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(Circulation Research. 2000;87:1048.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Integrated Ca²⁺ Signaling Between Smooth Muscle and Endothelium of Resistance Vessels

Yasuaki Yashiro, Brian R. Duling

From the First Department of Physiology (Y.Y.), Shinshu University School of Medicine, Matsumoto, Japan, and Department of Molecular Physiology and Biological Physics (B.R.D.), University of Virginia Health Sciences Center, Charlottesville, Va.

Correspondence to Dr Brian R. Duling, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, PO Box 800736, Charlottesville, VA 22908-0736. E-mail brd{at}virginia.edu

	Abstract

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Abstract—Cell-cell communication in the arteriolar wall was examined using the Ca²⁺-sensitive indicator fura-2 and the Ca²⁺ buffer BAPTA as means of measuring and buffering cellular Ca²⁺. The experiments focused on the role of endothelial cell [Ca²⁺]_i in modulating phenylephrine (PE)-induced contractions in in vitro arterioles of the hamster cremaster. Fura-2-AM and BAPTA-AM were applied intraluminally to accomplish endothelium-specific loading. PE was applied to short segments of arterioles using pressure-pulse ejection from a micropipette. Under control conditions at the site of stimulation, PE elicited a strong vasoconstriction preceded by an increase in endothelial cell [Ca²⁺]_i. A very small biphasic conducted response was observed at sites upstream from the stimulation site. BAPTA sharply reduced the measured Ca²⁺ response in the endothelium. This was associated with an enhanced local contractile response. In addition, the biphasic conducted response was converted into a strong conducted vasoconstriction. PE caused an initial rise in smooth muscle [Ca²⁺]_i at the stimulated site, which was followed by a rapid decrease below baseline. Endothelial cell loading of BAPTA had minimal effect on the initial [Ca²⁺]_i peak but eliminated the secondary decrease in smooth muscle [Ca²⁺]_i. Intraluminal application of charybdotoxin plus apamin mimicked the change in vasomotor state induced by BAPTA. These data lead us to hypothesize that, after smooth muscle stimulation, intercellular Ca²⁺ signaling between smooth muscle and endothelium causes a secondary rise in endothelial cell Ca²⁺, which triggers a hyperpolarizing event and initiates a conducted vasodilation. We conclude that smooth muscle and endothelium operate as a functional unit in these vessels.

Key Words: phenylephrine • gap junctions • endothelium • [Ca²⁺]_i • hyperpolarization

	Introduction

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Changes in arteriolar diameter spread bidirectionally from a localized site of stimulation. This coordination of arteriolar contraction along the vessel axis is achieved by a flow of current, apparently passing through gap junctions in one or more cells of the vascular wall.^{1 2 3 4 5} In arterioles composed of one layer of smooth muscle cells surrounding the endothelium, the close apposition of the 2 cell types also enables a direct communication, perhaps through myoendothelial gap junctions, with a resultant complex intercellular signaling pattern.^{2 6 7 8} The full contribution of myoendothelial gap junctions or other diffusible indicators to both local and conducted responses, however, is not well understood.

Phenylephrine (PE) is a potent vasoconstrictor, which acts in small arterioles via ₁-adrenoceptors on vascular smooth muscle (VSM). PE has in addition been shown to increase endothelial cell [Ca²⁺]_i in arterioles in vitro.⁶ It has been suggested that the increase in endothelial cell [Ca²⁺]_i might be due to secondary diffusion of Ca²⁺ or other messenger from smooth muscle to endothelium through myoendothelial gap junctions. The increase in endothelial cell [Ca²⁺]_i is capable of stimulating NO synthase, with a gradual onset over 1 to 2 minutes.^{6 9}

The goal of the present study was to determine whether the secondary rise in endothelial cell [Ca²⁺]_i might also modulate local and conducted responses through signaling processes other than NO production. Also, the onset of the NO production after PE stimulation is rather slow, and we wondered whether additional and more rapid signaling mechanisms might be activated after a brief stimulation with PE. We tested these ideas by damping changes in endothelial cell Ca²⁺ using selective chelation of endothelial Ca²⁺ with the Ca²⁺chelator BAPTA. We also examined the effect of blockers of calcium-activated K⁺ channels (K_Ca) on the PE-induced conducted response, given that K⁺ channels play a crucial role in regulating V_m, thus making them a prime candidate linking increases in [Ca²⁺]_i to changes in V_m.

