Published online before print June 5, 2003, doi: 10.1161/01.RES.0000080318.81205.FD
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2003 American Heart Association, Inc.
Integrative Physiology |
Homocellular Conduction Along Endothelium and Smooth Muscle of Arterioles in Hamster Cheek Pouch
Unmasking an NO Wave
From the John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Conn.
Correspondence to Steven S. Segal, PhD, The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Ave, New Haven, CT 06519-1403. E-mail sssegal{at}jbpierce.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We investigated roles for homocellular (endothelium or smooth muscle) and heterocellular (myoendothelial) conduction pathways along hamster cheek pouch arterioles in vivo (n=64; diameter, 33±1 µm). Endothelium-dependent and -independent vasoactive agents were delivered from micropipettes (0.5 or 1 second pulse) onto an arteriole while observing diameter changes at defined distances along the vessel. Acetylcholine (ACh) produced maximal diameter (63±1 µm) locally and vasodilation conducted rapidly (
10 µm response at 2 mm, <1 second). Responses to bradykinin (BK) were similar, whereas sodium nitroprusside produced maximal dilation locally without conduction. KCl evoked biphasic conduction of vasoconstriction and vasodilation, whereas phenylephrine (PE) produced conducted vasoconstriction. Disrupting the integrity of endothelium as a conduction pathway using focal light-dye treatment (LDT) abolished conducted vasodilation to BK and to KCl but not to ACh. Disruption of smooth muscle integrity with LDT abolished conducted vasoconstriction with no effect on conducted vasodilation. After LDT of respective cell layers at sites 1 mm apart, vasodilation to ACh conducted past disrupted smooth muscle or disrupted endothelium, but not beyond both sites in series. The loss of conduction after selective LDT indicates a lack of effective myoendothelial coupling along the arteriolar wall. During NO synthase inhibition (L-NA, 100 µmol/L), conducted vasodilation was abolished to BK and to KCl yet remained intact to ACh. However, after LDT of smooth muscle, L-NA inhibited conduction to ACh by 60%. Thus, conduction of vasodilation entails a wave of NO release along arteriolar endothelium that is masked when smooth muscle provides a parallel conduction pathway.
Key Words: microcirculation • conduction • endothelium • smooth muscle • nitric oxide
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Coordination of vasomotor activity within and among resistance microvessels is integral to the local control of tissue blood flow. Vasodilation or vasoconstriction initiated at a local site can conduct along arterioles for several millimeters1,2 and manifests the transmission of electrical signals from cell-to-cell through gap junction channels.3–6 Outstanding goals have been to define the role of endothelium and smooth muscle in the initiation and the conduction of vasomotor responses, and to resolve the nature of heterocellular communication along the arteriolar wall.4–12 In arterioles supplying the hamster cheek pouch, endothelium and smooth muscle monolayers can each provide a homocellular pathway for conduction.5,7 Nevertheless, heterocellular coupling is also apparent, particularly in vitro.8,11 Indeed, for isolated arterioles12 and feed arteries,6 electrical signals conduct readily between cell layers through myoendothelial gap junctions. However, there is a paucity of evidence for myoendothelial coupling in the intact system. In the present study, we investigated whether myoendothelial coupling enables conduction of vasomotor responses past sites of endothelium or smooth muscle disruption within the wall of arterioles controlling blood flow to the hamster cheek pouch.
The initiation and the conduction of vasodilation with acetylcholine (ACh) have consistently been shown to be independent of NO release.2,9,13–16 Throughout these studies, however, the integrity of both endothelium and smooth muscle layers has remained intact. Therefore, we also investigated whether redundancy in signaling pathways along the arteriolar wall may have obscured a role for NO in conducted vasodilation that has not previously been recognized. For this purpose, NO synthase (NOS) was inhibited in intact arterioles and after focal disruption of smooth muscle integrity. Our findings show an absence of effective myoendothelial coupling and that conduction along endothelium produces concomitant release of NO to relax arteriolar smooth muscle. However, this pathway is masked when smooth muscle provides a parallel conduction pathway, eg, in response to EDHF production with ACh as a stimulus.9,16
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hamster Cheek Pouch Preparation
Procedures were approved by the Animal Care and Use Committee of the John B. Pierce Laboratory. Male golden hamsters (102±1 g; n=64) were anesthetized with pentobarbital sodium (65 mg/kg, intraperitoneal injection) and tracheotomized (PE 190 tubing) to ensure airway patency. Esophageal temperature was maintained at 37°C to 38°C. A cannula (PE 50) was secured in the left femoral vein for fluid replacement and maintenance of anesthesia (10 mg pentobarbital mL-1 sterile saline, infused at 0.42 mL/h). At the end of the experiment, an overdose of pentobarbital was delivered intravenously.
