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Stroke. 1999;30:1125-1133

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(Stroke. 1999;30:1125-1133.)
© 1999 American Heart Association, Inc.

Original Contributions

P2u Receptor–Mediated Release of Endothelium-Derived Relaxing Factor/Nitric Oxide and Endothelium-Derived Hyperpolarizing Factor From Cerebrovascular Endothelium in Rats

Junping You, MD; T. David Johnson, PhD; Sean P. Marrelli, PhD; Jean-Vivien Mombouli, PhD Robert M. Bryan, Jr, PhD

From the Departments of Anesthesiology (J.Y., T.D.J., S.P.M., R.M.B.) and Medicine (J-V.M.) and the Graduate Program in Cardiovascular Sciences of the DeBakey Heart Center (S.P.M., R.M.B.), Baylor College of Medicine, Houston, Tex, and the Department of Internal Medicine, University of Lund (J.Y.), Lund, Sweden.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Background and Purpose—Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery (MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived hyperpolarizing factor (EDHF).

Methods—Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal perfusate to elicit dilations.

Results—Resting outside diameter of the MCAs in one study was 209±7 µm (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 µmol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to the right. Scavenging NO with hemoglobin (1 or 10 µmol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 µmol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 µmol/L UTP were accompanied by 13±2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28±2 to -41±1 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels, sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the large-conductance calcium-activated K channels, abolished the L-NAME–insensitive component of the dilation to UTP.

Conclusions—Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel.


Key Words: cerebrovascular circulation • endothelium-derived relaxing factor • endothelium, vascular • muscle, smooth • potassium channels • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The naturally occurring purine and pyrimidine phosphates—ATP, ADP, and UTP—dilate cerebral vessels by stimulating purinoceptors located on the endothelium.1 2 3 4 5 6 7 8 In the rat middle cerebral artery (MCA), ADP likely produces dilation through stimulation of the P2y (or P2Y1) purinoceptors with the subsequent synthesis and release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO).7 (The term EDRF/NO is used [in place of the commonly used term NO] to accurately reflect the fact that the molecular structure of this relaxing factor is not known. The relaxing factor responsible for stimulating guanylate cyclase in many circumstances may be an NO-containing compound. This is especially true in the cerebral circulation, where there is good evidence against the gas, NO, and evidence for a NO-containing compound.9 10 ) On the other hand, ATP and UTP dilate the rat MCA by stimulating P2u purinoceptors (likely P2Y2 subtype) with the subsequent release of EDRF/NO and possibly another endothelium-derived relaxing factor.7 NG-Nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, did not diminish the maximum dilation but did increase the concentration of UTP or ATP necessary to produce one half of the maximum dilation by {approx}10-fold. Either EDRF/NO was not completely blocked by L-NAME (10 µmol/L) or a relaxing factor other than EDRF/NO was also involved with the dilation.

In this study we tested the following hypothesis: The unknown relaxing factor involved with UTP-mediated dilations in rat MCAs is endothelium-derived hyperpolarizing factor (EDHF). EDHF, which is distinct from either EDRF/NO or prostacyclin, dilates vessels by hyperpolarizing the vascular smooth muscle through K channel activation.11 12 13 Given that EDHF has not been positively identified and that it may be more than a single compound,11 12 13 14 it would be difficult to positively determine the exact agent responsible for the unknown component of the dilation. Consequently, we asked whether this component of the UTP-mediated dilation had classic characteristics of EDHF.11 12 13 (1) Is it distinct from EDRF/NO? This issue is critical in light of a recent publication indicating that endothelial EDRF/NO may be difficult to block.15 (2) Is it distinct from prostacyclin or another cyclooxygenase metabolite? We have previously determined that the dilation did not involve a cyclooxygenase metabolite,7 and therefore we will not further address this issue in the present studies. (3) Does it hyperpolarize the vascular smooth muscle? (4) Are K channels involved in the dilation?


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The Animal Protocol Review committee at Baylor College of Medicine approved the experimental protocol. One hundred twenty male Long-Evans rats (weight, 250 to 350 g) were anesthetized with 3% isoflurane and decapitated. The brain was immediately removed and placed in cold (4°C) physiological salt solution (PSS). With the aid of a dissecting microscope, both MCAs were carefully harvested beginning at the circle of Willis and extending 6 to 8 mm distally. A section of the MCA (1 to 2 mm in length) was mounted in an arteriograph (Living Systems) as previously described.16 17 Micropipettes were inserted into both ends of each MCA and secured in place with nylon ties. Each MCA was bathed in PSS (37°C) that was equilibrated with a gas consisting of 20% O2/5% CO2 with a balance of N2.16 17 The pH of the bath was {approx}7.40, PCO2 {approx}35 mm Hg, and PO2 {approx}130 mm Hg.16

