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(Circulation Research. 1995;77:695-701.)
© 1995 American Heart Association, Inc.

Articles

L-Arginine–Induced Conducted Signals Alter Upstream Arteriolar Responsivity to L-Arginine

Mary D.S. Frame, Ingrid H. Sarelius

From the Department of Biophysics, University of Rochester (NY).

Correspondence to Mary D.S. Frame, PhD, Department of Biophysics, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642.

	Abstract

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Abstract Our purpose was to determine whether L-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg IP) hamsters (N=53). The upstream branch arterioles dilated significantly to locally applied L-arginine (100 µmol/L pipette concentration) only if the downstream branches (1400 µm away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, -3.8±1.5% (of baseline); third, +58.1±27%; first, +92±26% (n=5); with exposure order upstream to downstream: first branch, -0.4±3%; third, +5±11%; last, -5.6±7.5% (n=4). Thus, downstream preexposure to L-arginine altered the responsivity upstream to locally applied L-arginine. Downstream-applied L-arginine also induced a conducted vasodilation (+17.8±2.8%; n=14) 1327±166 µm upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or N-nitro-L-arginine (L-NNA, 100 µmol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of L-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1±2.6%, n=8) was also completely blocked by sucrose, halothane, or L-NNA. The change in responsivity upstream to locally applied L-arginine was not seen in the absence of a conducted vasodilation or when the conducted signal pathway was blocked after the conducted vasodilation was observed, and it could be triggered by a conducted response to acetylcholine as well as to L-arginine. Thus, the change in local responsivity upstream requires an ongoing conducted signal from downstream. Conducted signals likely play a dynamic role in the regulation of vascular responsivity within a defined microvascular region.

Key Words: conducted response • gap junction • vascular communication • flow regulation

	Introduction

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Vascular responses in the microcirculation are spatially predictable in response to metabolic stimuli, adrenergic stimuli at low concentrations,^{1 2} and agents acting through specific dilatory pathways.³ Coordination of responses is likely the function of a vascular communication system. One proposed form of vascular communication is conducted dilation, which is initiated by many agents; one of the best described is acetylcholine.^{4 5 6 7 8 9}

Flow distribution is likewise spatially predictable at the microvascular level, with more cell flow traveling into the upstream branches of a transverse feed arteriole compared with the downstream branches of the same vessel.^{3 10} Inhibition of both L-arginine and prostaglandin pathways significantly disrupts this spatially organized cell flow distribution.³ These observations suggest that the control of spatial differences in microvascular response and in flow distribution might be linked to L-arginine and/or prostaglandin metabolism.

We undertook the present study to determine whether a mechanism linked to L-arginine is involved in vascular communication. L-Arginine and prostaglandin pathways are each well-documented dilatory mechanisms.^{11 12 13 14 15} We chose to study L-arginine because preliminary experiments had suggested that it was involved in relaying information between downstream and upstream locations. We show that L-arginine exposure at a downstream site initiates conducted signals that control arteriolar responses upstream in two ways. First, L-arginine applied downstream by micropipette initiates a conducted vasodilation upstream, without inducing a local dilation at the downstream site of L-arginine exposure. Second, the ability of the upstream site to respond to L-arginine is significantly altered after downstream preexposure to L-arginine or acetylcholine. After the conducted vasodilation, a significant and persistent dilation to locally applied L-arginine is obtained at the upstream site; this action is not observed before the conducted response.