The cheek pouch was everted, pinned onto a transparent Plexiglas board, and superficial connective tissue was removed using microdissection. The preparation was superfused continuously (5 mL/min) with bicarbonate-buffered physiological saline solution (PSS; pH 7.4, 37°C) of the following composition (in mmol/L): 137.0 NaCl, 4.7 KCl, 1.2 MgSO4, 2.0 CaCl2, 18.0 NaHCO3 dissolved in deionized H2O (dH2O), and equilibrated with 5% CO2/95% N2. Salts were obtained from J.T. Baker or Sigma. The completed preparation was secured onto a fixed stage of an intravital microscope mounted on an X-Y translation platform and equilibrated at least 30 minutes.
Arterioles were observed using video microscopy.5,7,9 Internal diameter was measured continuously with a video micrometer (resolution, 
2 µm). Data were recorded onto a personal computer at 40 Hz; an event marker coincided with stimulus delivery. One arteriole was studied per hamster; those selected for experiments were typically second-order branches located at least 1 cm from tissue edges, devoid of adjacent venules, had regions 1- to 2-mm long with minimal branching, and constricted when the superfusion solution was equilibrated with 5% or 10% O2 (and 5% CO2, balance N2), which was sustained to promote vasomotor tone and the ability to study conducted vasodilation over several hours. Increasing arteriolar tone with oxygen does not affect conduction.2,17,18 Maximal diameter was measured during topical SNP (10 µmol/L; added to superfusate). Preparations that displayed vasomotion (<10%) were not used due to the uncertainty in measuring conducted responses when baseline diameter changes continuously.
Vasomotor Stimuli and Responses
Glass micropipettes were filled, secured in a micromanipulator, and positioned with the tip adjacent and perpendicular to the arteriolar wall. Acetylcholine (1 mol/L) and PE (0.5 mol/L) were microiontophoresed [500 ms, 500 or 1000 nA; tip internal diameter (ID) 1 µm]. Bradykinin (BK; 1 mmol/L) and SNP (1 mmol/L) were delivered as a pulse (typically 1 second, adjusted to produce maximal diameter at the site of delivery) using pressure ejection (28 to 35 kPa, tip ID, 2 to 3 µm); KCl (1 mol/L) was delivered similarly. The local response was evaluated at the micropipette tip (Figure 1) and conducted responses were evaluated at defined distances from the local stimulus.5,7 Vasomotor responses were quantified as follows: Diameter change=peak response diameter-baseline diameter. A separate pulse was delivered to observe each response; 2 to 3 minutes elapsed between each stimulus. Tachyphylaxis to respective stimuli was negligible.
|
Microejection of KCl readily produces conduction along the arteriolar wall that can be biphasic in nature,2,4,5 which may be attributable the transient increase and decline in [K+]o. The effect of defined [K+]o on arteriolar diameter was evaluated by equimolar substitution of KCl for NaCl in the superfusion solution.19 This was achieved by switching between reservoirs, which required 1 minute to reach a new steady state [K+]o over the tissue. For each [K+]o, diameter was recorded after 4 to 5 minutes of equilibration. Control [K+]o (4.7 mmol/L) was restored to return to control diameter between each increment in [K+]o.
Light-Dye Treatment (LDT)
Light-dye treatment was performed using fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin (3% solution)7 or to dextran (70 kDa, 0.67% solution)20 with similar results. For disruption of endothelium, dye was injected into the femoral vein (1 mL/kg). For disruption of smooth muscle, dye was perifused across the segment from a micropipette (10 µm tip ID).7,20 At the site of LDT, the arteriolar segment (length, 250 µm) was illuminated (excitation, 450 to 490 nm) through an immersion objective (40x; NA=0.75, Zeiss) using a 75-watt Xenon lamp. The site was centered 500 µm upstream from the original "local" site of stimulation. Local responses to ACh and to PE were evaluated before and during treatment until loss of dilation to ACh (with endothelium disruption) or loss of constriction to PE (with smooth muscle disruption).7,20 For dual LDT, arterioles with segments 
2 mm long were first evaluated for conducted responses to ACh in both directions. Light-dye treatment was then performed to disrupt smooth muscle at 500 µm and endothelium at 1500 µm. Responses to ACh were then re-evaluated in both directions. Selectivity of cellular disruption with LDT was confirmed by labeling with propidium iodide.7,20
Evaluating a Role for NO in Local and Conducted Vasodilation
To inhibit the activity of NOS, N
-nitro-L-arginine (L-NA, 100 µmol/L) was equilibrated in the superfusate, with the specificity of inhibition assessed by reversing the effects of L-NA after the addition of L-arginine (L-Arg, 1 mmol/L).2,9,15 The efficacy of NOS inhibition over time was ascertained by evaluating local responses to ACh and to BK. To evaluate whether NO was integral to the conduction of vasomotor responses along intact arterioles, stimuli were applied at 0 µm and conduction was evaluated at sites 500 and 1000 µm upstream along arterioles under control conditions and after equilibration with L-NA. In additional experiments, conduction was triggered by ACh in the middle of a 2-mm segment of the arteriole and evaluated for 1000 µm in each direction. Smooth muscle LDT was then performed midway along one direction, with the integrity of both cell layers maintained in the opposite direction. L-NA was than added to test whether NO release from endothelium would be unmasked by disrupting the smooth muscle conduction pathway.
