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Am J Physiol Heart Circ Physiol 290: H264-H271, 2006. First published August 26, 2005; doi:10.1152/ajpheart.00455.2005
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Initiation of remote microvascular preconditioning requires KATP channel activity

Lauren Mabanta, Patrick Valane, Jonathan Borne, and Mary D. Frame

Department of Biomedical Engineering, State University of New York Stony Brook, Stony Brook, New York

Submitted 5 May 2005 ; accepted in final form 22 August 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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The purpose of this study was to investigate vascular preconditioning of individual microvascular networks. Prior work shows that exposure of downstream arterioles to specific agonists preconditions upstream arterioles so that they exhibit an altered local vasoactive response [remote microvascular preconditioning (RMP)]. We hypothesized that mitochondrial ATP-sensitive K+ (KATP) channels were involved in stimulation of RMP. Arteriolar diameter (~15 µm) was observed ~1,000 µm upstream of the remote exposure site in the cheek pouch of pentobarbital sodium-anesthetized (70 mg/kg) male hamsters (n = 104); all agonists were applied via micropipette. RMP was initiated by application of pinacidil (Pin), diazoxide (DZ), sodium nitroprusside (SNP), or bradykinin (BK) to the downstream vessel. After 15 min, RMP was apparent at the upstream observation site from testing of local vasoactive responses to L-arginine. Pin, DZ, SNP, and BK each stimulated RMP. To evaluate a specific role for mitochondrial KATP channels in this response, 5-hydroxydecanoate was applied (via a 2nd pipette) during downstream stimulation with agonist. 5-Hydroxydecanoate blocked RMP initiated by Pin, DZ, or SNP, suggesting that mitochondrial KATP channels are involved before SNP signal transduction. To verify this, we applied N{omega}-nitro-L-arginine during DZ or SNP stimulation. RMP was blocked during SNP, but not during DZ, stimulation. Thus stimulation of the RMP response requires mitochondrial KATP channel activity after stimulation by nitric oxide donors.

nitric oxide; reactive oxygen; diazoxide

VASCULAR PRECONDITIONING is a protective response in which prior ischemic or pharmacological exposure confers an altered vascular response that prevents hypercontractility and the no-reflow phenomenon associated with reperfusion injury (5, 10, 11). There are differences due to the mode of stimulation (ischemic vs. pharmacological), with evidence for local, neurogenic, and humoral mechanisms (12, 17, 29, 52). In vivo work suggesting that preconditioning is an all-or-none phenomenon within the affected region of an organ supports the notion that local mechanisms are at play. Consistent with this notion are studies showing that preconditioning of one region of the heart does not protect "virgin" areas of the heart that were not preconditioned (38). Other data clearly show, for instance, that preconditioning of the gut will protect the heart; this is termed remote preconditioning between organs (51). The mechanism for remote preconditioning appears to have humoral and neurogenic components. The present study focuses solely on the local mechanisms within one organ in which preconditioning is a local phenomenon restricted to the preconditioned region. We have asked the following questions: What limits the anatomic extent of preconditioning? What is the "smallest vascular unit" that can be preconditioned?

In previous investigations deep within the peripheral circulation of the cremaster skeletal muscle or cheek pouch tissue, we showed that preconditioning can be achieved along one flow path, while neighboring flow paths remain unaffected (19, 23). The stimulus is a brief downstream remote exposure of a small arteriole to one of a growing list of agonists followed by a delay. Then the arterioles upstream along this flow path exhibit a significantly altered local vasoactive response. Although these upstream vessels were not directly exposed to the stimulus, they became preconditioned. Of importance for microvascular flow control, the extent of microvascular preconditioning is limited to a single terminal arteriolar network. That is, one initiating event will precondition only the vasculature within the anatomic boundaries of a network and will not extend beyond the borders of that defined network (23).