	Materials and Methods

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Preparation
Adult male Golden hamsters (N=53; 120±10 g; HSD:Syr) were anesthetized with pentobarbital sodium (70 mg/kg IP), tracheostomized, and maintained on a constant infusion of pentobarbital sodium (10 mg/mL at 0.56 mL/h) via a femoral venous catheter. Deep-body temperature was maintained between 37°C and 38°C. Mean arterial pressure was monitored via a left femoral arterial catheter and was constant for each preparation. The right cremaster muscle was prepared for in vivo microcirculatory observations, as previously described.^{1 16} This preparation was superfused with a bicarbonate-buffered physiologic salt solution (control superfusate) consisting of (in mmol/L) 132 NaCl, 9.4 KCl, 4 CaCl₂, 2.4 MgSO₄, and 20 NaHCO₃ (pH 7.4±0.5 at 37°C) and was observed by in vivo microscopic techniques.^{1 16}

Observation Site
The observation site was a transverse (feed) arteriole and its branches (Fig 1). This site was located in each preparation as described previously.^{1 16 17} The mean (±SD) interbranch lengths and the total length of the feed vessel are given in Fig 1.

View larger version (14K): [in this window] [in a new window]   Figure 1. Schematic of the experimental test site. This site was a transverse arteriole (third order, Wiedeman, Reference 18) and its branches, each a fourth-order arteriole. The interbranch segment lengths and the total length of the transverse arteriole are given in the figure (mean±SD, N=53 animals).

Experimental Protocol
During a 60-minute stabilization period, the presence of vasoactive tone (brisk dilation to topical 10^-4 mol/L adenosine) and oxygen sensitivity (constriction to 10% oxygen, equilibrated in the superfusate) was confirmed in three arterioles (two randomly chosen arterioles and one at the test site).

In all experiments, micropipettes were used to apply pharmacological agents to the external surface of localized areas of small arterioles, as described previously.^{1 3} The micropipette was placed within 15 to 25 µm of the vessel wall. The arteriolar diameter was recorded continuously for 5 minutes after initial pipette placement to verify a stable baseline diameter and then continuously during a 5-minute exposure to the pipette contents, and for 5 minutes after to verify vessel recovery. Flow out of the pipette was achieved by raising the driving pressure within the micropipette from -2 to +30 cm H₂O by switching between two separate manometer reservoirs. The calculated concentration at the vessel wall is one half of the concentration in the pipette, as described previously.¹ A tracer of 100 µmol/L FITC-dextran (4000 mol wt, Sigma Chemical Co) was added to each of the pipette solutions, and brief epifluorescence was used to verify exposure of the arteriole to the pipette contents; care was taken with pipette placement to ensure that only the test arteriole was exposed. Exposure of arterioles to control superfusate from the pipette produces diameter changes of <5%.^{1 3}

Washout and Diffusion Times
To estimate the time from initial downstream L-arginine exposure until the onset of conducted dilation upstream, we estimated the times for pipette washout and for diffusion from the pipette flow stream to the arteriole. The holding pressure in the pipette was negative (-2 cm H₂O, ie, the superfusate was sucked into the pipette tip). The time from when the pressure in the pipette was switched to +30 cm H₂O until verification of flow out of the pipette by epifluorescence of the FITC was 5 to 7 seconds (washout time). The pipette contents were observed to flow across the arteriole, but this flow path was a stream within the flowing superfusate, 15 to 25 µm above the test arteriole. The distance from the pipette to the vessel, usually between 15 and 25 µm, and the diffusion coefficient of 10^-6 cm²/s yielded diffusion times to the arteriole of 2.25 seconds or 6.25 seconds. Thus, the minimum delay time was 7 to 10 seconds from the time the manometer was switched to the high-pressure reservoir until the arteriole could first be exposed to the pipette contents. Thus, the responses we observed were too fast to be due to diffusion from the pipette to the observation site (430 minutes over a distance of 1600 µm), and the pipette contents flowed in the wrong direction for the response to be due to convection, as verified by the FITC flow path.

Protocol 1: Local Responses
Individual branch arterioles were exposed to L-arginine (100 µmol/L pipette concentration) and the response of that branch was observed. The first, third, and last branch arterioles were tested sequentially in the same preparation, alternating the exposure sequence to start either upstream or downstream; each vessel was exposed only once. The same pipette was used to apply L-arginine to each arteriole in a single preparation. In additional experiments, either the first or last branch was tested in a single preparation. The vessel was exposed three times; the pipette was raised and then lowered between exposures to parallel the sequence of pipette relocation used above.

