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(Circulation Research. 2001;89:716.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Endothelial G Protein ß-Subunits Trigger Nitric Oxide– but not Endothelium-Derived Hyperpolarizing Factor–Dependent Dilation in Rabbit Resistance Arteries

Philippe Véquaud, Eric Thorin

From the Institut de Cardiologie de Montréal, Centre de Recherche, Montréal, Québec, Canada.

Correspondence to Dr Eric Thorin, Institut de Cardiologie de Montréal, Centre de Recherche, 5000 rue Bélanger Est, Montréal H1T 1C8, Québec, Canada. E-mail thorin{at}icm.umontreal.ca

	Abstract

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Abstract—— A single subtype of heterotrimeric G protein–coupled receptor controls both nitric oxide (NO) (sensitive to L-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K⁺ and apamine) production by the vascular endothelium leading to dilation. We hypothesized that - and ß-subunits of the G protein serve as distinct intermediates to produce NO and EDHF. In pressurized resistance arteries, selective pinocytotic endothelial incorporation of specific antibodies (Abs) directed against _q/11-subunits abolished acetylcholine (Ach)-mediated dilation but failed to influence oxymetazoline (Oxy, ₂-adrenergic receptor agonist)-induced dilation. In contrast, _i1-2-subunit Abs prevented Oxy- but not Ach-induced dilation. Thus, as expected, endothelial muscarinic and ₂-adrenoceptors couple to G_q protein and G_i proteins, respectively. ß-subunit Abs reduced both Ach- and Oxy-induced dilation. The ß-subunit Abs abolished the nitro-L-arginine (L-NNA)-sensitive component but did not impair the high-external K⁺-sensitive component of the dilation induced by Ach and Oxy. Thus, G protein ß-subunits primarily accounted for NO production. Neutralization of Hsp90 and inhibition of the phospholipase C by U73122 (1 µmol/L) or intracellular Ca²⁺ buffering with BAPTA-AM (10 µmol/L) sharply reduced NO-dependent but not K⁺-sensitive dilation. In conclusion, mobilization of the G protein ß-subunit is pivotal to NO-dependent dilation triggered through muscarinic and ₂-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to ß-subunit Abs. Although not directly activating the NO pathway, -subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.

Key Words: G protein subunits • endothelium • nitric oxide • endothelium-derived hyperpolarizing factor • dilation

	Introduction

Top Abstract Introduction Material and Methods Results Discussion References

 
Signaling via the large family of G protein-coupled receptors (GPCRs) leads to a variety of cellular responses, ranging from regulation of intracellular level of AMPc to stimulation of gene transcription.¹ G proteins are made of 3 subunits, , ß, and . Overexpression of different subtypes of receptors and classes of G protein has been the preferred experimental strategy to assess receptor-to-G protein coupling.^2,3 Although G protein -subunits have been initially considered as the major determinant of intracellular pathway activation, it is now clear that activation of ß-subunits triggers specific intracellular responses.⁴ For instance, Gß-subunits have been shown to increase the activity of adenylate cyclase,^5,6 MAP kinases,⁷ phospholipase C,⁸ and phospholipase A₂.⁹ This implies that G protein subunits serve as pivotal determinants of downstream intracellular events.

Most of the attention has been directed at linking receptor activation to specific subtypes of -subunits of the G protein as _i, _s, and _q.^10–17 For example, using neutralizing antibodies (Abs) for specific subtypes of -subunits, Liao and Homcy¹⁰ demonstrated that ₂-adrenergic receptors (₂-ARs) coupled to G_i2-subunits in cultured endothelial cells. Furthermore, it was shown that angiotensin II–stimulated phospholipase D activity was reduced by Gß-subunit Abs in cultured smooth muscle cells and that the ß-subunit complex thus activated this enzyme.¹⁸ The differential production of endothelium-derived relaxing factors with respect to G protein coupling has not been considered earlier.

In this connection, it is now recognized that in vascular endothelial cells, a single subtype of receptor can lead to the production of both nitric oxide (NO) (sensitive to L-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K⁺ and apamine). This induces endothelium-dependent dilation.^19–23 This led us to hypothesize that the receptor-operated release of endothelium-derived relaxing factor reflects the specific coupling of G- or Gß-subunits to either NO or EDHF production. To address this issue, we have developed a unique model allowing selective endothelial incorporation of neutralizing Abs in pressurized resistance arteries and measurements of vascular diameter. Therefore, the specific objectives of the present study are (1) to investigate the differential contribution of G protein - and ß-subunits to NO- and EDHF-dependent dilation and (2) to elucidate the distinct intracellular effectors involved in these processes.