Luminal pressure of the MCAs was maintained at 85 mm Hg by raising reservoirs to the appropriate height above the MCAs.16 Luminal perfusion was adjusted to 100 µL/min by setting the 2 reservoirs at different heights. Pressure transducers on either side of the MCA provided a measurement of perfusion pressure across the MCA and pipettes. The vessels were magnified with an inverted microscope equipped with a video camera and monitor. Outside diameters of the MCAs were measured directly from the video screen. Agonists and other drugs were added either to the extraluminal bath (smooth muscle side) or to the PSS perfusing the lumen (endothelial side).

After mounting and pressurization, the MCAs developed spontaneous tone by constricting to {approx}75% of the initial diameter over the course of 1 hour. Experimental protocols were not initiated until the MCA diameter was stable over a 15-minute period.

Adding UTP to the luminal perfusate selectively stimulated endothelial purinoceptors.7 The change in MCA diameter was measured after exposure to UTP concentrations from 10-9 to 10-4 mol/L. Only 1 concentration-response curve was conducted for each MCA to avoid the risk of tachyphylaxis.

In 4 MCAs, membrane potential (Em) was measured in individual vascular smooth muscle cells with the use of glass microelectrodes filled with 3 mol/L KCl (impedances from 55 to 75 M{Omega}). The Em measurements were made in pressurized perfused MCAs mounted in the arteriograph so that diameters could be simultaneously recorded.18 The potential difference between the glass microelectrode and a reference electrode, placed in the bath of the arteriograph, was measured with a Dagan 8700 Cell Explorer with the output being displayed on a Tektronix 5223 digitizing oscilloscope. Micropipettes were made by pulling capillary tubing to a rapid taper (tip {approx}0.1 µm diameter) with the use of a model P-87 Brown-Flaming micropipette puller (Sutter). Primary criteria for a successful impalement included a sharp drop in voltage from baseline on entry of the microelectrode tip into the cell and no change in microelectrode resistance after exiting the cell. Em values from 5 different smooth muscle cells were averaged to obtain a single Em for a given condition in a single MCA. The number of observations (n) was the number of MCAs studied, not the number of impalements.

Drugs and Reagents
UTP, L-NAME, BaCl2, 4-aminopyridine, apamin, and KCl were purchased from Sigma Chemical Co. Glibenclamide, tetraethylammonium chloride (TEA), iberiotoxin, and charybdotoxin were purchased from Research Biochemicals Inc.

1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Tocris Cookson, Inc. Oxyhemoglobin was purchased from Calzyme Laboratories. ODQ was dissolved in ethanol; glibenclamide was dissolved in dimethyl sulfoxide; apamin was dissolved in 0.05 mol/L acetic acid solution. All other reagents and drugs were dissolved in distilled water.

L-NAME, oxyhemoglobin, and ODQ were added to both the abluminal bath and the luminal perfusate; UTP was added only to the luminal perfusate; all other drugs were added to the abluminal bath only. Vehicles for the blockers were given to the control MCAs. The composition of the PSS used to bathe the MCAs was previously described.16

Statistical Analysis
All data are presented as mean±SEM. For concentration-response curves to UTP, the results are presented as percentage of the maximum diameter of the MCAs and calculated by the following equation: % Maximum Diameter=[(DUTP–Dbase/(Dmax–Dbase)]x100, where Dmax is the maximum diameter of the MCA at 85 mm Hg, Dbase is the baseline diameter of the MCA before addition of UTP, and DUTP is the diameter of the MCA after the luminal administration of UTP. Dmax is the diameter of the MCA immediately after pressurization to 85 mm Hg and before development of spontaneous tone. Preliminary results demonstrated that Dmax, as calculated above, was identical to the diameter of the MCA in calcium-free buffer at 85 mm Hg.

For comparison of the concentration-response curves, repeated-measures ANOVA was used with a post hoc Student-Newman-Keuls test for comparison of individual data points. The acceptable level of significance was defined as P<0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Dilations to the luminal administration of UTP in control MCAs and after L-NAME (1 µmol/L to 1 mmol/L) are shown in Figure 1Down. Note that the dilations at 10-7 and 10-6 mol/L UTP (P<0.05 for each concentration compared with control) were either attenuated or abolished by L-NAME. However, the dilations to UTP in the presence of L-NAME were not altered at 10-5 or 10-4 mol/L UTP. The dilations in the presence of L-NAME were essentially identical regardless of the concentration (1000-fold increase in L-NAME concentration; Figure 1Down).