Protocol 2: Conducted Vasoregulation
While observing the first branch, a downstream branch was exposed to L-arginine, D-arginine, or acetylcholine (100 µmol/L pipette concentration for each). After the first minute, the downstream exposure site was briefly observed to verify exposure. Following the recovery period, the same pipette was moved and used upstream. The first branch was then exposed to the pipette contents and observed. Thus, both the conducted and upstream local responses were obtained for the same animal in each preparation. Alternately, the upstream site was exposed to L-arginine (100 µmol/L pipette concentration) and the downstream site was observed to evaluate the directionality of the conducted response.

Inhibitors of Conducted Signals
A second pipette was placed midway between the downstream exposure site and the upstream observation site. This pipette contained high-osmolar sucrose (control suffusate plus sucrose to equal 600 mOsm), halothane (0.0345%), or adenosine (10^-4 mol/L as a control for the local effect of the inhibitors). (Adenosine does not interfere with conducted responses,¹⁹ while high-osmolar sucrose and halothane are nonspecific inhibitors of gap junction signals.^{20 21} ) The middle of the feed vessel was exposed to sucrose, halothane, or adenosine for 5 minutes before downstream exposure to L-arginine or (separately) D-arginine. Flow out of this middle pipette was verified, and the local response to the blocker was obtained. Protocol 2 was completed first with and then without exposure to the blockers, with a washout period of 10 minutes. In preliminary experiments, 10 minutes was sufficient to demonstrate recovery from sucrose or halothane and observe a conducted response to acetylcholine. We also tested whether a specific inhibitor of L-arginine could block these responses by using a 5-minute preexposure to N-nitro-L-arginine (L-NNA, 100 µmol/L pipette concentration) in experiments with either L-arginine or acetylcholine. For the experiments with L-NNA, Protocol 2 was performed first without and then with L-NNA, because preliminary observations indicated that recovery from L-NNA was inconsistent.

Measurements
Vessel diameter (µm) was measured continuously from the recorded image with a modified video caliper (Colorado Video) calibrated with a videotaped stage micrometer and recorded on either a Kipp and Zonen chart recorder or computer disk using a video software program (Dataq Instruments, Inc). Diameter measurements were reproducible to ±0.6 µm, which is 1% to 2% of the diameter. Distances (µm) between branches and from the exposure site to observation site were measured on sequential video fields.

Calculations
As a criterion for stability of each preparation, the baseline diameter was determined for the 5-minute baseline period. Only preparations in which the diameter did not change by more than 5% during this time period were used for analyses; 3 of 56 preparations were discarded using this criterion. The baseline diameter used for a reference was the average diameter during the 2-minute interval before exposure to pipette contents. The test diameter was the peak diameter during exposure to pipette contents; for the conducted responses this was limited to the peak diameter during the first 60 seconds of exposure. The diameter change (response) was calculated as the percent change in diameter from baseline: [(test diameter-baseline diameter)/baseline diameter]x100.

Statistical Analyses
The calculated values were pooled by position and test condition to determine the population means and standard errors (SEM), and n is indicated in the figures, tables, or text. Statistical analyses evaluated whether the mean of the response for an individual protocol represented a significant change from baseline by t tests, as appropriate.²² For all statistics, differences were considered significant at P.05.