	Materials and Methods

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Procedures and protocols used in this study were approved by the University of Virginia Animal Care and Use Committee. In all experiments, male Golden hamsters (138±3 g, n=40; Charles River Breeding Laboratories, Wilmington, Mass) were anesthetized with pentobarbital sodium (50 mg/10 g, IP), and the cremaster muscle was excised, freed of connective tissue, and spread out in a refrigerated (4°C) dissection chamber. Arterioles (59±2 µm, n=40, resting diameter) were isolated, cannulated, superfused at a rate of 2 mL/min, and observed using video microscopy.^{6 10}

To stimulate the vessels, a pipette (tip diameter 5 µm) was positioned near the vessel (within 20 µm), and agonists were pressure-pulse ejected (20 psi) onto the abluminal surface of the vessel using a Pneumatic PicoPump (PV 820, World Precision Instruments). Under conditions used in these experiments, fluorescent dye (fluorescein, molecular weight 332.31) ejected from the stimulating pipette moved <200 µm in the upstream direction.

Fluorescence Imaging
For measurement of endothelial cell [Ca²⁺]_i, the Ca²⁺-sensitive indicator fura-2 was selectively loaded into endothelial cells by intraluminal perfusion as described previously.¹¹ Vessels were illuminated with epifluorescence (125-W xenon arc lamp) at a magnification of x900. Fura-2 was excited at 340 and 380 nm, and the emission light was sampled at 510 nm with an intensified charge-coupled device camera (XR GenIII, Stanford Photonics). The images were recorded, digitized, and stored with the MetaFluor system (Universal Imaging Corp) for subsequent [Ca²⁺]_i estimation from a region of interest.¹²

For measurement of smooth muscle [Ca²⁺]_i, VSM of isolated arterioles was selectively loaded by flowing fura-2–acetoxymethyl ester (AM) in the superfusion solution.¹³ Images of the fluorescent arteriole were acquired using an identical acquisition routine as described above for the endothelial loading.

Protocols
After the equilibration period, changes in arteriolar diameter in response to a short pulse of PE (1 mmol/L pipette concentration, 0.5- to 1.0-second pulse) were observed at the site of the stimulating pipette (designated as "local" in the figures) and at sites 500 and 1000 µm upstream from the stimulating pipette. Responses were then re-examined after the following treatments: (1) prazosin superfusion (10 nmol/L), (2) no intraluminal flow, (3) superfusion with N^G-nitro-Ld-arginine methyl ester (L-NAME, 10 µmol/L for >20 minutes), (4) buffering of endothelial cell Ca²⁺ with luminal application of BAPTA-AM (10 µmol/L) for 15 to 30 minutes followed by a 20-minute wash, and (5) perfusion with iberiotoxin (100 nmol/L) or charybdotoxin (CTX; 100 nmol/L) in combination with apamin (500 nmol/L).

Changes in endothelial or smooth muscle cell [Ca²⁺]_i in response to the PE pulse were measured at the site of stimulation before and after the intraluminal BAPTA treatment. Changes in diameter and endothelial [Ca²⁺]_i were also measured during cumulative application of PE (10^-7 to10^-5 mol/L, 4 minutes each) to either the perfusion or superfusion solution and after passive vasoconstriction induced by a sudden pressure drop from 40 to 0 mm Hg.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Effect of PE Pulses on Local and Conducted Responses of Cremasteric Arterioles
During the equilibration period, arterioles developed tone to roughly 50% of their maximal diameter (110±2 µm to 59±2 µm, n=40). Typical responses of an isolated arteriole to a PE pulse are shown in Figure 1A. At the site of stimulation, PE (0.5- to 1.0-second pulse, n=25) caused a vasoconstriction (Figures 1A and 1B). At the 2 upstream sites (500 and 1000 µm), on the other hand, a very small biphasic response was observed (constriction followed by dilation). Peak conducted constrictions were 5.1±1.0% (500 µm, P<0.01) and 3.6±0.8% (1000 µm, P<0.01) of their baseline diameter, respectively. The conducted dilation was relatively larger, reaching 15.0±2.0% (500 µm, P<0.01) and 13.8±2.1% (1000 µm, P<0.01). Vessel diameter returned to baseline level within 1 minute after the onset of stimulation. Pretreatment with the ₁-adrenoceptor blocker prazosin produced a 77% attenuation of the PE-induced vasoconstriction at the site of stimulation and completely eliminated conducted responses at the 2 upstream sites (Figure 1B). In addition, neither absence of intraluminal flow nor NO blockade with L-NAME had any inhibitory effect on the pattern of PE-induced local and conducted responses (Figure 2).