Data Analysis
Local and conducted responses were evaluated before and after LDT using paired t tests and analysis of variance with Tukey tests for post hoc comparisons. Differences were considered statistically significant with P<0.05. Summary data are mean±SEM; n indicates number of arterioles.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Control Responses
Resting diameter after equilibration averaged 44±1 µm and elevating superfusate O2 constricted arterioles to 33±1 µm (P<0.01, n=61). Maximal diameter of arterioles during topical SNP was 65±1 µm. When delivered from a micropipette, each stimulus (with the exception of SNP, which produced local but not conducted vasodilation) evoked reproducible and characteristic changes in diameter along the arteriolar wall (Figure 1). The amplitudes and durations of local responses were typically
2-fold greater than respective conducted responses (Figure 1). Irrespective of the stimulus, observation site, or experimental intervention, the interval between the onset of local and conducted responses was consistently <1 second. Both ACh and BK produced vasodilation and PE produced vasoconstriction. When KCl was ejected from a micropipette for at least 1 second, vasoconstriction occurred first, was sustained for the stimulus (up to 15 seconds; Figure 2A), and was followed by transient vasodilation. With a 0.5-second pulse, only vasodilation was observed. A 1-second pulse was used thereafter and both vasoconstriction and vasodilation conducted with high fidelity (Figure 1). Nifedipine (1 µmol/L; Sigma) inhibited the constrictor component (Figure 2B) while increasing resting diameter by 7±1 µm (n=5). During sustained elevations in [K+]o, arterioles dilated at 10 to 20 mmol/L and constricted at 40 to 100 mmol/L (Figure 2B). Stimuli delivered from micropipettes at 1000 µm (and at 500 µm before LDT) produced local responses at respective sites that were not different from those recorded at 0 µm before and after LDT (data not shown). Moving a micropipette >100 µm away from an arteriole eliminated responses, confirming the localized nature of each stimulus. Based on the maintenance of vasomotor tone and conducted vasodilation to ACh, preparations remained stable for 5 hours.
|
Light-Dye Treatment of Endothelium
For stimuli delivered at 0 µm with responses observed at 1000 µm, disruption of endothelium at 500 µm had no effect on conducted vasoconstriction to either PE or to KCl. However, conducted vasodilation in response to BK and to KCl were abolished (Figure 3). In contrast, conducted vasodilation evoked by ACh remained intact at 1000 µm. The reduction in conducted vasodilation at 500 µm (Figure 3) was associated with partial loss of tone in the segment of LDT,7 where resting diameter increased from 26±2 to 46±2 µm (P<0.05, n=10).
|
For stimuli delivered at the site of LDT, vasodilation to ACh was abolished locally along with the conducted response at 1000 µm. Local constriction to PE increased from -21±2 µm (control) to -29±2 µm (P<0.05, n=6), whereas conducted vasoconstriction at 1000 µm remained intact (-8±2 µm). After LDT, the local response to BK converted from vasodilation (22±3 µm) to vasoconstriction (-13±3 µm; P<0.05, n=6) that did not conduct. KCl evoked vasoconstriction (local, -16±3 µm) that readily conducted (-14±1 µm; n=6), with no evidence of the vasodilatory component. Local dilation to SNP was reduced after LDT (8±2 versus 20±1 µm; P<0.05, n=10) in association with the partial loss of tone.