	Results

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Local Dilation to L-Arginine Requires Downstream Preexposure
Three branch arterioles were exposed to L-arginine in each experiment (see schematic, Fig 1). The baseline diameters were 13.1±2.0 µm (first branch, mean±SEM), 11.3±1.9 µm (third), and 15.9±2.1 µm (last). When the exposure order was last, third, and first branches (downstream to upstream exposure, Fig 2A), the last branch did not change significantly in diameter (-3.8±1.5% of baseline), but the upstream branches each dilated (third, +58.1±27%; first, +92±26%; n=5). The actual diameter changes were -1.42±0.6 µm (last), +5.0±1.8 µm (third), and +10.8±2.5 µm (first). The time from L-arginine exposure to the onset of this dilation was 71±22 seconds for the third branch and 71.2±23 seconds for the first branch. The dilation persisted for the duration of L-arginine exposure (5 minutes), and the vessel recovered to its baseline within 2 minutes after L-arginine exposure ended. However, when the exposure order was first, third, and last (upstream to downstream exposure, Fig 2B), none of the branches dilated to L-arginine (first, -0.4±3%; third, +5±11%; last, -5.6±7.5%; n=4). Thus, downstream preexposure to L-arginine changed the responsivity of the upstream branches to L-arginine.

View larger version (14K): [in this window] [in a new window]   Figure 2. Bar graphs showing local responses of the branch arterioles to micropipette-applied L-arginine (100 µmol/L). The exposure order was last, third, and then first branch (A, n=5) or first, third, and then last branch (B, n=4). The data are mean±SEM of the diameter change from baseline; dilations are positive changes. *Significant changes from baseline.

This change in responsivity was specific for the biologically active L-isomer, as D-arginine had no effect. After preexposure of the last branch to D-arginine, the local response of the first branch to D-arginine was -3.4±3.7% (n=3). Furthermore, the change in responsivity upstream was not due to multiple exposures of the tissue to L-arginine, because repeated exposure of the first branch did not induce a dilation at the first branch (first exposure, -1.0±6.3%; second exposure, -0.7±5.6%; third exposure, -7.2±6%; n=4). Likewise, repeated exposure of the last branch did not induce a dilation at the last branch (first exposure, -5.3±7%; second exposure, +0.6±3%; third exposure, +1.3±8%; n=4). Therefore, this unidirectional response required preexposure of a downstream branch to elicit a persistent change in the responsivity upstream, indicating communication between the downstream and upstream locations.

L-Arginine Induces a Unidirectional Conducted Vasodilation
L-Arginine was applied downstream while viewing the first branch, a distance of 1327±166 µm upstream (n=14). The peak dilation at the first branch was +17.8±2.8%, a significant dilation from the baseline diameter of 14.1±1.6 µm. The onset of this dilation was 11.8±1.1 seconds after the pipette delivery was initiated, ie, faster than diffusion (see "Materials and Methods"). Fig 3 shows a conducted dilation to L-arginine from a single experiment. An initial dilation usually lasted 20 to 30 seconds from onset to recovery; a similar response was observed in the first branch and second branches and feed vessel segments associated with them. In some preparations there was a secondary dilation that followed, dilating the vessel an additional 5% to 10%. The secondary dilation, when present, persisted for several minutes (downstream exposure time was 5 minutes).

View larger version (14K): [in this window] [in a new window]   Figure 3. Raw data from a single experiment illustrating the diameter of the upstream branch during baseline and downstream (700 µm) exposure to L-arginine (100 µmol/L). The baseline arteriolar diameter was 16.5±0.5 µm, and the peak diameter was 19.2 µm (+16.4%). The onset of dilation occurred 5 seconds after the onset of L-arginine delivery downstream.

The conducted dilation was unidirectional; the last branch did not dilate (-9.5±4%) when the first branch was exposed to L-arginine 1003±137 µm upstream (n=5). Conducted dilation was L-arginine specific; there was no significant diameter change at the first branch when the last branch was exposed to D-arginine (+0.7±3.6%, n=3) 950±118 µm downstream. Thus, in the absence of a local dilation, L-arginine induced a unidirectional conducted dilation.