Importantly, repeated local stimuli do not precondition. Instead, a single remote stimulus does precondition (23). Hence, "remote microvascular preconditioning" (RMP) refers to an altered vasoactive response that is initiated downstream and observed upstream along the same flow path. There is a key difference between RMP and the classic remote/propagated/conducted vasoactive responses (7, 34, 36, 45). Propagated responses are brief, lasting for the duration of the remote exposure. In contrast, the RMP response appears long after the stimulus has ceased and alters subsequent vasoactive responses upstream for hours.

RMP is a complex and integrative microvascular response that may be related to organ-level vascular preconditioning. The early phase of organ-level preconditioning occurs within minutes after the initial event and is followed several hours later by a late phase (3, 40). Microvascular dysfunction, especially compromised endothelial cell function, accounts for altered vasoactive responses in organ-level vascular preconditioning (11, 12, 49). Although it is recognized that differences exist between ischemic and pharmacologically induced vascular preconditioning, common initiation factors include the involvement of nitric oxide (NO), superoxide, and ATP-sensitive K+ (KATP) channels (27, 30). To evaluate whether RMP and vascular preconditioning share a common basis, we tested the hypothesis that RMP is initiated by NO and KATP channels.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animal model. With approval from the Stony Brook University Animal Care and Use Committee, adult male Golden (HSD:Syr) hamsters (n = 104, 81 ± 12 days old, 115 ± 14 g body wt, 54 ± 2% hematocrit) were anesthetized with pentobarbital sodium (70 mg/kg ip), tracheostomized, and maintained on constant infusion of pentobarbital sodium (10 mg/kg at 0.50 ml/h ip). Body temperature was maintained at 37–38°C by conductive and convective heat sources. The left cheek pouch was prepared for in vivo microcirculatory observations (48). The preparation was continuously superfused with bicarbonate-buffered saline containing (in mmol/l) 132 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 20 NaHCO3 (equilibrated with 5% CO2-95% N2, pH 7.4 at 36°C). All chemicals were obtained from Sigma-Aldrich Chemical (St. Louis, MO) unless otherwise noted.

The microcirculation was observed with bright-field microscopy (modified E400 Nikon upright microscope) with a x10 or x60 SWI (Olympus) objective. Video images were produced using an intensified charge-coupled device camera system (Solamere Technology Group, Salt Lake City, UT) and displayed on a Panasonic black-and-white monitor. During a 60-min stabilization period, arteriolar tone was verified by dilation to topically applied 10–2 M adenosine and constriction to 10% O2, which were added to the suffusate. Arteriolar networks fed by an arcade arteriole (Fig. 1) were chosen for study. The central feed of the network provides flow to several branch arterioles that directly flow into capillary networks; the feed arteriole with its arteriolar branches is the arteriolar network (2, 18, 20).



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  		Fig. 1. Schematic of arteriolar network illustrates remote microvascular preconditioning (RMP) response. Local responses were obtained by applying drugs to location A and noting responses at location A. Remote responses were obtained by applying drugs to location B and noting responses 851 ± 424 (SD) µm (n = 197) upstream at location A. RMP was initiated by brief application of remote stimulus to location B, and after 15 min, local responses were altered at location A. Left: control response (local constriction to 10–4 M L-Arg) before RMP; middle: propagated vasodilation to 10–4 M sodium nitroprusside (SNP); right: RMP response (local dilation to 10–4 M L-Arg). Repeated exposure to L-Arg at location A did not induce dilation (23). Local vasoactive response to L-Arg was altered in a striking and predictable fashion when network was preconditioned.

 
Two types of vasoactive responses were examined: local and remote. For local responses, the test agents were applied via micropipette to location A (Fig. 1), which is the entrance to the network, and observations were made at location A. For remote responses, the test agents were applied to location B, which is the distal end of the network, and observations were made upstream at location A.

