions. The dilations in the module inflow and branch arterioles were not blocked by the gap junction uncoupler beta -GA, and a depolarizing solution of high KCl did not block the dilation of the module inflow arteriole. Also, the conducted dilation in the branch arteriole is attenuated by L-NNA, whereas L-NNA does not interfere with the dilation in the module inflow arteriole. Together, these findings suggest that there are multiple signaling pathways by which conducted dilations can be initiated in these microvessels.


    ACKNOWLEDGEMENTS

We are very grateful to Dr. Coral L. Murrant for critical reading of the manuscript, helpful discussions, and for generation of Fig. 1. We also thank Patricia A. Titus for technical assistance.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-56574.

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. §1734 solely to indicate this fact.

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

Received 11 June 1999; accepted in final form 21 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baez, S. An open cremaster preparation for the study of blood vessels by in vivo microscopy. Microvasc Res 5: 384-394, 1973[ISI][Medline].

2.   Berg, BR, Cohen KD, and Sarelius IH. Direct coupling between blood flow and metabolism at the capillary level in striated muscle. Am J Physiol Heart Circ Physiol 272: H2693-H2700, 1997[Abstract/Free Full Text].

3.   Berg, BR, and Sarelius IH. Functional capillary organization in striated muscle. Am J Physiol Heart Circ Physiol 268: H1215-H1222, 1995[Abstract/Free Full Text].

4.   Charles, A. Intercellular calcium waves in glia. Glia 24: 39-49, 1998[ISI][Medline].

5.   Davidson, JS, Baumgarten IM, and Harley EH. Reversible inhibition of intercellular communication by glycyrrhetinic acid. Biochem Biophys Res Commun 134: 29-36, 1986[ISI][Medline].

6.   Dhein, S. Gap junctions in the cardiovascular system: pharmacological and physiological modulation. Trends Physiol Sci 19: 229-241, 1998.

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Am J Physiol Heart Circ Physiol 278(6):H1916-H1923
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