Inhibition of Conducted Signals
L-Arginine Conducted Vasodilation
The conducted dilation to L-arginine could be totally blocked by high-osmolar sucrose (600 mOsm), by halothane (0.0345%), or by L-NNA (100 µmol/L) directed (by micropipette) to the feed vessel midway between the downstream L-arginine exposure site and the upstream observation site (Table 1). Each of these agents also induced diameter changes extending 200 µm to either side of the blocker pipette or caused diameter changes upstream. Sucrose induced a local dilation of 50% but did not significantly change the diameter at the first branch (-0.34±3.0%). Halothane induced a local dilation of 30% and a gradually appearing dilation upstream of +25.4±9% (at 5 minutes). L-NNA induced a local constriction of 30% but no sustained diameter change at the first branch (+1.7±9%). To rule out that a diameter change (and not these agents) had blocked the conducted dilations we used adenosine. Adenosine (100 µmol/L) induced a local dilation (157±50%; n=5) extending 200 µm to either side of the adenosine pipette and a lesser but significant sustained dilation of +20±5% at the first branch. The conducted dilation to L-arginine remained significant both in the presence and absence of adenosine (Table 1). The time to onset of conducted dilation to L-arginine was 10.1±2 seconds with adenosine. Thus, the conducted dilation to L-arginine was not blocked or delayed by a generalized dilation of the vessel but in contrast was significantly blocked by a high-osmolar sucrose solution, by halothane, or by L-NNA.

View this table: [in this window] [in a new window]   Table 1. Inhibition of Conducted Vasodilation

Acetylcholine Conducted Vasodilation
Application of acetylcholine (100 µmol/L) induced a local dilation (+120±31%) extending 200 µm to either side of the acetylcholine micropipette and also induced a significant conducted dilation at the first branch (+18.1±2.6%), a distance of 1207±155 µm upstream (n=8). The baseline diameter was 18.6±1.5 µm. The time to onset of the conducted dilation was 11.7±1.1 seconds. As with L-arginine, the initial transient dilation to acetylcholine lasted 30 seconds, and the secondary dilation, when it occurred, persisted for several minutes. The conducted dilation to acetylcholine could be totally blocked by sucrose (600 mOsm), halothane (0.0345%), or L-NNA (100 µm) (Table 1).

Conducted Signals Alter Responsivity Upstream
We used three series of experiments to evaluate how an L-arginine–induced conducted signal could alter the upstream responsivity to L-arginine. The first series tested whether a local dilation to L-arginine could occur without a conducted signal. The downstream site was preexposed to L-arginine while the middle region was exposed to sucrose, halothane, or L-NNA (separately) to prevent a conducted signal from being received upstream. While continuing exposure to the blocker, the first branch was exposed to L-arginine. Table 2 shows that high-osmolar sucrose, halothane, and L-NNA each inhibited the dilation to local L-arginine upstream. The altered responsivity was not due to nonspecific diameter changes induced by the blockers, because the upstream local dilation to L-arginine remained significant in the presence of adenosine (Table 2). Thus, when the signal for conducted dilation was blocked, the change in upstream responsivity to L-arginine was prevented.

View this table: [in this window] [in a new window]   Table 2. Inhibition of Altered Responsivity Upstream by Blocking Conducted Signals (After the Conducted Responses Shown in Table 1A)

In the second series of experiments, we determined whether the altered local responsivity upstream required a transient signal from downstream during downstream exposure or required a signal from downstream that persisted after the downstream exposure ended. In this protocol, a conducted dilation to L-arginine (+17.9±2.7%, n=4; Fig 4) was first obtained (1125±75 µm from the first branch). After downstream exposure to L-arginine ended, sucrose was applied to the middle region of the feed vessel (500±44 µm from the first branch). L-Arginine exposure upstream during sucrose block did not induce a local diameter change upstream (-3.5±4.1%). After recovery from sucrose, both the conducted (+17.9±2.7%) and the subsequent upstream local (+29±6%) dilations were significant (Fig 4). Thus, a signal pathway must be open for longer than the initial downstream preexposure in order to alter the local responsivity to L-arginine upstream.