Light-Dye Treatment of Smooth Muscle
For stimuli delivered at 0 µm with responses observed at 1000 µm, disruption of smooth muscle at 500 µm had no effect on conducted vasodilation in response to ACh or BK, or to the dilatory component of the biphasic response to KCl (Figure 4). However, conduction of vasoconstriction in response to PE was abolished, along with the constrictor component of the biphasic response to KCl (Figure 4).
|
At the site of LDT, the loss of all responses confirmed disruption of smooth muscle integrity by LDT. With stimulation at 500 µm and observations at 1000 µm, conduction of vasoconstriction in response to PE (-11±2 µm, n=5) or to KCl (-14±2 µm, n=6) was abolished by LDT. However, the conduction of vasodilation (pre- versus post-LDT, respectively) in response to ACh (14±2 versus 16±2 µm, n=6), to BK (13±2 versus 12±2 µm; n=5), or to KCl (10±3 versus 13±1 µm; n=6) remained intact.
Dual Light-Dye Treatment
Under control conditions, the conduction of vasodilation in response to ACh was equally effective in both directions for at least 2000 µm (Figure 5). After LDT, control diameter (30±2 µm; n=7) was maintained along arterioles except for constriction (to 21±3 µm; P<0.01) of the segment with smooth muscle disruption and dilation (to 54±2 µm; P<0.01) of the segment with endothelium disruption. When ACh was applied at 0 or 2000 µm, conduction of vasodilation in the middle of the vessel (observed at 1000 µm) from either direction through the first site of LDT was not different from control (Figure 5). However, conduction was ineffective beyond the second site of LDT in both directions. In 5 of these experiments, conducted responses were abolished beyond the second site of LDT. In 2 remaining experiments, the conducted response (4±1 µm) was diminished by 60%.
|
In additional control experiments (n=4), conducted vasodilation through two sites of LDT in the same cell layer (ie, at 500 and 1500 µm in endothelium or in smooth muscle) remained intact along 2000 µm. In one arteriole studied along a total distance of 4000 µm, conduction initiated in the middle of the arteriole was blocked at 2000 µm in the direction of dual LDT (as in Figure 5) yet remained intact for the same distance in the opposite (untreated) direction.
Inhibition of NO Production
Under control conditions, ACh and BK increased local arteriolar diameter by 25 to 30 µm (Figure 1). Within 10 minutes of adding L-NA to the superfusate, arteriolar diameter decreased from 35±1 to 24±1 µm (n=17, P<0.01). Local dilation to ACh was reduced to 21±3 µm (P<0.05) after 25 minutes and stabilized thereafter. Local dilation to BK was attenuated progressively through 15 minutes of L-NA; by 25 minutes, the response reversed to vasoconstriction (-8±6 µm), which increased to -14±3 µm at 35 minutes. Remaining experiments equilibrated with L-NA for at least 30 minutes.
In the presence of L-NA, conducted vasodilation to ACh was maintained but was abolished to BK (Figure 6), and the ensuing vasoconstriction to BK did not conduct. For KCl, conducted vasodilation was also abolished and the local dilatory response was attenuated by 47%. In contrast, conducted vasoconstriction to PE or to KCl remained intact. Attenuation of the local vasoconstriction to PE (Figure 6) was associated with the reduction in baseline diameter. Each effect of L-NA was reversed within 10 minutes of adding L-Arg. Maximal diameter with topical SNP was unaffected by L-NA.
|
Inhibition of NO Production With Disruption of Smooth Muscle Conduction
After smooth muscle disruption at 1500 µm, the conduction of vasodilation in response to ACh delivered at 1000 µm was not different from control in either direction along the arteriole (ie, at 0 or 2000 µm; Figure 7). With the addition of L-NA, however, conducted vasodilation was attenuated by 60% beyond the site of smooth muscle LDT (eg, at 2000 µm) while being maintained in the direction that both cell layers remained intact (eg, at 0 µm; Figure 7). This effect of L-NA also reversed with L-Arg.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study has investigated cellular signaling pathways underlying the conduction of vasomotor responses along arterioles controlling blood flow in the cheek pouch of anesthetized hamsters. Distinct sites of endothelium or smooth muscle were selectively disrupted alone or in combination with the inhibition of NOS activity. Conduction was initiated with endothelium-dependent vasodilators (ACh, BK), with a smooth muscle agonist (PE), and independent of membrane receptors (KCl). We show for the first time that conduction of hyperpolarization along arteriolar endothelium5 produces concomitant release of NO that relaxes smooth muscle cells along the vessel wall. This "wave" of NO is essential to conduction with BK but is masked when smooth muscle provides a parallel conduction pathway, which occurs when ACh is the stimulus (Figure 8). Remarkably, the biphasic response to KCl is explained by initiation and conduction of vasoconstriction along smooth muscle and of vasodilation along endothelium. These complementary signaling pathways illustrate both selectivity and redundancy in mechanisms by which cell-to-cell conduction can coordinate vasomotor responses in arteriolar resistance networks. Our findings indicate that myoendothelial coupling observed in vitro6,11,12,21,22 is not manifest under the conditions of the present experiments and substantiate that endothelium and smooth muscle can each provide an independent, homocellular pathway for conduction in vivo.5,7
|