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  		Figure 1. Diameter change of rat MCAs in response to luminal applications of UTP in controls and after inhibition of NO synthase with L-NAME (1 µmol/L to 1 mmol/L). Diameters for each group before administration of UTP: control, 209±7 µm (n=10); 1 µmol/L L-NAME, 173±6 µm (n=5); 10 µmol/L L-NAME, 168±5 µm (n=17); 100 µmol/L L-NAME, 179±6 µm (n=5); 1 mmol/L L-NAME, 197±10 µm (n=3). With the use of 2-way repeated-measures ANOVA, there was a significant group effect (P<0.0001) and a significant interaction between group and concentration (P<0.001). *P<0.05 compared with all other groups at the same concentration (Student-Newman-Keuls method).

Figure 2Down shows the effects of oxyhemoglobin, a scavenger of EDRF/NO, alone or in combination with L-NAME (10 µmol/L) on UTP-mediated dilation. Oxyhemoglobin (10 µmol/L) altered the dilation to UTP in a manner similar to that of L-NAME (Figure 2aDown), that is, the dilations at 10-7 mol/L UTP (P=NS) and 10-6 mol/L UTP (P<0.05) were attenuated by oxyhemoglobin. With the use of repeated-measures ANOVA, the control and oxyhemoglobin groups reached statistical significance (P<0.04), and there was a significant interaction between groups and UTP concentration (P=0.007). Dilations to UTP in the presence of L-NAME and oxyhemoglobin were not significantly different from the dilations in the presence of L-NAME alone (Figure 2bDown).



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  		Figure 2. Effects of oxyhemoglobin (Hb), a scavenger of NO, alone (a) or in combination with 10 µmol/L L-NAME (b) on UTP-mediated dilation. Diameters for each group before administration of UTP: control, 216±6 µm (n=6); 10 µmol/L Hb, 205±8 µm (n=5); L-NAME, 164±7 µm (n=5); L-NAME+1 µmol/L Hb, 173±11 µm (n=5); L-NAME+10 µmol/L Hb, 164±12 µm (n=5). The control and Hb groups (a) were significantly different (P<0.04), and there was a significant interaction between group and concentration of UTP (P<0.0001, 2-way repeated-measures ANOVA). *P<0.05 compared with dilation in the control group with 10-6 mol/L UTP (Student-Newman-Keuls method).

Figure 3Down shows the effects of ODQ, a guanylate cyclase inhibitor, alone or in combination with L-NAME (10 µmol/L) on UTP-mediated dilation. ODQ, at concentrations of either 1 or 10 µmol/L, altered the dilation to UTP in a manner similar to that of L-NAME (P=0.0002 compared with control; Figure 3aDown). In the presence of L-NAME, ODQ did not further suppress the dilations to UTP (Figure 3bDown).



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  		Figure 3. Effects of ODQ, a guanylate cyclase inhibitor, alone (a) or in combination with 10 µmol/L L-NAME (b) on UTP-mediated dilation. ODQ, at concentrations of either 1 or 10 µmol/L, altered the dilation to UTP. With the use of 2-way repeated-measures ANOVA, there was a significant group effect (P<0.0001) and significant interaction between group and concentration of UTP (P=0.0002). Diameters for each group before administration of UTP: vehicle control, 168±6 µm (n=10); 1 µmol/L ODQ, 171±13 µm (n=6); 10 µmol/L ODQ, 158±3 µm (n=4); L-NAME, 165±3 µm (n=5); L-NAME+1 µmol/L ODQ, 157±5 µm (n=6). *P<0.05 compared with dilation at corresponding UTP concentration in vehicle control group; +P<0.05 compared with dilation at the corresponding UTP concentration in the other 2 groups.

Figure 4Down shows mean MCA diameters (n=3, top) and Em (n=4, bottom) before and after dilations to 10-5 mol/L UTP in L-NAME–treated (10 µmol/L) vessels. Diameter and Em were measured simultaneously from the same MCAs with 1 exception, in which case the diameter measurement was not obtained. The MCAs significantly dilated 107±1 µm after the administration of UTP (P=0.007). The Em of the vascular smooth muscle significantly hyperpolarized by 13±2 mV (n=4; P=0.009).



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  		Figure 4. Mean diameter of MCAs (n=3, top) and Em (n=4, bottom) in MCAs before and after dilations to 10-5 mol/L UTP in vessels treated with 10 µmol/L L-NAME. Note that in 1 MCA the diameter was not simultaneously measured with the Em because of technical difficulties. *P<0.01 compared with the corresponding measure in MCAs treated with L-NAME alone.