View larger version (25K): [in this window] [in a new window]   Figure 4. Bar graphs showing the upstream diameter changes (mean±SEM, n=4) during downstream exposure to L-arginine (100 µmol/L, open bars, Conducted) and then during upstream exposure to L-arginine (100 µmol/L, hatched bars, Upstream Local). High-osmolar sucrose was applied only during the first upstream exposure to L-arginine (Sucrose); otherwise, no sucrose was used (No Sucrose). *Significant changes from baseline.

In the third series of experiments, we tested whether acetylcholine could induce the altered responsivity to L-arginine upstream. Fig 5 (left) shows that before downstream exposure the first branch (as expected) did not dilate to locally applied L-arginine (-3.4±1%, n=3). Acetylcholine induced a significant conducted vasodilation (+26±6%) 1264±477 µm downstream. The subsequent upstream local dilation to L-arginine was now significant (+33.0±12%). For comparison, Fig 5 (right) shows the baseline local response to L-arginine (-5.75±7%, n=4). L-Arginine applied 1780±539 µm downstream induced a significant conducted vasodilation (+16.3±6%); the subsequent local dilation to L-arginine upstream was significant (+30.3±10%). Thus, a conducted signal induced by either L-arginine or acetylcholine could trigger the altered responsivity to locally applied L-arginine upstream.

View larger version (25K): [in this window] [in a new window]   Figure 5. Bar graphs showing the upstream diameter changes (mean±SEM) during upstream exposure to L-arginine (100 µmol/L; filled bars, Local [before]), downstream exposure to acetylcholine (100 µmol/L; left, n=3) or L-arginine (100 µmol/L; right, n=4) (open bars, Conducted), and then repeated upstream exposure to L-arginine (hatched bars, Local [after]). *Significant changes from baseline.

	Discussion

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We show that a conducted signal initiated by L-arginine has two effects: First, it induces a dilation at a "remote" site and not at the site exposed. Second, there is a subsequent change in the responsivity of the remote location that allows a dilation to locally applied L-arginine. We initially wondered whether this response was due to the extracellular buffering effect of 0.1 mmol/L of added basic amino acid (L-arginine). We therefore tested D-arginine, which is not transported but would provide the same buffering. However, D-arginine had no effect; it neither initiated a conducted signal nor affected the upstream responsivity. Therefore, these observations appear specific to an actively metabolized amino acid.

Conducted Signal: Similarities Between Responses to L-Arginine and Acetylcholine
The conducted vasodilation to L-arginine is similar to the conducted vasodilation to acetylcholine (as demonstrated in the present study and in References 7, 8, and 23^{7 8 23} through 27). These "remote" dilations (1) have a rapid onset that cannot be explained by diffusion of the pipette contents to the remote site, (2) are transient, (3) are inhibitable by (nonspecific) inhibitors of gap junction signaling, and (4) are inhibitable by a specific arginine analogue antagonist. The inhibitors were applied to the middle of the feed vessel to block the signal transmission pathway and thus cannot be used to evaluate an effect on signal initiation (downstream) or on the remote dilation itself (upstream). Acetylcholine is known to activate calcium-calmodulin–dependent nitric oxide (NO) synthase, which uses L-arginine as a substrate to form NO.^{14 28 29} By using L-arginine, we may have bypassed other receptor-linked effects of acetylcholine and directly caused an increase in NO.