View larger version (26K): [in this window] [in a new window]   Figure 1. Figure 1. Time course of changes in vessel diameter in response to abluminal pressure-pulse application of PE (1 mmol/L, 0.5- to 1.0-second pulse). A, Typical trace of changes in vessel diameter in response to PE in an isolated perfused arteriole. PE elicited a large decrease in diameter at the site of stimulation (local) and small biphasic changes (constriction followed by dilation) at 2 upstream sites. B, Summary data of changes in vessel diameter in response to PE (n=25) in isolated perfused arterioles. Pretreatment with prazosin (10 nmol/L, n=4) significantly reduced the local response and eliminated the conducted responses to PE. Diameters are normalized as a percentage of baseline diameters before PE stimulation. Values are mean±SE. *Significant difference (P<0.05) from initial diameter.

View larger version (18K): [in this window] [in a new window]   Figure 2. Figure 2. Lack of dependency of PE-induced responses on flow or NO production. L-NAME (10 µmol/L, n=5) was perfused in the bath for 20 minutes before the onset of PE stimulation. Neither 0 flow (n=3) nor L-NAME had any inhibitory effect on the PE-induced conducted vasodilation.

Effects of Endothelial Ca²⁺ Buffering on the PE-Induced Conducted Response
The Table shows estimated [Ca²⁺]_i before and after BAPTA treatment. Intraluminal BAPTA-AM application caused an initial vasoconstriction (20% to 40%). Within 20 minutes after washout of luminal BAPTA-AM, the vessel returned to the control diameter (63.6±2.8 µm before versus 65.8±3.7 µm after the BAPTA treatment, n=5). Typical responses of an isolated arteriole to PE after the BAPTA treatment are shown in Figure 3A. At the site of stimulation, there was an increase in magnitude and a substantial increase in the duration of vasoconstriction. At the upstream sites, the dilator component was eliminated, and PE caused only a conducted constriction that developed slowly as compared with the rapid and strong constriction observed at the local site. Note that the local constriction began to return to baseline while the conducted constrictions were still increasing. Diameters gradually returned to baseline over a period of 2 minutes after the onset of stimulation. Averaged data for 5 vessels are summarized in Figure 3B.

View this table: [in this window] [in a new window]   Table 1. Estimated [Ca2+]i Before and After Intraluminal Perfusion With BAPTA-AM

View larger version (29K): [in this window] [in a new window]   Figure 3. Figure 3. Effect of endothelial cell Ca2+ buffering on the local and conducted responses to pressure-ejected PE (1.0-second pulse, n=5). Endothelial cells of the isolated arterioles were loaded with BAPTA by luminal perfusion of the ester. Values are mean±SE. Significant differences (P<0.05) between control and BAPTA treatment groups.

Changes in Endothelium and Smooth Muscle [Ca²⁺]_i Before and After Endothelial Cell BAPTA Treatment
Application of PE caused a sharp, transient increase in the endothelial cell fura-2 fluorescence ratio at the site of stimulation; this peaked 6 seconds after the onset of stimulation (Figure 4A). The increase in endothelial cell [Ca²⁺]_i in response to PE was greatly attenuated after the BAPTA treatment. PE caused a similar rapid increase in the smooth muscle cell [Ca²⁺]_i, which then decreased rapidly below baseline (Figure 4B). The nadir in the Ca²⁺ signal occurred at 12 seconds (arrow in Figure 4B), after which [Ca²⁺]_i gradually returned to the initial level. After endothelial cell BAPTA loading, the initial rate of change of smooth muscle [Ca²⁺]_i in response to PE was unaltered, but the Ca²⁺ response was prolonged. In addition, the maximal constriction in response to PE in arterioles of which the smooth muscle cells were loaded with fura-2 (upper right panel, Figure 4), was slightly smaller compared with those of which the endothelial cells were loaded with fura-2 (61.1±7.0% versus 76.5±2.3%, lower right panel, Figure 4). The duration was also shorter. These changes may be the result of slight buffering of smooth muscle Ca²⁺ by fura-2. In both treatment groups, however, endothelial BAPTA treatment caused an enhanced vasoconstriction in response to PE, similar to that observed in the arterioles not loaded with fura-2 (Figure 3).