	Material and Methods

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Vascular Preparation
Experiments were conducted, as previously described,²⁴ on isolated resistance arteries (200 µm) of the mesenteric bed of male New Zealand White rabbits (weight range: 2.5 to 3.5 kg; Charles River Laboratories, St Constant, Quebec, Canada). The procedures and protocols were in accordance with our institution guidelines and the Guide for the Care and the Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23). Rabbits were anesthetized with an intravenous injection of sodium pentobarbitol (65 mg · kg^-1) and exsanguinated. The mesenteric bed was harvested and a fifth branch of the mesenteric artery was dissected out and placed in ice-cold physiological salt solution (PSS) containing indomethacin (10 µmol/L). A 2- to 3-mm length arterial segment was isolated and pressurized at 60 mm Hg.²⁴

Incorporation of Antibodies Inside Endothelial Cells
This method is based on osmotic lysis of pinocytic vesicules, previously described by Okada and Rechsteiner.²⁵ We used the Influx pinocytic cell-loading reagent kit (Molecular Probes). Pressurized vessels were first intraluminally perfused (2 µL · min^-1) with a hyperosmotic solution containing the Abs for 60 minutes. Abs were used at a dilution ranging from 1:1000 to 1:100. Arteries were then perfused with the hypo-osmotic solution for 10 minutes. Finally, vessels were perfused with normal PSS followed by a 30-minute equilibration period. Experiments performed without Abs in the hyperosmotic solution are identified as vehicle experiments. In control experiments, vessels were not subjected to the internalization protocol. Internalization of FITC-labeled IgG was restricted to the endothelium, as revealed by confocal microscopy imaging (Figure 1). Furthermore, none of the Abs used in this study altered smooth muscle reactivity to phenylephrine (PE), confirming the selective incorporation of the Abs in the endothelium (Table).

View larger version (105K): [in this window] [in a new window]   Figure 1. Confocal images of isolated pressurized resistance arteries subjected to the protocol of internalization in the presence of FITC-labeled IgG (1:100 dilution). A, Penetration of the IgG was restricted to the endothelial layer. B, Internalization was homogeneously distributed to the endothelium.

View this table: [in this window] [in a new window]   Table 1. Percent Reduction in Internal Diameter in the Various Experimental Conditions

Experimental Protocols
The relative contribution of NO and EDHF to agonist-mediated dilation in no-flow conditions was determined after exposure to N-nitro-L-arginine (L-NNA) (10 µmol/L) or KCl-PSS (25 mmol/L). In vehicle conditions, the dilation induced by acetylcholine (Ach) in the presence of L-NNA was blocked by further addition of 25 mmol/L KCl-PSS. On the other hand, the dilation induced by Ach in the presence of 25 mmol/L KCl-PSS was blocked by further addition of L-NNA (data not shown). To determine the G protein subtype coupled to muscarinic and ₂-ARs, cumulative concentration-response curves were constructed with Ach (0.1 nmol/L to 30 µmol/L) or oxymetazoline (Oxy) (0.1 nmol/L to 30 µmol/L) on preconstricted vessels (PE, 10 µmol/L). These experiments were repeated after incorporation of the anti–-subunit Ab. Similarly, the effect of the anti–-subunit Ab on substance P-induced dilation was investigated.

To determine the involvement of G protein ß-subunit in NO- or EDHF-mediated dilation to the agonists, experiments were repeated after endothelial incorporation of anti–Gß-subunit Abs in the absence or presence of L-NNA (10 µmol/L) or KCl-PSS (25 mmol/L).

We then investigated the contribution of Hsp90, phospholipase C (PLC), and intracellular free Ca²⁺ in NO- and EDHF-mediated dilation. Selective endothelial inhibition of the PLC was achieved by intraluminal perfusion with U73122 (1 µmol/L during 30 minutes) or its inactive analogue, U73343. On the other hand, buffering of endothelial cell intracellular Ca²⁺ was achieved by intraluminal perfusion of BAPTA-AM (10 µmol/L) during 25 minutes followed by a 30-minute washout period.