There is evidence that conducted signals are transmitted via a membrane potential–dependent mechanism within the vascular wall.²³ This finding is consistent with inhibition of conducted responses by gap junction inhibitors (as shown in the present study and in References 4⁴ -8, 20, and 21). However, there appears to be a component of either signal initiation or signal transmission that is insensitive to the membrane potential.²³ Our data showing inhibition by L-NNA support the presence of a potential-insensitive component in signal transmission. L-NNA neither changes the membrane potential³⁰ nor affects changes in the membrane potential that occur with acetylcholine exposure.³¹ Yet L-NNA blocks both local³² and conducted (present study) dilations to acetylcholine. Because L-NNA has no effect on L-arginine entry into the cell,³³ these effects are likely via specific inhibition of the action of L-arginine. An attractive explanation is that the signal transmission mechanism therefore involves NO, perhaps as a paracrine response. In contrast, signal initiation does not appear to be via NO, because sodium nitroprusside does not initiate a conducted dilation.²⁶

Conducted Signal: Differences Between Responses to L-Arginine and Acetylcholine
The conducted vasodilation to L-arginine differs from the conducted vasodilation to acetylcholine and a variety of other mediators,^{7 8 23 24 25 26 27} in at least two respects. (1) The conducted vasodilation to L-arginine does not include a local dilation at the downstream site of exposure, supporting the idea that the local and conducted signaling pathways are separate.⁹ This phenomenon may also explain a controversy regarding the inconsistent effects of L-arginine on isolated vessel segments^{9 13 34 35} compared with the consistent decrease in vascular resistance obtained with intravenous L-arginine.³⁶ (2) The conducted vasodilation to L-arginine was only seen upstream with downstream exposure (ie, unidirectional), whereas the conducted vasodilation to acetylcholine is observed in either direction (eg, References 7 and 8^{7 8} ).

Unidirectionality
Unidirectional coupling of gap junction signals is described for neuroglial cells.³⁷ This possibility in the vasculature would indicate a hierarchy for the control of gap junctions (perhaps conducted signals) within the vascular tree. However, unidirectional coupling appears to be a rare physiological occurrence.^{4 6} Alternatively, an inhibitor of conducted vasodilation may be released intravascularly upon L-arginine exposure. This inhibitor would be carried downstream and thus block manifestation of the dilation at the downstream observation point.

Another possibility is that the ability to send or receive the signal (electrotonic or paracrine) is related to the local pressure or flow conditions. Conducted responses are blocked during very low transmural pressure, achieved with total occlusion of the inflow arteriole.³⁸ In our preparation perfusion is maintained, and the resting tone along the transverse feed arteriole is uniform¹ ; the transmural pressure is likely uniform as well. Thus, we have no evidence to support a role for differential pressure in the unidirectionality of these responses. However, higher flow rate (wall shear stress) has been linked to a higher L-arginine metabolism and NO production in endothelial cell culture,³⁹ isolated vessels,⁴⁰ and in vivo.³¹ Wall shear stress upregulates NO synthase.^{31 39 41} The steepest effect of wall shear stress on NO production occurs at low (<10 dynes/cm²) wall shear stress,⁴⁰ and cGMP production increases with shear up to 40 dynes/cm².⁴² The prevailing wall shear stress at the upstream branches of this vascular site is 20 to 25 dynes/cm², roughly four times greater than the wall shear stress downstream (5 to 10 dynes/cm²).^{3 10} Thus, despite the fact that these are all fourth order arterioles, their wall shear stress environment is different, dictated by their location, and we can hypothesize that their NO synthase contents are likely different, making their ability to respond via this pathway quite different as well. We speculate that the ability to send or receive a conducted signal may be a function of the amount of NO-generating ability present at the two locations. Clearly, more work is required to determine the mechanism for the initiation, transmission, and manifestation of the conducted response.

Change in Upstream Responsivity
We report a new phenomenon in this study. Downstream exposure to a conducted response mediator alters the upstream local responsivity to L-arginine. This phenomenon suggests that a conducted signal has upregulated a specific biochemical pathway in the vasculature. We find the most interesting aspect of this novel response to be its link to conducted signals. The increased responsivity to local L-arginine required a conducted signal; it did not occur when a conducted response was blocked nor when the signaling pathway was blocked even after the conducted signal was received, and it could be triggered by two different mediators of conducted responses. The initial upregulation of this local responsivity requires no more than 20 minutes from the time of the downstream preexposure until the upstream branches dilate to local L-arginine (see "Materials and Methods"). This time frame is much slower than would be expected for simple enzyme phosphorylation and is more consistent with the time required for de novo protein synthesis or extensive protein modification.⁴³ Furthermore, repeated exposure of the downstream region seems to increase the responsivity of the upstream site to local L-arginine (compare Fig 2A with Table 2).