When 30 mmol/L KCl was added to the abluminal bath to negate any effects of K channels,19 the L-NAME–insensitive component of the UTP-mediated dilation was significantly attenuated (Figure 5Down). The addition of 10 µmol/L glibenclamide or 75 µmol/L BaCl2, blockers of ATP-sensitive and inward-rectifier K channels, respectively, had no effect on the L-NAME–insensitive component of the UTP-mediated dilation (Figure 6Down). 4-Aminopyridine (3 mmol/L), a blocker of the delayed-rectifier K channels, slightly but significantly altered the dilations to luminal UTP (Figure 6Down; group effect, P=0.017; interaction between group and UTP concentration, P=0.047). TEA (either 3 or 10 mmol/L) attenuated the L-NAME–insensitive component of the UTP-mediated (10-5 mol/L) dilation; however, 1 mmol/L TEA had no significant effect on the response (Figure 7aDown). TEA is considered selective for the calcium-activated K (KCa) channels at 1 and 3 mmol/L but is considered a nonspecific inhibitor at 10 mmol/L.20 Apamin (either 1 or 3 µmol/L), a selective blocker of the small-conductance KCa channels, attenuated the L-NAME–insensitive component of the UTP-mediated dilation (Figure 7bDown).



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  		Figure 5. Effects of 30 mmol/L KCl (abluminal bath) on the dilations produced by the luminal administration of UTP in L-NAME (10 µmol/L)–treated MCAs. KCl negates the effects of K channels. Diameters for each group before administration of UTP: L-NAME control, 168±5 µm (n=17); L-NAME+30 mmol/L KCl, 154±10 µm (n=7). *P<0.05 compared with dilation at the corresponding UTP concentration in L-NAME control MCAs (repeated-measures ANOVA followed by Student-Newman-Keuls method).



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  		Figure 6. Effects of addition of 10 µmol/L glibenclamide (Glib), 75 µmol/L BaCl2, or 3 mmol/L 4-aminopyridine (4-AP), blockers of ATP-sensitive, inward-rectifier, and delayed-rectifier K channels, respectively, on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs. Diameters for each group before administration of UTP: L-NAME control, 168±5 µm (n=17); L-NAME+10 µmol/L Glib, 166±14 µm (n=5); L-NAME+75 µmol/L BaCl2, 172±10 µm (n=5); L-NAME+3 mmol/L 4-AP, 156±5 µm (n=6). *P<0.05 compared with dilation at the corresponding UTP concentration in L-NAME control MCAs (repeated-measures ANOVA followed by Student-Newman-Keuls method).



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  		Figure 7. Effects of TEA (a) or apamin (b) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs. Diameters for each group before administration of UTP: (a) L-NAME control for TEA, 164±4 µm (n=22); L-NAME+1 mmol/L TEA, 182±9 µm (n=5); L-NAME+3 mmol/L TEA, 168±8 µm (n=6); L-NAME+10 mmol/L TEA, 147±5 µm (n=7); (b) L-NAME control for apamin, 170±4 µm (n=25); L-NAME+1 µmol/L apamin, 166±7 µm (n=11); L-NAME+3 µmol/L apamin, 157±2 µm (n=6). There was a significant group effect for the TEA and apamin studies (P<0.0001 and P=0.0007, respectively) and a significant interaction between group and concentration of UTP for the TEA and apamin studies (P<0.0001 and P=0.012, respectively). *P<0.05 compared with dilation at corresponding UTP concentration in the corresponding control group (L-NAME alone) (repeated-measures ANOVA followed by Student-Newman-Keuls method).

Figure 8Down shows that charybdotoxin, a blocker of the large-conductance KCa channels, completely abolished the dilation to UTP in MCAs treated with L-NAME. Iberiotoxin, a blocker of the large-conductance KCa channels, at concentrations of 50 and 100 nmol/L attenuated the dilations to UTP in MCAs treated with L-NAME (Figure 9aDown). However, the effects of iberiotoxin at either concentration were quite variable. Figure 9bDown and 9cDown shows mean responses for the L-NAME control and individual responses for the MCAs treated with 50 and 100 nmol/L iberiotoxin, respectively. In some vessels, iberiotoxin (either 50 or 100 nmol/L) substantially blocked the dilation to UTP, while in other vessels there appeared to be no block at all.



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  		Figure 8. Effects of charybdotoxin (CHTX) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs. Diameters for each group before administration of UTP: L-NAME control, 167±4 µm (n=12); L-NAME+100 nmol/L CHTX, 186±14 µm (n=6). *P<0.05 compared with dilation at the corresponding UTP concentration in L-NAME control MCAs (repeated-measures ANOVA followed by Student-Newman-Keuls method).