Micropipette techniques. Micropipettes (15- to 20-µm tip diameter) were pulled (Kopf Instruments), backfilled with test solutions, and placed within 25 µm of the arteriolar wall. Flow from the micropipette was pneumatically controlled (model PLI-100, Medical Systems, Greenvale, NY) using minimal ejection pressure (typically 0.2–0.4 psi); balance pressure was set so that the solutions were not ejected from the pipette in the holding configuration. Each micropipette solution contained 25 µM fluorescein-labeled dextran (4,000 mol wt FITC-dextran) as a flow marker to verify that the arteriolar segment was exposed to the pipette contents. The FITC-dextran flow path was observed using a B1E dilter set (Chroma Technology, Brattleboro, VT). Because of the placement of the micropipette, geometry of the chosen sites, and continuous flow of suffusate over the tissue (5 ml/min), the micropipette's contents were washed away from location A during drug application to location B and were not recirculated. Test application times were typically 60 s (unless noted otherwise) and were always preceded by a 30-s baseline period.

Protocol 1: initiation of RMP. After a 30-s stable baseline diameter, the initiating agonist was applied via micropipette for 60 s to location B downstream, while location A upstream was observed. Each network received only one concentration of one initiating agonist; up to four networks were examined per animal to obtain concentration-response relations, which were paired within animals. The four networks were chosen so that the entrances to the networks were 500–1,000 µm from each other; frequently, the networks arose along the same arcading arteriole. The initiating agents were three NO donors, sodium nitroprusside (SNP, 10–4 M), 3-morpholinosydnonimine (SIN-1, 10–3 M), and (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2 diolate (DETA-NO, 10–3 M); a receptor-linked vasodilator acting through NO and cGMP, bradykinin (BK, 10–6 M); a nonhydrolyzable cGMP analog, 8-bromo-cGMP (10–7–10–3 M); a nonspecific KATP channel opener, pinacidil (10–8–10–4 M); and a mitochondrial KATP channel-specific agonist, diazoxide (10–6–10–4 M). Each agent stimulated a remote vasodilation that was observed during the 60-s exposure period but did not persist after the test agent was removed. After 15 min, the RMP response was tested according to protocol 3. Combinations of RMP initiation and test agents are shown in Table 1.


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  		Table 1. RMP initiation and test agents

 
Protocol 2: inhibition of RMP during the downstream stimulus. To block RMP, inhibitor agents were applied via a second micropipette to location B downstream for 5 min before and during RMP initiation (protocol 1). Then the inhibitor and test agents were removed. Thus inhibitors were applied only during the initiation phase and not during the 15-min delay. The inhibitor agents were as follows: superoxide dismutase (SOD) + catalase (Cat, 60 U/ml each) to block all sources of reactive oxygen [reactive oxygen species (ROS)]; N{omega}-nitro-L-arginine (L-NNA, 10–4 and 10–5 M), an inhibitor of NO formation through NO synthase; and 5-hydroxydecanoate (5-HD, 10–5–10–3 M), a specific antagonist for the mitochondrial KATP channel.

Protocol 3: testing for RMP at location A. Before any remote vasoactive stimulus (e.g., protocol 1 or 2), the local vasoactive response of the RMP test agent was ascertained at location A to determine the control response for that network. After ≥5 min, protocol 1 or 2 was performed. After 15 min, location A was again exposed to the RMP test agent. The RMP test agents that were applied to location A were as follows: L-arginine (10–4 M, 5 min), adenosine (10–5 M, 60 s), and SNP (10–5 M, 60 s). Prior work has shown that an altered vasoactive response to L-arginine applied via micropipette to the upstream observation site is a rigorous and reproducible test to determine whether preconditioning has occurred. Before preconditioning, the initial diameter change in response to L-arginine is not significant; after the downstream initiation of RMP and the delay period, L-arginine stimulates a significant dilation (19, 20, 23). Because the present study examines the mechanism of initiation of RMP, we have focused on the L-arginine response before and after the stimulus.