View larger version (43K): [in this window] [in a new window]   Figure 4. Figure 4. Time course of changes in [Ca2+]i and vessel diameter in response to abluminal pressure-pulse application of PE (1 mmol/L, 0.5- to 1.0-second pulse). A, Changes in endothelial [Ca2+]i and vessel diameter in isolated perfused arterioles the endothelial cells of which were loaded with fura-2 (n=7). B, Changes in smooth muscle [Ca2+]i and vessel diameter in isolated perfused arterioles the smooth muscle cells of which were loaded with fura-2 (n=5). Values are mean±SE. Significant differences (P<0.05) between control and BAPTA treatment groups.

Figure 5 is an alternative presentation of the relations between diameter and estimated [Ca²⁺]_i. This presentation emphasizes the differences between the 2 cell types and the effects of BAPTA. When the vessel is stimulated, the rise in smooth muscle cell [Ca²⁺]_i precedes the contractile response, and the initial rise in the smooth muscle cell [Ca²⁺]_i is both faster and greater than that in the endothelial cell (Figure 5, compare O_VSM–a with O_Endo–d). As the smooth muscle [Ca²⁺]_i rises to roughly 60% above control, the rate of contraction accelerates even though [Ca²⁺]_i begins to decrease (Figure 5, a–b). Throughout the contractile cycle, the endothelial cell [Ca²⁺]_i lags behind the diameter change rather than anticipating it as in the smooth muscle (compare a–b with d–e). Note the rapid acceleration in recovery of the endothelial cell [Ca²⁺]_i during a period in which there is little change in diameter (e–f). In the case of the BAPTA-loaded vessels, the endothelial Ca²⁺ shows a much smaller loop; ie, the endothelial cell Ca²⁺ correlates very poorly with the diameter changes. After BAPTA, the smooth muscle loop is minimally altered, although contraction occurs with a smaller change in Ca²⁺ (g–h). The similarity in the size of the calcium swings in smooth muscle during stimulation before and after treatment suggests that the BAPTA loading was relatively selective for endothelium. The diameter-Ca²⁺ loop for the endothelium is virtually eliminated, whereas the loop for the VSM is altered relatively little.

View larger version (29K): [in this window] [in a new window]   Figure 5. Figure 5. Comparison of the relative changes in diameter and [Ca2+]i for the 2 cell types in response to PE before and after BAPTA treatment. Changes in [Ca2+]i were plotted against changes in diameter for the 2 cell types using data from Figure 4. In each case the control condition is the 100%-to-100% locus. The intent of the figure is to emphasize the difference between calcium changes in smooth muscle and endothelium and to show the efficacy and selectivity of BAPTA treatment. Early response of smooth muscle is an elevation of calcium preceding the change in diameter (O–a). Note that during this phase there is a very modest change in endothelial cell Ca2+ (O–d). During the time that smooth muscle calcium is elevated, endothelial cell calcium continues to rise slowly (d–e). The figure also emphasizes the selectivity of the BAPTA buffering.

In another set of experiments, PE was applied selectively to either the adventitial or luminal side of an arteriole to establish that PE has minimal direct effect on the endothelial cell [Ca²⁺]_i. The efficacy of intraluminal application of PE was compared with that of adventitial application. As expected from prior experiments on cheek pouch arterioles, the endothelium of cremasteric arterioles acted as a significant barrier to PE movement.¹⁴ Luminal application of PE never produced a dilation, and the constrictor response was shifted from an EC₅₀ on the adventitial side of 5x10^-6 to 5x10^-4 when PE was applied to the luminal side of the vessel (data not shown). As would be anticipated, the degree of Ca²⁺ rise correlated well with the level of vasoconstriction induced by PE. Possible involvement of the diameter change per se on estimates of [Ca²⁺]_i was also assessed by reducing intraluminal pressure from 40 to 0 mm Hg. The resultant passive collapse of the vessel reduced arteriolar diameter by 54.8±3.8% of baseline but elicited only 18.8±2.4 nmol/L increase in apparent endothelial cell [Ca²⁺]_i (n=3).