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  		Figure 9. Effects of iberiotoxin (IBTX) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs. Mean responses are provided in a; mean responses for the L-NAME control and the individual responses with 50 and 100 nmol/L IBTX are provided in b and c, respectively. Diameters for each group before administration of UTP: L-NAME control, 218±14 µm (n=14); L-NAME+50 nmol/L IBTX, 218±7 µm (n=7); L-NAME+100 nmol/L IBTX, 230±13 µm (n=7). *P<0.05 compared with dilation at the corresponding UTP concentration in L-NAME control MCAs (repeated-measures ANOVA followed by the Student-Newman-Keuls method).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The purpose of the present investigation was to determine the unknown relaxing factor associated with the stimulation of purinoceptors with UTP on cerebrovascular endothelium. This study demonstrated that the L-NAME–insensitive component of the UTP-mediated dilation can be attributed to EDHF. This conclusion is based on the following: (1) The unknown relaxing factor was not EDRF/NO. (2) The unknown relaxing factor was not a cyclooxygenase metabolite such as prostacyclin.7 (3) The dilation produced by the unknown relaxing factor was accompanied by hyperpolarization of the vascular smooth muscle. (4) The dilation and hyperpolarization were accompanied by K channel activation.

L-NAME–Insensitive Component of UTP-Mediated Dilation Is Not Attributed to EDRF/NO
Stimulation of purinoceptors on cerebrovascular endothelium by ATP and UTP dilates the rat MCA through a mechanism involving EDRF/NO, since L-NAME abolished or attenuated the dilation at lower agonist concentrations.7 However, at higher concentrations of UTP or ATP (>10-6 mol/L), L-NAME (10 µmol/L) had no effect on the dilation.7 Either another relaxing factor was involved at the higher concentrations of agonists, or we had not adequately blocked NO synthase in the original study.7 This issue was critical in light of a recent publication indicating that endothelial EDRF/NO may be difficult to block.15

The present study conclusively demonstrates that (1) EDRF/NO was blocked, for all practical purposes, and (2) a relaxing factor other than EDRF/NO was involved in the UTP-mediated dilation. This conclusion is based on 3 findings. First, dilations to 10-5 and 10-4 mol/L UTP were maintained in the presence of 1 µmol/L, 10 µmol/L, 100 µmol/L, or 1 mmol/L L-NAME. If NO synthase was not sufficiently inhibited by 10 µmol/L L-NAME,7 then it would be expected that a 10- or 100-fold concentration increase of L-NAME (10-4 or 10-3 mol/L, respectively) would abolish or at least attenuate the UTP-mediated dilations.15 Since this did not occur (Figure 1Up), we conclude that NO synthase was essentially blocked. Second, oxyhemoglobin, a scavenger of EDRF/NO, attenuated the dilation to UTP in a manner similar to that of L-NAME (Figure 2aUp). Furthermore, oxyhemoglobin, in combination with L-NAME, did not further attenuate the dilation to UTP more than L-NAME alone (Figure 2bUp). If NO synthesis was not completely blocked by L-NAME, then oxyhemoglobin should have abolished or further attenuated the dilation to UTP. Third, ODQ, an inhibitor of guanylate cyclase,21 attenuated the dilations to UTP in a manner similar to that of L-NAME (Figure 3aUp). EDRF/NO dilates cerebral vessels by stimulation of soluble guanylate cyclase.21 22 In combination with L-NAME, ODQ had no further effects on inhibiting the dilation to UTP than did L-NAME alone (Figure 3bUp). Thus, it appears clear that NO synthase was adequately blocked and that a relaxing factor, other than EDRF/NO, must be involved in the UTP-mediated dilations in rat MCAs.

L-NAME–Insensitive Component of the UTP-Mediated Dilation Is Attributed to EDHF
Ever since Furchgott and Zawadzki23 reported the existence of endothelium-derived relaxing factor, later identified as EDRF/NO,24 25 the role of endothelium in the regulation of vascular tone has been an important and fruitful area of investigation. As the field progressed, it soon became apparent that EDRF/NO could not explain all endothelium-dependent relaxations. Even when one considered the family of vasodilatory metabolites of the cyclooxygenase pathway, at least 1 other relaxing factor derived from the endothelium had to be postulated.26 This relaxing factor became known as endothelium-derived hyperpolarizing factor (EDHF).11 12 14 26 EDHF is defined as a relaxant, released from the endothelium, that is distinct from both EDRF/NO and prostaglandins and that dilates vessels by hyperpolarizing the vascular smooth muscle.12 The hyperpolarization is a result of activation of K channels (for comprehensive reviews, see References 1111 –14 and 26).