Data acquisition and statistics. The distance between locations A and B was determined on sequential video fields for each network. All diameter changes were obtained online using a video caliper system (Microvascular Research Institute, College Station, TX) and a software data acquisition system (Strawberry Tree, Workbench, Sunnyvale, CA) calibrated with a micrometer. The diameter change is reported as the raw diameter or the fractional change in diameter from baseline: (peak – baseline)/(maximum – minimum), where the maximum and minimum diameter were obtained with 10–2 M adenosine and 10% O2 during testing for tone. The diameter changes were evaluated with Student's t-test for paired comparisons, ANOVA for repeated measures, or linear regression to determine statistical differences; {alpha} was set to 0.05 (47).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Arteriolar network and RMP. The 851 ± 424 (SD)-µm-long central feed of the arteriolar networks (n = 197 in 104 animals) examined in this study can be classified as A4 or A5. At the entrance of the feed, the baseline diameter was 10 ± 4.3 µm, the minimal diameter (10% O2) was 7 ± 2 µm, and the maximal diameter (10–2 M adenosine) was 16 ± 6 µm (location A, Fig. 1). Three to seven arteriolar branches arise from the central feed; each branch arteriole terminates in groups of capillaries (2). RMP was tested within networks by micropipette application of the initiating agonist to location B (protocol 1), removal of the micropipette, and 15 min later another micropipette application of the agonist to location A (protocol 3). A temporal delay has always been essential for observation of RMP. Also, repeating L-arginine at location A does not induce the RMP response; RMP must be initiated from the remote location downstream.

Although numerous vasoactive responses can be altered by RMP, we limited the agents to investigate the initiation phase of this response. A rigorous and reproducible positive test for RMP is an altered vasoactive response to locally applied L-arginine (micropipette at location A). Figure 1 illustrates the change in vasoactive response to L-arginine when SNP is used to stimulate RMP. The combinations of agents are shown in Table 1.

Requirement for ROS and NO for initiation of preconditioning. NO and some form of ROS are required to initiate RMP (Fig. 2). If either pathway is blocked, preconditioning does not occur. For these experiments, preconditioning was stimulated by micropipette application of SNP to location B downstream (protocol 1). The inhibitors were applied via a second micropipette directed to location B, according to protocol 2. After 15 min, RMP was tested with micropipette application of L-arginine to location A upstream (protocol 3). In the presence of SOD + Cat (60 U/ml each, n = 6) or L-NNA (10–5M, n = 6) at location B, no preconditioning occurred (Fig. 2B). Thus NO can initiate the RMP response; however, endogenous NO and ROS are required to complete the RMP initiation.



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  		Fig. 2. Fractional diameter changes. A: propagated vasoactive responses, i.e., remote diameter changes at location A during downstream application of 10–4 M SNP to location B. B: RMP responses. After 15 min, local diameter changes were measured at location A during application of 10–4 M L-Arg to location A. Two networks were studied per animal: without inhibitors (control) and with 10–5 M N{omega}-nitro-L-arginine (L-NNA, n = 6 animals) or superoxide dismutase (SOD) + catalase (Cat, 60 U/ml each, n = 6 animals). Inhibitors were applied to location B for 5 min before and during SNP exposure.

 
Figure 2A shows the propagated vasodilatory response to SNP applied to location B. When SNP was applied downstream to location B, there was a concurrent propagated dilation at location A that persisted only during the 60-s SNP exposure downstream. Figure 2A shows that the propagated dilation to SNP was unaffected by L-NNA or SOD + Cat downstream. Thus each SNP stimulus at location B initiated two distinct responses: one immediate and one delayed. During the SNP exposure, there was an immediate propagated dilation (Fig. 2A); this initiating stimulus also preconditioned the network after a delay (Fig. 2B).