Bidirectional electrical coupling between smooth muscle and endothelium implies that respective cell layers comprise an electrical syncytium.22 In isolated arterioles8,12 and feed arteries,6,23 intracellular recordings from endothelium and smooth muscle indicate that myoendothelial gap junctions enable hyperpolarization and depolarization to spread readily between cell layers and to thereby coordinate vasomotor responses along resistance microvessels. However, our finding that LDT at a single site could eliminate conducted vasodilation (with endothelium disruption; Figure 3) or conducted vasoconstriction (with smooth muscle disruption; Figure 4) is not consistent with effective electrical coupling between respective cell layers. Further, after dual LDT, the ability of vasodilation to conduct past either smooth muscle or endothelial disruption, but not beyond both sites in series (Figure 5), provides further evidence against effective heterocellular coupling along arterioles in the superfused cheek pouch.5,7,24 In light of differences reported for myoendothelial coupling between studies performed in vivo and those performed using isolated vessels, we suggest that heterocellular coupling in the arteriolar wall may be modulated by experimental conditions. It may also be possible to regulate myoendothelial coupling and/or conduction in a stimulus-specific manner.23
The biphasic response to microejection of KCl is explained by the initiation and conduction of vasoconstriction along smooth muscle and of vasodilation along endothelium (Figures 3 and 4 
). Thus, the initial vasoconstriction reflects depolarization and activation of voltage-gated Ca2+ channels in smooth muscle, as predicted by a Nernst effect and confirmed here by the inhibition of vasoconstriction with nifedipine (Figure 2). However, with membrane potential of -30 to -35 mV,5,9 arteriolar dilation when raising [K+]o to 10 to 20 mmol/L is not consistent with a passive effect on cellular excitability. In turn, we propose that as the local elevation in [K+]o dissipates, vasoconstriction can give way to vasodilation on activation of KIR25,26 channels or Na+/K+ ATPase activity.27 This interpretation is also consistent with observing only vasodilation in response to the shortest pulse of KCl (Figure 2). Although further experiments will be required to resolve the contributions of KIR channels or Na+/K+ ATPase activity to arteriolar dilation in the cheek pouch, our finding that respective components of the biphasic response to KCl are conducted along distinct cellular pathways provides additional evidence that smooth muscle and endothelium are not directly coupled to each other in vivo.5,7,24 Thus, according to the integrity of respective cell layers and changes in local milieu, elevating [K+]o can differentially activate arteriolar endothelium and smooth muscle to initiate the conduction of vasodilation and vasoconstriction, respectively.
Unmasking a Role for NO in Conducted Vasodilation
In response to ACh, a role for NO has been investigated through competitive inhibition of NOS activity using analogues of L-arginine.9,11,13,15,16 Consistent with these earlier studies, conducted vasodilation to ACh was maintained during treatment with 100 µmol/L L-NA, despite significant vasoconstriction and attenuation of the local response (Figure 6). At the same time, L-NA eliminated conducted vasodilation to BK and to KCl (Figure 6) and reversed the BK response to vasoconstriction that did not conduct, suggesting that smooth muscle contraction occurred without a significant change in membrane potential; eg, through release of Ca2+ from intracellular stores or changes in Ca2+ sensitivity of regulatory proteins. Our finding that the actions of L-NA were reversed with a 10-fold excess of L-arginine supports the specificity of NOS inhibition. Further, the differential inhibition of conducted vasodilation to BK and to KCl, but not to ACh, indicates that vasoconstriction itself is not the mechanism by which L-NA exerted its effect. Moreover, arteriolar conduction is maintained with similar levels of vasoconstriction during elevated superfusate PO2.2,17,18 Thus, for two vasodilator responses triggered independent of muscarinic receptors, smooth muscle relaxation along arterioles is explained by NO release along endothelium. Indeed, the actions of L-NA mimic those seen after disruption of endothelium integrity (Figure 3). The observation that conducted vasoconstriction to PE or to KCl was unaffected by L-NA or by disruption of endothelium strengthens the conclusion that smooth muscle alone mediates this response.7
Arteriolar conduction is often studied along segments 1 mm long.4,5,7–9,13 The present experiments establish that conduction is bidirectional and equivalent for distances of at least 2 mm (Figure 5). Thus, with ACh delivered at the center of a 2-mm segment and LDT performed midway along a 1-mm segment in one direction, the 1-mm segment in the opposite direction provided a corresponding control (Figure 7). In this manner, conducted responses to ACh were compared at equivalent distances along the same vessel in the presence of L-NA both with and without smooth muscle disruption. Our finding that the conducted response was inhibited (reversibly) by L-NA only beyond the segment of smooth muscle disruption lends further support to the conclusion that conduction along the endothelium generates the release of NO (Figure 8). In response to ACh, the endothelium can also release a signal that can initiate conducted vasodilation along smooth muscle, which we attribute to production of EDHF9,16 (Figure 8B) and the induction of hyperpolarization along this parallel conduction pathway.5,7 In contrast to ACh, neither BK nor KCl appears to initiate conduction along the smooth muscle layer.