We believe that the L-NAME–resistant component of the UTP-mediated dilation in the rat MCA is mediated by EDHF. Our conclusion is based on the following: First, this component is independent of EDRF/NO (see above) or cyclooxygenase metabolites.7 Second, this component of the UTP-mediated dilation is accompanied by hyperpolarization of the vascular smooth muscle (Figure 4Up). Third, the dilation was dependent on the activation of K channels (Figures 5 through 9UpUpUpUpUp ). The fact that 30 mmol/L KCl (Figure 5Up) or 10 mmol/L TEA (Figure 7aUp) blocked or attenuated the L-NAME–insensitive component of the UTP-mediated dilation indicates that K channels were involved.19 20 27 KCl negates any involvement of K channels by making the Nernst potential for K+ and the Em of the smooth muscle equal. Thus, opening of K channels would produce no net movement of K+ and no change in the Em. TEA at a concentration of 10 mmol/L is a general blocker of K channels.20 27 The L-NAME–insensitive component of the UTP-mediated dilation fits the criteria for EDHF, defined in the preceding paragraph. We therefore conclude that the unknown relaxing factor, elicited by stimulating P2u receptors on the endothelium with UTP, is EDHF.

There have been only a few reports of EDHF associated with agonist-mediated dilations in cerebral arteries. EDHF may be released from endothelium in rabbit MCA, guinea-pig basilar artery, and human pial artery after stimulation with acetylcholine or substance P.28 29 30 We have now added to this list by demonstrating the release of EDHF from cerebrovascular endothelium of the rat MCA. There have been other published reports in cerebral arteries that might involve EDHF4 31 32 33 34 ; however, involvement of a cyclooxygenase metabolite was not ruled out. For our discussion, the term EDHF is reserved for a substance that differs from a cyclooxygenase metabolite (and EDRF/NO).12

EDHF may not be a single agent but rather a diverse class of agents, all of which open K channels.12 14 The exact agent might vary with species and/or vessel. Candidates for EDHF include the following: (1) epoxyeicosatrienoic acids, a cytochrome P450 metabolite of arachidonic acid; (2) anandamide, an endogenous agonist of the cannabinoid receptors; (3) hydrogen peroxide; (4) hydroxyl radicals; (5) superoxide anions; or (6) CO.12 14 35 36 37 38 Although many, if not most, investigators believe that EDHF and EDRF/NO are different entities, EDHF may not be distinct from EDRF/NO in some vessels. Cohen et al15 reported that an inadequate block of NO synthase could lead to a false conclusion regarding the existence of an EDHF distinct from EDRF/NO. For the rat MCA, we are able to rule out an inadequate block of NO synthase (see above). The identity of EDHF in the rat MCA is presently not known and awaits further studies for its identification.

Identification of the type of K channel involved with the EDHF-mediated dilation by UTP in the rat MCA is not straightforward. We are able to immediately rule out the ATP-sensitive and inward-rectifier K channels since neither 10 µmol/L glibenclamide nor 75 µmol/L BaCl2 had any effect on the L-NAME–insensitive component of the dilation (Figure 6Up).39 40 41 42 43 The delayed-rectifier K channels do not appear to be a major factor, since 4-aminopyridine, a selective blocker of this channel, had only a slight effect on the UTP-mediated dilations (Figure 6Up). However, the use of 4-aminopyridine as a general inhibitor of all delayed rectifiers has been questioned.28 44 45 It is therefore possible that delayed-rectifier K channels, not sensitive to 4-aminopyridine, could be involved. On the other hand, the fact that TEA (3 mmol/L), apamin (1 or 3 µmol/L), or iberiotoxin (50 or 100 nmol/L) attenuated the dilation while charybdotoxin (100 nmol/L) completely abolished the L-NAME–insensitive component of the UTP-mediated dilation points to involvement of the KCa channels. KCa channels, although a distinct class of K channels, belong to the delayed rectifier K channel superfamily.46 Apamin is a blocker of the small-conductance KCa channels,41 and charybdotoxin and iberiotoxin are blockers of the large-conductance KCa channels.41