Multiple forms of NO donors can initiate the RMP response and induce propagated vasodilation. Propagated vasodilation was observed at location A during downstream stimulation: 0.84 ± 0.18 (fractional change, mean ± SE) with 10–4 M SNP (n = 6), 0.81 ± 0.16 with 10–3 M SIN-1 (n = 6), and 0.79 ± 0.12 with 10–3 M DETA-NO (n = 7). After 15 min, the RMP response to L-arginine was positive (dilation) at location A: 0.9 ± 0.08 for SNP-initiated RMP, 0.93 ± 0.04 for SIN-1-initiated RMP, and 0.88 ± 0.11 for DETA-NO-initiated RMP. The control response to L-arginine before exposure to NO donors was not significant (–0.24 ± 0.22, mean ± SD, pooled data, n = 19). BK is well described to stimulate NO production and generate an NO-dependent dilation. BK (10–6 M, n = 4 animals and 8 networks) stimulated a local dilation at location B (0.29 ± 0.08) and a propagated vasodilation at location A (0.26 ± 0.05). After 15 min, the RMP response was evident at location A (L-arginine, 10–4 M), causing a dilation of 0.28 ± 0.04, which was blocked by L-NNA at a second network in the same preparation (10–5 M, 0.05 ± 0.1). Thus BK-stimulated NO production can precondition the arteriolar network.

To determine whether NO was initiating RMP through a cGMP-mediated pathway, we tested a nonhydrolyzable analog, 8-bromo-cGMP. First, we characterized the local and propagated vasodilation to 8-bromo-cGMP (n = 4). Neither response was robust, and the propagated vasodilation was stimulated at a lower dose than the local dilation (Fig. 3A). From this information, we chose 10–4 M 8-bromo-cGMP to test for the RMP response. Figure 3B shows the variability in the propagated vasodilation with 8-bromo-cGMP compared with the paired RMP response (n = 15 networks in 7 animals). Because of the variability, the RMP response was not significant for the population; however, in some animals a large RMP response was obtained. There was no correlation between magnitude of propagated vasodilation and RMP response (r = 0.11, P = 0.08, n = 15). Thus, although cGMP may participate in the RMP response, it cannot alone always stimulate it.



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  		Fig. 3. Fractional diameter changes. A: propagated and local dilation to 8-bromo-cGMP (8-Br-cGMP). {circ}, Remote diameter changes at location A during downstream 8-bromo-cGMP application to location B; {blacksquare}, local dilation at location B during 8-bromo-cGMP application to location B (n = 4). B: propagated vasodilation induced by 10–4 M 8-bromo-cGMP in 15 networks (n = 7). After 15 min, RMP response was obtained as local diameter change at location A during 10–4 M L-Arg application to location A. Lines connect data obtained in the same network for comparison of independent variability. {square}, Mean ± SE.

 
To further test the role of ROS in initiation of the RMP response, we examined the end effect using other vasodilators, selectively replacing L-arginine at location A with SNP or adenosine. By this method, we tested whether RMP altered the local dilation to SNP or adenosine and whether some form of ROS was required. For these experiments, high SNP was still used to initiate preconditioning at location B. Experiments were performed at two vascular networks per animal in the absence and presence of SOD + Cat (60 U/ml each, 2nd micropipette at location B). The high SNP concentration at location B always stimulated a propagated vasoactive dilation (without SOD + Cat, 0.82 ± 0.27%, mean ± SD, pooled data), which was not statistically different from the response with SOD + Cat (0.62 ± 0.16%, P = 0.41 by paired t-test). At 15 min after the stimulation at location B, we applied 10–5 M SNP (n = 6) or, in separate animals, 10–5 M adenosine (n = 6) to location A and examined the local vasoactive response. Submaximal concentrations were determined previously by this laboratory (41). Figure 4 shows the local responses to SNP or adenosine at location A before (control) and 15 min after RMP without and with ROS inhibition. RMP enhanced dilation to SNP and attenuated dilation to adenosine; ROS inhibition blocked each of these effects.



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  		Fig. 4. Fractional diameter changes in response to locally applied 10–5 M SNP (n = 8 networks in 6 animals) or 10–5 M adenosine (n = 10 networks in 6 animals) at location A. RMP was initiated by application of 10–4 M SNP alone (RMP) or with SOD + Cat (60 U/ml each) to location B. At 15 min after remote stimulus with SNP at location B, local response to SNP or adenosine was again tested at location A. RMP alone and RMP with SOD + Cat are paired data from separate vascular networks in the same animal. *P < 0.05 vs. Before (i.e., control responses obtained before RMP).