The distinction between conducted vasodilation triggered by ACh as compared with BK may be explained by differences in respective signaling pathways28–30 and whether eNOS is activated alone or in conjunction with cytochrome P-450 enzymes to produce EDHF.9,16 During L-NA treatment, the residual conduction to ACh after smooth muscle disruption (Figure 5) may reflect incomplete inhibition of NOS, although the concentration of inhibitor used here (100 µmol/L) is 2- to 10-fold greater than used in previous studies.9,11,13,15,16 It is also possible that there is residual release of another autacoid along the arteriolar wall in response to ACh. Indeed, the combination of L-NA+17-octadecynoic (a cytochrome P-450 antagonist) suppressed conducted vasodilation to ACh by 80%,9 which is greater than the 60% inhibition observed here with L-NA alone. Although indomethacin alone has no effect on conduction,9 prostaglandins may further contribute to the residual response.16 Nevertheless, and despite such redundancy in signaling pathways for endothelium-dependent vasodilation, the present experiments are the first to resolve a novel and integral role for NO in conducted vasodilation that has been masked in previous studies using ACh as the stimulus.2,9,13–16 In turn, we propose that the role of endothelium or smooth muscle in providing a conduction pathway can be determined by whether NO and/or EDHF, respectively, are released in response to an endothelium-dependent vasodilator (Figure 8B).
Nitric oxide donors delivered as a brief local stimulus have proven to be ineffective in generating conducted responses,9,13,16,31 as confirmed here with SNP. In light of the present findings, we propose that eNOS is activated rapidly and transiently32 as hyperpolarization travels along the endothelium,5 eg, through promoting the driving force for calcium entry.33 This prediction is supported by the common requirement for integrity of the endothelium to conduct vasodilation in response to BK, to KCl, and to ACh after smooth muscle disruption.
In summary, this study has revealed an integral role for NO in mediating conducted vasodilation in arterioles of the hamster cheek pouch. With conduction along the endothelium in response to BK and to KCl, a "wave" of NO is released along the arteriolar wall that is integral to vasodilation. In response to ACh, conduction is triggered in parallel along the smooth muscle layer, masking the NO-dependent pathway that is resolved here by disrupting smooth muscle integrity in conjunction with NOS inhibition. Both the initiation and conduction of vasoconstriction are inherent to smooth muscle and independent of endothelium. In such fashion, respective cell layers can provide parallel pathways for conduction that are distinct as well as complementary. These findings imply that the consequences of vascular pathology on blood flow control in arteriolar networks will depend not only on stimulus specificity but also on the functional integrity of each cell layer.
|
Acknowledgments |
|---|
This study was supported by grant RO1-HL41026 from the National Institutes of Health and by the Heritage Affiliate of the American Heart Association, Postdoctoral Fellowship 9920265T (I.S.B.). Dr Robin Looft-Wilson provided valuable comments and critical review of this work.
|
Footnotes |
|---|
Original received October 9, 2002; resubmission received March 11, 2003; revised resubmission received May 23, 2003; accepted May 23, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Segal SS, Damon DN, Duling BR. Propagation of vasomotor responses coordinates arteriolar resistances. Am J Physiol. 1989; 256: H832–H837.[Medline] [Order article via Infotrieve]
2. Hungerford JE, Sessa WC, Segal SS. Vasomotor control in arterioles of the mouse cremaster muscle. FASEB J. 2000; 14: 197–207.
3. Segal SS, Duling BR. Flow control among microvessels coordinated by intercellular conduction. Science. 1986; 234: 868–870.
4. Xia J, Duling BR. Electromechanical coupling and the conducted vasomotor response. Am J Physiol. 1995; 269: H2022–H2030.[Medline] [Order article via Infotrieve]
5. Welsh DG, Segal SS. Endothelial and smooth muscle cell conduction in arterioles controlling blood flow. Am J Physiol. 1998; 274: H178–H186.[Medline] [Order article via Infotrieve]
6. Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control. Circ Res. 2000; 87: 474–479.