The term "EDHF" used in this study refers to the KCl- and apamine-sensitive, L-NNA- and indomethacin-resistant component of endothelium-dependent vasodilation.^20,26–29

Statistical Analysis
Only two vessels were used from each rabbit. Only one protocol was performed per vessel. n refers to the number of rabbits used in each protocol. Half-maximum effective concentrations (EC₅₀) of Ach and Oxy were measured from each individual dose-response curve using a logistic curve-fitting program (Allfit, Dr Deléan, University of Montréal). The pD₂ value is the negative log of the EC₅₀. Results were expressed as mean±SE. At the end of the protocol, the maximal diameter (D_max) was determined by changing the PSS to a Ca²⁺-free PSS containing sodium nitroprusside (10 µmol/L) and EGTA (1 mmol/L).

For each protocol, basal diameter in no-flow condition was determined at the end of the 30-minute equilibration period. Myogenic tone (at 60 mm Hg) represents the difference between the D_max and the basal diameter, and is expressed as a percentage of the D_max. PE-induced constriction is expressed as a percentage of the D_max. Agonist-induced dilation is expressed as a percentage of the initial PE-induced constriction.

Statistical differences between two groups of data were determined using an unpaired Student’s t test. For multiple comparisons, an ANOVA was used followed by a Scheffé’s F test. A P valueh0.05 was considered significant.

Drugs
Ach, apamine, indomethacin, L-NNA, Oxy, phenylephrine (PE), substance P, and sodium nitroprusside (SNP) were purchased from Sigma. BAPTA-AM, U73122, and U73343 were purchased from Calbiochem. All drugs were added directly to the vessel chamber and final concentration is given. They were prepared daily in distilled water except for indomethacin and the U compounds, which were prepared in ethanol (final dilution of ethanol was 0.1%). BAPTA-AM was prepared according to a method developed by Yashiro and Duling³⁰ and used at 10 µmol/L; 5 mg BAPTA was dissolved in 1 mL of hygroscopic DMSO (stable under argon) and vortex. From this solution, 6.1 µL was added to 4 mL of Ca²⁺ PSS containing 1% (vol/vol) albumin. Equimolar amounts of NaCl were replaced with KCl to prepare K⁺-rich solution (KCl-PSS). Antibodies were purchased from Calbiochem and Transduction Laboratories (Hsp90). Their characteristics were as follows: G_q/11-subunit Abs (Internal, 283-300); G_i1- and G_i2-subunit Abs (C-terminal, 345-354 and 346-355); Gß_common-subunit Abs (Internal, 127-139); and Hsp90 (clone 68, C-terminal, 586-732).

	Results

Top Abstract Introduction Material and Methods Results Discussion References

 
Characterization of Ach- and Oxy-Induced Endothelium-Dependent Dilation
The relative contribution of NO and EDHF to agonist-mediated dilation was determined after exposure to L-NNA (10 µmol/L) or KCl-PSS (25 mmol/L). The residual dilation to Ach or Oxy after L-NNA was blocked by 25 mmol/L KCl-PSS. Apamine (0.1 µmol/L) alone also prevented L-NNA-resistant dilation (-0.4±2.6% dilation, n=6, P<0.05) as compared with 93±1% dilation in the absence of apamine (n=6 per group). In separate experiments, the residual dilation to Ach or Oxy in the presence of 25 mmol/L KCl-PSS was abolished by L-NNA, consistent with the involvement of NO. Thus, in isolated pressurized resistance arteries, receptor-operated dilation involves both NO-dependent mechanism and a KCl- and apamine-sensitive process resembling the effects of EDHF.

Incorporation Protocol (Vehicle)
In control conditions, Ach (0.1 nmol/L to 30 µmol/L, n=6) induced a concentration-dependent dilation with a pD₂ value of 6.2±0.3 and a maximal dilation (E_max) of 72±11%. This dilatory response was not altered by the incorporation protocol (vehicle, n=5; pD₂=6.6±0.1, E_max=69±13%). Similarly, Oxy-induced dilation (0.1 nmol/L to 30 µmol/L, n=6) was not affected by the incorporation protocol (control: pD₂=5.9±0.2, E_max=44±8%, n=7; vehicle: pD₂=5.8±0.2, E_max=47±2%, n=6). Neither myogenic tone nor PE-induced contractions were altered by vehicle (Table).