Interestingly, the altered responsivity may only be related to conducted dilation and not conducted constriction, such as occurs with norepinephrine (NE).¹⁹ In a previous study,¹ we determined the local response of the branch arterioles to NE using one concentration of NE per preparation and alternating the exposure order between branches, as shown in Fig 2. Unlike L-arginine exposure (current study), the response to NE¹ was unrelated to exposure order for the concentration range of 10^-9 to 10^-3 mol/L. Thus our data suggest that conducted dilatory pathways are involved in modulating the local responsivity at remote sites.

Vascular Communication and Coordinated Vascular Responses
Small arterioles from similar tissues have the same pharmacological capacity to respond to specific stimuli.^{11 13 44} It is known that by varying the transmural pressure (ie, initial load) or the resting diameter (ie, initial length) the response of an individual vessel to a specific stimulus is altered.^{13 45 46} In the whole animal, these factors are considered to be nonuniform from vessel to vessel, which may in part account for the wide range of responses observed in vivo to low concentrations of a specific tissue-wide stimulus.^{1 47} This range of responses, however, is not due to random variability but instead is predictable with respect to spatial location.^{1 2 3} The response patterns are different for receptor-mediated and metabolic stimuli,^{1 2} and specific patterns of response are directly related to the way blood flow is distributed.³ Such an organized system likely involves some means of vascular communication to coordinate upstream versus downstream responses with respect to the local conditions. We find it interesting that L-arginine, involved in flow-dependent responses,^{11 12 13 14 15} also initiates a conducted signal.

We show that the responses initiated by L-arginine extend up to a maximum of 3190 µm upstream (in that experiment conducted dilation was +19%; local dilation, +15%). Several questions arise. Do these pathways have an active role in the control of vascular responses (eg, blood flow regulation) in the terminal arteriolar bed? How would this pathway be triggered physiologically? For example, could activation of the I_K.S potassium channel⁴⁸ such as occurs in response to increased wall shear stress⁴¹ trigger these responses? This would imply that flow changes at one site may control vascular responses, and responsivity, at remote sites. There is growing evidence that capillaries can integrate local stimuli and transiently affect upstream arteriolar resistance^{49 27} and that arteriolar stimuli can summate via conduction pathways to transiently influence upstream diameters.¹⁹ Our data further suggest that the conducted signals have the potential to continually provide a dynamic input to persistently regulate vascular responsivity within a defined microvascular region.

In summary, this study demonstrates that L-arginine induces a conducted dilation without a local dilation. The conducted dilation to both L-arginine and acetylcholine can be blocked by high-osmolar sucrose or halothane (both nonspecific blockers of gap junctions) or by L-NNA (a specific inhibitor of L-arginine). Therefore, the mechanism of this conducted response may involve both gap junction and paracrine communication. We further show that after the conducted dilation is observed upstream (ie, a message is received), the responsivity of the upstream vessel to local L-arginine is altered, now allowing a dilation to upstream locally applied L-arginine. This change in local responsivity requires a conducted signal (by L-arginine or acetylcholine), is not seen when the conducted signal is blocked, and is not seen when the conducted signal pathway is blocked after a conducted response is observed. Thus, the conducted signal pathway may be an ongoing dynamic communication pathway that coordinates vascular responses within a defined microvascular region.

	Acknowledgments

 
This work was supported by a grant from the American Heart Association (91-074G) and National Institutes of Health grants HL-29929 and HL-07220.

Received April 25, 1994; accepted May 25, 1995.

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