7. Bartlett IS, Segal SS. Resolution of smooth muscle and endothelial pathways for conduction along hamster cheek pouch arterioles. Am J Physiol Heart Circ Physiol. 2000; 278: H604–H612.
8. Xia J, Little TL, Duling BR. Cellular pathways of the conducted electrical response in arterioles of hamster cheek pouch in vitro. Am J Physiol. 1995; 269: H2031–H2038.[Medline] [Order article via Infotrieve]
9. Welsh DG, Segal SS. Role of EDHF in conduction of vasodilation along hamster cheek pouch arterioles in vivo. Am J Physiol Heart Circ Physiol. 2000; 278: H1832–H1839.
10. Little TL, Xia J, Duling BR. Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall. Circ Res. 1995; 76: 498–504.
11. Dora KA, Doyle MP, Duling BR. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc Natl Acad Sci U S A. 1997; 94: 6529–6534.
12. Yamamoto Y, Klemm MF, Edwards FR, Suzuki H. Intercellular electrical communication among smooth muscle and endothelial cells in guinea-pig mesenteric arterioles. J Physiol. 2001; 535: 181–195.
13. Doyle MP, Duling BR. Acetylcholine induces conducted vasodilation by nitric oxide-dependent and -independent mechanisms. Am J Physiol. 1997; 272: H1364–H1371.[Medline] [Order article via Infotrieve]
14. Delashaw JB, Duling BR. Heterogeneity in conducted arteriolar vasomotor response is agonist dependent. Am J Physiol. 1991; 260: H1276–H1282.[Medline] [Order article via Infotrieve]
15. Segal SS, Welsh DG, Kurjiaka DT. Spread of vasodilatation and vasoconstriction along feed arteries and arterioles of hamster skeletal muscle. J Physiol. 1999; 516: 283–291.
16. Hoepfl B, Rodenwaldt B, Pohl U, De Wit C. EDHF, but not NO or prostaglandins, is critical to evoke a conducted dilation upon ACh in hamster arterioles. Am J Physiol Heart Circ Physiol. 2002; 283: H996–H1004.
17. Segal SS, Duling BR. Conduction of vasomotor responses in arterioles: a role for cell-to-cell coupling? Am J Physiol. 1989; 256: H838–H845.[Medline] [Order article via Infotrieve]
18. Kurjiaka DT, Segal SS. Interaction between conducted vasodilation and sympathetic nerve activation in arterioles of hamster striated muscle. Circ Res. 1995; 76: 885–891.
19. Duling BR. Effects of potassium ion on the microcirculation of the hamster. Circ Res. 1975; 37: 325–332.
20. Emerson GG, Segal SS. Endothelial cell pathway for conduction of hyperpolarization and vasodilation along hamster feed artery. Circ Res. 2000; 86: 94–100.
21. Yashiro Y, Duling BR. Integrated Ca2+ signaling between smooth muscle and endothelium of resistance vessels. Circ Res. 2000; 87: 1048–1054.
22. von der Weid PY, Beny JL. Simultaneous oscillations in the membrane potential of pig coronary artery endothelial and smooth muscle cells. J Physiol. 1993; 471: 13–24.
23. Emerson GG, Neild TO, Segal SS. Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine. Am J Physiol Heart Circ Physiol. 2002; 283: H102–H109.
24. Chen Y, Rivers RJ. Arteriolar occlusion causes independent cellular responses in endothelium and smooth muscle. Microcirculation. 2002; 9: 353–362.[CrossRef][Medline] [Order article via Infotrieve]
25. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol. 1995; 268: C799–C822.[Medline] [Order article via Infotrieve]
26. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev. 2001; 81: 1415–1459.
27. Weston AH, Richards GR, Burnham MP, Feletou M, Vanhoutte PM, Edwards G. K+-induced hyperpolarization in rat mesenteric artery: identification, localization and role of Na+/K+-ATPases. Br J Pharmacol. 2002; 136: 918–926.[CrossRef][Medline] [Order article via Infotrieve]
28. Delmas P, Wanaverbecq N, Abogadie FC, Mistry M, Brown DA. Signaling microdomains define the specificity of receptor-mediated InsP3 pathways in neurons. Neuron. 2002; 34: 209–220.[CrossRef][Medline] [Order article via Infotrieve]
29. Vequaud P, Thorin E. Endothelial G protein ß-subunits trigger nitric oxide-but not endothelium-derived hyperpolarizing factor-dependent dilation in rabbit resistance arteries. Circ Res. 2001; 89: 716–722.