Our data further support the idea that the large-conductance KCa channel is "atypical" or "nonclassic." First, 1 mmol/L TEA did not alter the dilation to UTP in L-NAME–treated MCAs. TEA at this concentration should partially or fully block the KCa channels20 27 40 41 and therefore should block or attenuate the dilation. Second, while charybdotoxin completely blocked the dilation (Figure 8Up), the response with iberiotoxin was quite variable (Figure 9Up). Iberiotoxin is a very specific and potent inhibitor of KCa channels.41 On the other hand, charybdotoxin affects certain delayed-rectifier K channels in addition to blocking KCa channels.28 44 45 Given that our results are not entirely consistent with a "typical" or "classic" KCa, we conclude that the KCa channel must be atypical or nonclassic. Atypical K channel involvement has also been reported for EDHF-mediated dilations by others. While individual K channel blockers, including apamin, charybdotoxin, and iberiotoxin, were ineffective in inhibiting the dilation (or relaxation) by EDHF, the combination of apamin and charybdotoxin, but not apamin and iberiotoxin, completely abolished the dilation produced by EDHF.28 44 45 47

In attempting to explain the type of K channel involved with the L-NAME–insensitive component of the UTP-mediated dilation, we offer 2 possible explanations. First, the UTP-mediated dilation in the rat MCA might involve both a small-conductance (apamin-sensitive) and a large-conductance (charybdotoxin-sensitive) KCa channel. The large-conductance KCa channel would be nonclassic, as discussed above. A second possibility would be the involvement of a single nonclassic K channel sensitive to charybdotoxin, apamin, and TEA (3 and 10 mmol/L but not 1 mmol/L) and somewhat sensitive to iberiotoxin. A nonclassic K channel having similarities to both delayed-rectifier and KCa channels has been postulated to be involved with the EDHF-mediated dilations.44 Our results do not exclude this channel type.

The natural agonists for the endothelial P2u purinoceptors in cerebral vessels in vivo are ATP and UTP in the plasma.48 49 50 51 These agonists appear to stimulate only the P2u receptor subtype in rat MCAs without affecting the P2y (P2Y1) subtype, which is also present on the endothelium.7 Since UTP may be more selective at the P2u site than ATP,52 UTP was the agonist of choice in the present study. UTP in plasma is derived from endothelium and platelets.49 51 53 ATP in plasma is derived from endothelium, platelets, and erythrocytes.51 54 55 56 It appears that UTP and ATP can reach concentrations of {approx}5 and 50 µmol/L, respectively, in plasma.49 50 51 However, there is reason to believe that after platelet aggregation, the concentrations of these nucleotides at the endothelial wall (receptor site) could approach or even reach concentrations in the millimolar range.48 Thus, these agonists may reach concentrations necessary to stimulate the synthesis and release of not only EDRF/NO (10-8 to 10-6 mol/L for either agonist) but also EDHF (above 10-6 mol/L for either agonist) (Figures 1 through 3UpUpUp).7

Although stimulation of endothelial purinoceptors is known to dilate peripheral and cerebral vessels by the release of EDRF/NO and/or prostacyclin,1 50 our laboratory is the first to conclusively demonstrate that EDHF is associated with purinoceptors57 (see also Reference 5858 ). Thus, stimulation of purinoceptors by UTP on endothelium of the rat MCA dilates the vessel by the release of EDRF/NO at concentrations up to 10-6 mol/L UTP and EDHF (likely in combination with EDRF/NO) at higher concentrations. One or more K channels are associated with the EDHF-mediated dilation. Although stimulation of some receptors, including P2y purinoceptors, on cerebrovascular endothelium leads to dilation exclusively by the release of EDRF/NO or prostacyclin, stimulation of P2u purinoceptors by UTP also involves EDHF in the rat MCA. The extent to which EDHF is associated with other endothelial receptor systems in the cerebral circulation is not presently known. EDHF may be an important but understudied relaxing factor released from the endothelium of cerebral arteries.


*    Acknowledgments
 
This study was supported by National Institute of Neurological Disorders and Stroke grants (PO1 NS-27616 and RO1 NS-37250) and by a National Institutes of Health training grant (T32HL07816).


*    Footnotes
 
Reprint requests to Robert M. Bryan, Jr, Department of Anesthesiology, Room 434D, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

Received November 3, 1998; revision received January 12, 1999; accepted February 3, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

Frank M. Faraci, PhD, Guest Editor

Department of Internal Medicine, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
Endothelium plays a major role in regulation of vascular tone.1 A key endothelium-derived relaxing factor in the cerebral circulation is NO (or a closely related, NO-containing compound).1 For example, relaxation of the carotid artery, large cerebral arteries, and cerebral microvessels in response to acetylcholine (the classic endothelium-dependent agonist) is mediated predominantly by NO.1 2 Relaxation of human cerebral arteries in response to endothelium-dependent agonists is also mediated in large part by NO.3 4

In addition to NO, endothelium may produce other relaxing factors, including prostacyclin and one or more endothelium-derived hyperpolarizing factors (EDHFs).5 Many studies define EDHF as an endothelium-derived relaxing factor that produces hyperpolarization of vascular muscle but which is not NO or a product of cyclooxygenase. In contrast to NO, which produces relaxation of cerebral vascular muscle by activation of soluble guanylate cyclase,6 7 EDHF produces hyperpolarization and relaxation of vascular muscle by activation of potassium channels. In comparison with NO, much less is known regarding the functional importance of EDHF in the cerebral circulation.