 
Pharmacological evidence of requirement for mitochondrial KATP channels. Data summarizing KATP channel involvement in the preconditioning response are shown in Figs. 5–7. Figure 5 shows that pinacidil initiates RMP in a concentration-dependent fashion from 10–7 M (no effect) to 10–4 M. For the RMP response, the dose-response curve to the individual data provided a lower limit of –0.4 ± 0.1, an upper limit of 0.46 ± 0.036, and an EC50 of 6.4 ± 1.4 x 10–7 M for 10–7–10–4 M pinacidil (n = 7 animals, 14 networks). Pinacidil application to location B also stimulated a propagated vasodilation that was dose dependent and was stimulated at a lower pinacidil concentration than was used to stimulate the RMP response. Fitting a dose-response curve to the individual propagated vasodilation data, we obtained a lower limit of –0.07 ± 0.25, an upper limit of 0.90 ± 0.36 (10–8–10–4 M, n = 7 animals, 14 networks), and an EC50 of 1.8 ± 2.7 x 10–6 M for pinacidil. Thus KATP channels initiate the immediate propagated vasodilation beginning at lower concentrations and the RMP response at larger doses.



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  		Fig. 5. Fractional diameter changes at location A during remote application of 10–8–10–4 M pinacidil to location B. Preconditioning was initiated by pinacidil at location B (propagated vasodilation to pinacidil; n = 29 networks in 21 animals). RMP response to local 10–4 M L-Arg applied to location A was tested 15 min after pinacidil. EC50 for remote dilation to pinacidil (propagated vasodilation to pinacidil, 1.8 ± 2.7 µM) was not different from that for pinacidil initiation of RMP response (0.64 ± 0.14 µM).

 


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  		Fig. 7. Fractional remote and RMP diameter changes at location A. Remote responses were stimulated with 10–4 M SNP alone, SNP + 10–4 M 5-HD, 10–4 M diazoxide, and diazoxide + 10–4 M L-NNA. After 15 min, RMP response was determined for each condition using 10–4 M L-Arg. One condition was tested per network (n = 16 networks in 4 animals). *P < 0.05 compared with no inhibitor.

 
Diazoxide and 5-HD were used to test specific involvement of mitochondrial KATP channels in the RMP response. 5-HD was applied via micropipette to location B according to protocol 2. The propagated vasodilation to diazoxide was concentration dependent, and 10–4 M 5-HD did not completely block the propagated vasodilation (Fig. 6A). A lower concentration of 5-HD (10–5 M) had no effect on the propagated vasodilation to diazoxide (10–6 M, +0.43 ± 0.08). A higher concentration of 5-HD (10–3 M) partially blocked the propagated vasodilation to diazoxide (10–4 M, +0.11 ± 0.08, n = 5). 5-HD alone did not stimulate a remote diameter change (–0.22 ± 0.41, mean ± SD, n = 16).



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  		Fig. 6. Fractional diameter changes at location A during remote dilation to diazoxide (A) and diazoxide initiation of RMP response to L-Arg (B). A: 10–6–10–4 M diazoxide alone (control remote dilation) or with 10–4 M 5-hydroxydecanoate (5-HD, in a separate micropipette) was applied to location B, and remote diameter change at location A was observed. B: 15 min later, local response to 10–4 M L-Arg was determined at location A. Control RMP, with diazoxide alone initiating RMP response, was compared with diazoxide and 5-HD initiation of RMP. One diazoxide concentration was tested per network; data were pooled from 60 networks in 24 animals. *P < 0.05 compared with control responses without 5-HD. #P < 0.05 compared with baseline diameter.