30. Lagaud GJ, Skarsgard PL, Laher I, van Breemen C. Heterogeneity of endothelium-dependent vasodilation in pressurized cerebral and small mesenteric resistance arteries of the rat. J Pharmacol Exp Ther. 1999; 290: 832–839.
31. Kurjiaka DT, Segal SS. Conducted vasodilation elevates flow in arteriole networks of hamster striated muscle. Am J Physiol. 1995; 269: H1723–H1728.[Medline] [Order article via Infotrieve]
32. Malinski T, Taha Z. Nitric oxide release from a single cell measured in situ by a porphyritic-based microsensor. Nature. 1992; 358: 676–878.[CrossRef][Medline] [Order article via Infotrieve]
33. Luckhoff A, Busse R. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pflugers Arch. 1990; 416: 305–311.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  C. de Wit Different pathways with distinct properties conduct dilations in the microcirculation in vivo Cardiovasc Res, October 31, 2009; (2009) cvp340v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. G. Bohlen, X. Zhou, J. L. Unthank, S. J. Miller, and R. Bills Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1337 - H1346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. T. Tran, E. J. Vigmond, F. Plane, and D. G. Welsh Mechanistic basis of differential conduction in skeletal muscle arteries J. Physiol., March 15, 2009; 587(6): 1301 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. F. Figueroa and B. R. Duling Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H2001 - H2007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Arciero, B. E. Carlson, and T. W. Secomb Theoretical model of metabolic blood flow regulation: roles of ATP release by red blood cells and conducted responses Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1562 - H1571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Parkington, M. Tare, and H. A. Coleman The EDHF Story: The Plot Thickens Circ. Res., May 23, 2008; 102(10): 1148 - 1150. [Full Text] [PDF]
|
|
|
|
|
|
T. M. Griffith Which Connexins Connect? Circ. Res., December 7, 2007; 101(12): 1219 - 1221. [Full Text] [PDF]
|
|
|
|
|
|
Y. N. Tallini, J. F. Brekke, B. Shui, R. Doran, S.-m. Hwang, J. Nakai, G. Salama, S. S. Segal, and M. I. Kotlikoff Propagated Endothelial Ca2+ Waves and Arteriolar Dilation In Vivo: Measurements in Cx40BAC GCaMP2 Transgenic Mice Circ. Res., December 7, 2007; 101(12): 1300 - 1309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Wolfle, V. J. Schmidt, B. Hoepfl, A. Gebert, S. Alcolea, D. Gros, and C. de Wit Connexin45 Cannot Replace the Function of Connexin40 in Conducting Endothelium-Dependent Dilations Along Arterioles Circ. Res., December 7, 2007; 101(12): 1292 - 1299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. F. Figueroa, C.-C. Chen, K. P. Campbell, D. N. Damon, K. H. Day, S. Ramos, and B. R. Duling Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone? Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1371 - H1383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Rodenwaldt, U. Pohl, and C. de Wit Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2341 - H2348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Uhrenholt, T. L. Domeier, and S. S. Segal Propagation of calcium waves along endothelium of hamster feed arteries Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1634 - H1640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Domeier and S. S. Segal Electromechanical and pharmacomechanical signalling pathways for conducted vasodilatation along endothelium of hamster feed arteries J. Physiol., February 15, 2007; 579(1): 175 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. F. Figueroa, B. E. Isakson, and B. R. Duling Vascular Gap Junctions in Hypertension Hypertension, November 1, 2006; 48(5): 804 - 811. [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Brekke, W. F. Jackson, and S. S. Segal Arteriolar smooth muscle Ca2+ dynamics during blood flow control in hamster cheek pouch J Appl Physiol, July 1, 2006; 101(1): 307 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Siegl, M. Koeppen, S. E. Wolfle, U. Pohl, and C. de Wit Myoendothelial Coupling Is Not Prominent in Arterioles Within the Mouse Cremaster Microcirculation In Vivo Circ. Res., October 14, 2005; 97(8): 781 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Looft-Wilson, G. W. Payne, and S. S. Segal Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle J Appl Physiol, September 1, 2004; 97(3): 1152 - 1158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Clifford and Y. Hellsten Vasodilatory mechanisms in contracting skeletal muscle J Appl Physiol, July 1, 2004; 97(1): 393 - 403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Sandow, R. Looft-Wilson, B. Doran, T.H. Grayson, S. S. Segal, and C. E. Hill Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries Cardiovasc Res, December 1, 2003; 60(3): 643 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fleming Bobbing Along on the Crest of a Wave: NO Ascends Hamster Cheek Pouch Arterioles Circ. Res., July 11, 2003; 93(1): 9 - 11. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||