The preceding study provides evidence that uridine triphosphate (UTP) produces relaxation of the middle cerebral artery through endothelium-dependent mechanisms. Vasorelaxation in response to lower concentrations of UTP was mediated by NO and activation of soluble guanylate cyclase. In contrast, relaxation of this artery in response to higher concentrations of UTP appears to be mediated by an EDHF. At least two aspects of the present study are noteworthy. First, the authors tried very hard to eliminate any contribution of NO and prostacyclin and thus provide stronger evidence that the factor being studied was an EDHF. This latter point is important because recent evidence suggests that it can be difficult to completely inhibit production of NO by endothelium in some blood vessels.8 Second, the authors demonstrated that relaxation of the middle cerebral artery in response to UTP was associated with hyperpolarization of vascular muscle and attenuated by KCl (which prevents membrane hyperpolarization) or inhibitors of potassium channels. Activation of potassium channels in vascular muscle is generally believed to mediate relaxation in response to EDHF.

An important unanswered question in this study, and studies of EDHF in general, relates to the identity of the factor(s). Candidate factors have included epoxyeicosatrienoic acids (EETs) and anandamide (products of metabolism of arachidonic acid), and potassium ion.2 5 9 Recent studies suggest that EETs and anandamide do not account for EDHF-mediated responses in cerebral arteries.7 10 11 Although small-to-moderate increases in the concentration of extracellular potassium produce membrane hyperpolarization and relaxation of cerebral arteries, this response is mediated by activation of a barium-sensitive potassium channel (an inward-rectifier potassium channel).2 This latter characteristic not consistent with the pharmacological profile obtained for EDHF in the present study.

Thus, although the identity of EDHF(s) in the cerebral circulation remains unclear, findings such as those in the present study illustrate that EDHF may contribute to regulation of cerebral vascular tone under some conditions. It is noteworthy that recent evidence suggests EDHF may become functionally more important in disease states that are associated with impairment of the NO/cGMP signaling pathway.2

Received November 3, 1998; revision received January 12, 1999; accepted February 3, 1999.


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Faraci FM, Heistad DD. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev.. 1998;78:53–97.[Abstract/Free Full Text]
  2. Faraci FM, Sobey CG. Role of potassium channels in regulation of cerebral vascular tone. J Cereb Blood Flow Metab.. 1998;18:1047–1063.[Medline] [Order article via Infotrieve]
  3. Onoue H, Kaito N, Tomii M, Tokudome S, Nakajima M, Abe T. Human basilar and middle cerebral arteries exhibit endothelium-dependent responses to peptides. Am J Physiol.. 1994;267:H880–H886.[Abstract/Free Full Text]
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  5. Vanhoutte PM. Old-timer makes a comeback. Nature.. 1998;396:213–216.[Medline] [Order article via Infotrieve]
  6. Sobey CG, Faraci FM. Effects of a novel inhibitor of guanylyl cyclase on dilator responses of mouse cerebral arterioles. Stroke.. 1997;28:837–843.
  7. Petersson J, Zygmunt PM, Jonsson P, Hogestatt ED. Characterization of endothelium-dependent relaxation in guinea pig basilar artery: effect of hypoxia and role of cytochrome P450 mono-oxygenase. J Vasc Res.. 1998;35:285–294.[Medline] [Order article via Infotrieve]
  8. Cohen RA, Plane F, Najibi S, Huk I, Malinski T, Garland CJ. Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery. Proc Natl Acad Sci U S A.. 1997;94:4193–4198.
  9. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature.. 1998;396:269–272.[Medline] [Order article via Infotrieve]
  10. Dong H, Waldron GJ, Cole WC, Triggle CR. Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery. Br J Pharmacol.. 1998;123:821–832.[Medline] [Order article via Infotrieve]
  11. Petersson J, Zygmunt PM, Jonson P, Hogestatt ED. Involvement of derivatives of arachidonic acid in endothelium-dependent relaxations mediated by EDHF in the guinea pig basilar artery. Br J Pharmacol.. 1997;122:401P. Abstract.



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