 
The RMP response 15 min later was positive for diazoxide (Fig. 6B). The RMP response was sensitive to 5-HD (10–4 M) at the lower concentrations of diazoxide. A lower concentration of the inhibitor 5-HD (10–5 M) blocked only the RMP response stimulated by 10–6 M diazoxide, whereas the higher concentration of 5-HD (10–3 M) blocked the RMP response by 10–4 M diazoxide (–0.23 ± 0.29, n = 5). Thus the RMP response was completely blocked by inhibition of mitochondrial KATP channels in the presence of propagated vasodilation.

The effect of 5-HD (10–4 M) was tested on the pinacidil (10–5 M)-initiated responses for 12 networks in 3 additional animals. The propagated vasodilation to pinacidil (+0.62 ± 0.05) was suppressed but not completely blocked by this high dose of 5-HD (+0.24 ± 0.06). However, the subsequent RMP response (dilation to L-arginine, +0.52 ± 0.08) was prevented (with 5-HD, –0.24 ± 0.11). The control response to L-arginine for these networks was –0.31 ± 0.11. Thus, as with diazoxide, the pinacidil-stimulated RMP response was blocked in the presence of propagated vasodilation.

To determine whether NO initiated the RMP response through activation of KATP channels, we tested SNP in the presence of 5-HD and diazoxide in the presence of L-NNA. 5-HD (or L-NNA) was applied via a separate micropipette for 5 min before the agonist and during agonist exposure downstream, according to protocol 2. In the presence of 5-HD, SNP did not initiate the RMP response, and the propagated vasodilation was significantly attenuated (Fig. 7). In contrast, in the presence of L-NNA, diazoxide stimulated the propagated vasodilation and the RMP response.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary key finding of this study is the involvement of NO and mitochondrial KATP channels in RMP. The second key finding is that the remote stimulation initiates two separate signals with different concentration dependence and KATP channel sensitivity for the immediate propagated vasodilation compared with the delayed RMP.

Not all remote vasoactive agonists will induce RMP (19). Equally important, some remotely applied agonists do not display an apparent remote propagated vasoactive response, yet that stimulus does induce RMP. The present study supports the notion that individual remote stimuli are capable of stimulating more than one type of signaling pathway. In the case of NO donors or BK, one signal is an immediate acute propagated vasodilation that lasts for the duration of the downstream exposure. The second signal precipitates RMP. The present study examines only the initiation of RMP, not transmission or the end effect. Exogenous NO can initiate RMP, and endogenous NO and ROS are required for RMP. NO does not appear to be exclusively acting through cGMP, and the ROS source remains to be determined. However, mitochondrial KATP channel activity appears to be required for the RMP response. It is not clear whether KATP channels are an essential component for the immediate acute propagated vasodilation.

Our previous data showed that only remote dilation, and not RMP, was blocked by inhibition of the sulfonylurea receptor (19); this was consistent with involvement of sarcoplasmic KATP channel activity, specifically, or hyperpolarization, in general, in the remote vasodilation. In the present study, we tested pinacidil, which activates the sarcoplasmic and mitochondrial KATP channels (4, 31). On the basis of the difference in efficacy of pinacidil for the remote RMP responses, we tested whether different populations of KATP channels may be involved in the two responses. The more specific agonist-antagonist pair of diazoxide and 5-HD was used to investigate the role of mitochondrial KATP channels directly. Our data suggest that diazoxide stimulates both responses at 10–6 M (although this was the lowest concentration tested). More definitively, the data with the mitochondrial KATP antagonist 5-HD showed that the acute remote vasodilation was not completely blocked when the RMP response was prevented for SNP, pinacidil, or diazoxide. On the basis of the specificity to block the RMP response, we conclude that the RMP response does require mitochondrial KATP channels. This conclusion is consistent with many studies of organ-level preconditioning that support an essential role for mitochondrial KATP channel activity in preconditioning (13, 14, 26, 33).