Role of K⁺ Channel Activation in PE-Induced Conducted Responses
To test for a role of endothelial K_Ca channels in the PE-induced conducted response, vessels were perfused with a variety of blockers of Ca²⁺-sensitive potassium channels. Iberiotoxin alone or in combination with apamin (500 nmol/L),blockers of large-conductance and small-conductance K_Ca channels, respectively, had no inhibitory effect on the PE-induced local and conducted responses (Figure 6). Prior work by others^{15 16 17 18 19} had shown that in many vascular beds and cell preparations, a combination of CTX and apamin blocks endothelium-derived hyperpolarizing factor (EDHF). In these vessels, we found that perfusion of a combination of CTX (100 nmol/L) plus apamin (500 nmol/L) did not significantly alter control vascular diameter (54.1±4.8 µm control versus 53.2±4.4 µm during CTX plus apamin treatment, n=6). However, as shown in Figure 6, CTX plus apamin significantly enhanced the PE-induced vasoconstriction at the stimulated site and converted the dilatory component of the conducted response to a small conducted constriction.

View larger version (23K): [in this window] [in a new window]   Figure 6. Figure 6. Effects of luminal application of KCa blockers on the PE-induced diameter changes in isolated arterioles. Values are mean±SE. Significant differences (P<0.05) between control and KCa blockade groups. §Significant diameter reduction (P<0.05) from initial diameter at the upstream sites. IbTX indicates iberiotoxin.

	Discussion

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We have previously shown that contractile agonists that are known to act primarily on smooth muscle cells of arterioles (PE and KCl) cause an elevation in endothelial [Ca²⁺]_i in addition to the expected rise in smooth muscle [Ca²⁺]_i.⁶ With stimuli of 2 minutes’ duration, the contraction of the arteriole was associated with a rise in endothelial cell [Ca²⁺]_i and an accompanying increase in the synthesis of NO. However, NO synthase activation in arterioles is evident only after 15 to 30 seconds of stimulation.^{6 9} Delayed production of NO after endothelial cell stimulation has also been shown in rat mesenteric artery.²⁰ We interpret the foregoing data as showing that the frequent myoendothelial gap junctions in arterioles allow the rising Ca²⁺ in smooth muscle to generate a diffusion gradient that drives Ca²⁺ into endothelial cells, thereby initiating the synthesis of NO. It should be recognized that our methodology cannot distinguish simple calcium diffusion from the action of other second messengers such as inositol triphosphate (IP₃). However, given the very short distances involved and the molecular weights of Ca²⁺ and IP₃, the most likely species to diffuse over these short times would seem to be Ca²⁺.

In the present study, we demonstrated that brief application of PE induces a biphasic conducted response and that the rise in smooth muscle [Ca²⁺]_i is associated with a rise in endothelial [Ca²⁺]_i. Thus, the slow rise in NO production previously reported^{6 9} is anticipated by the biphasic conducted vasomotor responses observed here (Figure 2). We therefore hypothesized that different Ca²⁺-dependent signaling pathways are responsible for the complex conducted responses. Because conducted vasomotion is well predicted by changes in membrane potential,^{1 3} the biphasic responses seen at upstream sites (Figure 3) were probably due to an initial depolarization followed by a hyperpolarization. Our data are consistent with the idea that the hyperpolarization is mediated by an activation of one or more K⁺ channels that are sensitive to CTX plus apamin.^{15 16 17 18 19} The relative efficacy of BAPTA in buffering endothelial cell Ca²⁺ and modifying the response supports the idea that the functional change in Ca²⁺ occurs in the endothelium rather than the smooth muscle (Figures 5 and 6).