Regarding the remote vasodilation, we question whether a nonspecific effect of the drugs caused hyperpolarization, which then induced the remote vasodilation. This is consistent with the abundant literature showing a link between hyperpolarization and remote vasodilation (6, 9, 15, 25); depolarization has similarly been demonstrated to occur with many remote vasoconstrictor agents (46). Lin and Duling (32) additionally tested remote vasoconstrictor responses with ischemia but did not test recovery of responses after the single ischemic event. Although we used diazoxide as a specific inhibitor of mitochondrial KATP channels, there are reports that diazoxide also blocks succinic dehydrogenase (SDH), thus poisoning the mitochondrial pathways (43, 44). A confounding finding is that the "specific" inhibitor of SDH, a neurotoxin, 3-nitroproprionic acid, is reported to also inhibit the mitochondrial KATP channel directly, thereby causing a preconditioning protective effect (28, 37); there does not appear to be a specific pharmacological tool to test the involvement of SDH separately from mitochondrial KATP channels.

Vascular preconditioning has been studied extensively in many contexts, including ischemic preconditioning of the myocardium, ischemia-reperfusion injury, and pharmacological preconditioning (11, 12, 27, 30). A common thread in understanding the effect of vasoregulatory dysfunction with early preconditioning has been linked to adenosine, NO pathways, superoxide, and KATP channels (1, 30, 33, 35, 39). The present study shows the involvement of these agents in the RMP response and raises the following three points regarding vascular preconditioning.

Initiation of preconditioning occurs with a brief localized signal. The physiological response of RMP can be triggered by a brief localized downstream stimulus. The region of the blood vessel that is exposed to the test agents is not more than 200 µm in axial length, as defined previously using these techniques (41). Location B is a terminal arteriole that dilates in response to NO donors or adenosine, as defined previously for this network location (18). Thus endothelium and vascular smooth muscle are present at the initiating location. The smooth muscle is likely discontinuous; downstream from location B are the capillary networks, which is confirmed in each preparation.

RMP requires vascular communication between locations B and A. The observation of vascular preconditioning upstream of the stimulation point, progressively along the central arteriolar feed of the network, demonstrates the importance of vascular communication in preconditioning and provides the microvascular locations that will be altered by RMP. Previously, we showed that remote vascular communication between locations B and A is essential for the SNP-stimulated RMP response (21, 23); the transmission mechanism is beyond the scope of the present study. The data support the notion that the arteriolar network is a functional unit (8, 16, 18, 24, 42, 48, 50).

The RMP response is not all or none; rather, it is a gradual response to specific agonists. By altering the initiation stimulus, the RMP response is graded. A higher concentration of the initiation agent induces a larger RMP response (Figs. 5 and 6). Furthermore, the magnitude of the RMP response is greater with NO donors than with KATP channel agonists. This illustrates optimal activation of the RMP response with NO, although inhibition of KATP channels blocks the NO-initiated response.

Our goal is to determine whether the RMP response is related to organ-level vascular preconditioning. Endogenous NO is required to initiate RMP. The sequence of signal transduction shows that NO must act before KATP channel involvement. Thus the initiation of RMP is similar to organ-level vascular preconditioning. Importantly, the initiation site consists of the terminal arteriolar branches of a defined network, transmission of a signal must occur in the retrograde direction along the network, and the resulting preconditioning is a graded response dependent on the stimulus and stimulus strength. The significance of these findings suggests that this defined network may be the smallest intact group of blood vessels that can be preconditioned.


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 ABSTRACT
 METHODS
 RESULTS
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This study was supported by the American Physiological Society Frontiers in Physiology Program (J. Borne) and National Heart, Lung, and Blood Institute Grant HL-55492 and American Heart Association Grant EI0040197 (M. D. Frame).


   ACKNOWLEDGMENTS
 
Portions of this study have appeared in abstract form (22).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Frame, Dept. of Biomedical Engineering, SUNY Stony Brook, Stony Brook, New York 11794 (e-mail: mframe{at}notes.cc.sunysb.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.


   REFERENCES
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 DISCUSSION
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 REFERENCES
 

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