Effect of Endothelial Ca²⁺ Buffering on the PE-Induced Conducted Response
Luminal treatment with BAPTA-AM significantly reduced the Ca²⁺ response of endothelial cells at the site of stimulation in response to PE and at the same time enhanced the response of the smooth muscle. Moreover, the conducted dilation was converted to a conducted constriction. The data support our hypothesis that an elevation of endothelial [Ca²⁺]_i triggers the PE-induced conducted vasodilation. Functional data support the idea that BAPTA was largely confined to the endothelium; BAPTA neither altered the baseline arteriolar diameter nor reduced the baseline smooth muscle [Ca²⁺]_i (see Table). In addition, the initial rise in smooth muscle [Ca²⁺]_i in response to PE was slightly enhanced, not inhibited by the BAPTA treatment (Figure 4B). Although BAPTA treatment in our protocol failed to completely clamp the endothelial [Ca²⁺]_i (Figure 4A), the level of Ca²⁺ buffering seems to have been sufficient to eliminate the subsequent production of hyperpolarizing signals. We did find that prolongation of the incubation (>30 minutes) or higher BAPTA concentration often caused loss of myogenic tone, suggesting an entry of BAPTA into smooth muscle in addition to endothelial cells.

The pattern of [Ca²⁺]_i change in response to PE was clearly different between the 2 cell types (Figure 4). We speculate that an additional delay in the attainment of peak Ca²⁺ in the endothelium might be a diffusional delay, but as mentioned above, the involvement of a second messenger such as IP₃ cannot be excluded. A more subtle component of the late elevation of endothelial [Ca²⁺]_i might also be due to Ca²⁺ influx as a result of hyperpolarization of endothelial cells triggered by opening of K_Ca channels.²¹ The rapid decrease of smooth muscle [Ca²⁺]_i from its peak value, on the other hand, may be attributed to smooth muscle hyperpolarization and subsequent inhibition of voltage-gated Ca²⁺ channels in smooth muscle.

Characteristics of the Hyperpolarizing Signal
Figure 3 shows clearly that BAPTA loaded selectively into endothelial cells converts a small biphasic conducted vasomotor response into a large constriction. We hypothesize that the PE-induced conducted dilation was triggered by endothelial cell hyperpolarization induced by activation of K_Ca channels. This is supported by the facts that BAPTA was largely confined to the endothelium and that luminally applied K_Ca blockers eliminated the dilation response. The effect of K_Ca blockers was likely to have been limited to endothelium, because they did not alter vascular diameter significantly and because their structure would suggest lower membrane permeability. It remains to be shown (1) how K_Ca channels were activated and (2) how the hyperpolarizing signal was transmitted to the smooth muscle. The simplest explanation is that an elevated endothelial Ca²⁺ may directly activate K_Ca in endothelium to induce transmembrane currents that trigger endothelial cell hyperpolarization. The hyperpolarizing current could be transmitted to smooth muscle through myoendothelial gap junctions.⁸ Alternatively, a recent report suggested that elevated K⁺ in the subintimal space of the vessel, resulting from opening of endothelial cell K⁺ channels, might promote smooth muscle hyperpolarization through activation of inward rectifier K⁺ channels and/or Na⁺-K⁺-ATPase in smooth muscle.¹⁵

Certainly, the hyperpolarization could be due to the production of an as-yet-unidentified EDHF. The production and release of EDHF is thought to require an increase in [Ca²⁺]_i of the endothelium,²² consistent with the finding in the present study. EDHF is thought by some to comprise one or more metabolites of arachidonic acid produced through cytochrome P-450.^{23 24} The evidence for this has been presented both in large arteries and in hamster cremasteric arterioles.²⁵ Further investigation is required to clarify the mechanism of hyperpolarizing events involved in the PE-induced conducted dilation in this study.

The K_Ca blockers failed to convert the conducted dilation into as large a conducted constriction as did the BAPTA treatment. The incomplete effect of the K_Ca blockers may be related to the fact that the abluminal surface of the endothelium was not directly exposed to the blockers, ie, only a fraction of the channels were blocked. Alternatively, part of the conducted vasodilatation may be triggered by other calcium-sensitive K⁺ channels or by an unknown endothelial K_Ca-independent mechanism.

It is also important to recognize that many other K⁺ channel blockers may influence the responses that we observe. The changes in the pattern of the vasomotor responses at the conducted site, compared with the local site, are likely to involve reactive alterations in the activation of other K⁺ channels as the conducted signal moves down the length of the vessel. Clearly a more thorough investigation with other blockers is warranted to address this issue.

Characterization of the PE-Induced Conducted Constriction After Endothelial Ca²⁺ Buffering
After BAPTA treatment, PE caused a conducted vasoconstriction. There are several possible pathways that might be involved in the PE-induced depolarization. These include (1) activation of nonspecific cation channels,²⁶ (2) calcium-activated chloride channels,^{27 28 29} and (3) inactivation of K_Ca channels³⁰ and/or ATP-sensitive potassium channels.³¹ In contrast to the rapid vasoconstriction at the local site, however, the PE-induced conducted constriction, and thus, we infer, the conducted depolarization after the BAPTA treatment, developed rather slowly. Such slow depolarization might have been due to activation of smooth muscle K_Ca channels,³² thus buffering some other depolarizing signals. Also, it is known that highly localized Ca²⁺ release from sarcoplasmic reticulum (Ca²⁺ sparks) can regulate myogenic tone though activation of K_Ca channels in rat cerebral arteries.^{33 34} Whether such mechanisms also play a role in agonist-induced local and conducted responses in arterioles remains to be clarified.

It is worth noting that the presence or absence of a conducted response is also highly dependent on concentration and duration of exposure to the agonist. Thus, a lower concentration of PE (10 µmol/L in the pipette) failed to induce conducted responses, although it elicited substantial local vasoconstriction (data not shown). In contrast, a higher concentration of PE (1 mmol/L in the pipette) was able to induce the consistent biphasic conducted response, as reported here. Although the pipette concentration was high, local smooth muscle concentration would have been much lower and only transiently expressed. A possibility that PE acted directly on endothelium would be excluded because, as pointed out above, the increase in endothelial [Ca²⁺]_i in response to PE was remarkably less when the drug was applied intraluminally than when applied extraluminally. In addition, the efficacy of low-dose prazosin blockade (Figure 1B) argues strongly in favor of specific effects of PE.

The concentration dependence of the PE response is compatible with the idea that the relative contribution of electromechanical coupling or pharmacomechanical coupling to a response can be affected by either duration or concentration of the drugs applied.³⁵ In the present study, 1 mmol/L PE in the pipette induced strong and uniform vasoconstriction extending 100 µm upstream from the stimulated site. It is thus conceivable that local stimulation of a wide range of smooth muscle cells was required for endothelial cells to produce hyperpolarizing signals enough for causing conducted vasodilation.

Obviously, a key remaining question concerns the physiological relevance of the responses we observe and, in fact, of the conducted response in general. Large, transient elevations of neurotransmitters are common at nerve terminals. The use of micropipettes may mimic this situation. Alternatively, it may be that drug microapplication such as we have used serves as a tool that allows one to disclose the inherently complex, multicellular signaling that exists in the arteriolar wall.

In summary, in small hamster cremasteric arterioles in vitro, local stimulation with PE causes a graded ₁-dependent constriction and a biphasic conducted response. We hypothesize that the integrated arteriolar response is a synthesis of multiple processes initiated in at least 2 cell types (Figure 7). The result is a complex response composed of competing dilatory and constricting responses. We propose that the conducted dilatory component is secondary to Ca²⁺ signaling, perhaps via Ca²⁺ diffusion from smooth muscle to endothelium through myoendothelial gap junctions. The increase in endothelial [Ca²⁺]_i induces hyperpolarizing signals in endothelium, including activation of K_Ca channels. These K_Ca-dependent hyperpolarizing signals sum with the initial depolarizing signals of smooth muscle to produce a biphasic conducted response. Thus, the endothelium may play multiple roles as a coupled negative feedback system modulating VSM responses to agonist-induced vasoconstriction in arterioles.

View larger version (32K): [in this window] [in a new window]   Figure 7. Figure 7. Schematic of hypothesized interactions of Ca2+-dependent pathways on the PE-induced local and biphasic conducted responses in hamster cremasteric arterioles. "Ion channels" in this figure represent channels involved in the PE-induced depolarization, including nonselective cation channels, chloride channels, and potassium channels (see Discussion). PMC indicates pharmacomechanical coupling, EMC, electromechanical coupling, VOC, voltage-operated Ca2+ channels, SR, sarcoplasmic reticulum; and EC, endothelial cell. Openings connecting PE, VSM, lumen, and EC indicate gap junctions.

	Acknowledgments

 

This work was supported by NIH Grants HL53318 and HL12792. We are grateful to D.N. Damon for technical assistance and valuable discussions.

	Footnotes

 
Received September 7, 2000; revision received September 29, 2000; accepted September 29, 2000.

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