Endothelial Incorporation of G Protein -Subunit Antibodies
Incorporation of anti–G_q/11 Abs at a dilution of 1/100 substantially reduced Ach-induced dilation without affecting Oxy-induced dilation (Figure 2). At lower dilutions, the Ab dose dependently reduced (P<0.05) Ach-induced dilation. A 1/100 dilution of Abs was used in all subsequent experiments. Incorporation of anti–G_i1-2 Abs had no effect on Ach-induced dilation (Figure 3). As for Ach, the anti–G_q/11 Ab prevented substance P-induced dilation, whereas the anti–G_i1-2 Ab had no effect (online Figure 1, available in the data supplement at http://www.circresaha.org). In contrast, Oxy-induced dilation was reversed to a contraction (P<0.05) by anti–G_i1-2 Abs but was not significantly affected by anti–G_q/11 Abs (Figure 4). None of the Abs altered myogenic tone or PE-induced contraction (Table).

View larger version (23K): [in this window] [in a new window]   Figure 2. Concentration-dependent inhibitory effect of the anti–Gq/11-subunit antibody (n=5 per group) on the maximal dilation (30 µmol/L) induced by Ach and Oxy. Experiments were performed in the presence of indomethacin (10 µmol/L). *P<0.05 compared with vehicle conditions; P<0.05 compared with dilution 1:1000;P<0.05 compared with dilution 1:500.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effect of endothelial incorporation of anti–Gq/11-subunit (n=8) and anti–Gi1-2-subunit (n=9) antibodies on Ach-induced dilation of mesenteric arteries preconstricted by PE (10 µmol/L). Experiments were performed in the presence of indomethacin (10 µmol/L). *P<0.05 compared with vehicle conditions;P<0.05 compared with anti–Gi1-2-subunit Ab conditions.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of endothelial incorporation of anti–Gi1-2-subunit (n=5) and anti–Gq/11-subunit (n=5) antibodies on Oxy-induced dilation of mesenteric arteries preconstricted by PE (10 µmol/L). Experiments were performed in the presence of indomethacin (10 µmol/L). *P<0.05 compared with vehicle conditions;P<0.05 compared with anti–Gq/11-subunit Ab conditions.

Endothelial Incorporation of G Protein ß-Subunit Antibodies
Incorporation of anti-Gß_common-subunit Abs reduced both Ach-and Oxy-induced dilation (Figures 5 and 7). Maximal dilation to Ach and Oxy was reduced (P<0.05) from 69±13% and 47±2% to 44±7% and 28±3%, respectively. The residual dilation to Ach and Oxy persisting after the incorporation of Gß-subunit Abs was prevented (P<0.05) by 25 mmol/L KCl-PSS (Figure 5 and online Figure 2). Conversely, the residual dilation to Ach and Oxy persisting after the incorporation of Gß-subunit Abs was not altered by L-NNA (Figure 6 and online Figure 3). Thus, Gß-subunit Abs selectively prevented Ach- and Oxy-induced NO-dependent dilation. Incorporation of Gß-subunit Abs increased myogenic tone but failed to influence PE-induced contractions (Table). Gß-subunit Abs did not influence L-NNA- and high-external K⁺-induced contraction.

View larger version (34K): [in this window] [in a new window]   Figure 5. Ach-induced dilation in the absence (vehicle, n=6) or in the presence of endothelial incorporation of anti–Gßcommon-subunit antibodies (n=6). Experiments were reproduced in the presence of a hyperpotassic solution alone (KCl-PSS, 25 mmol/L, n=6) or combined with the Ab (n=6). Experiments were performed in the presence of indomethacin (10 µmol/L). *P<0.05 compared with vehicle conditions;P<0.05 compared with anti–Gß-subunit Ab conditions.

View larger version (32K): [in this window] [in a new window]   Figure 6. Ach-induced dilation in the absence (vehicle, n=6) or in the presence of endothelial incorporation of anti–Gßcommon-subunit antibodies (n=7). Experiments were reproduced in the presence of L-NNA alone (10 µmol/L, n=6) or combined with the Ab (n=6). Experiments were performed in the presence of indomethacin (10 µmol/L). *P<0.05 compared with vehicle conditions.

View larger version (28K): [in this window] [in a new window]   Figure 7. Ach-induced dilation in the absence (n=7) and in the presence of U73122, a PLC inhibitor, either alone (n=7) or combined with L-NNA (10 µmol/L, n=7) or a hyperpotassic solution (K+; 25 mmol/L KCl-PSS, n=7). *P<0.05 compared with L-NNA and K+ conditions.

Involvement of PLC in NO-Mediated Dilation
Pressurized arteries were treated intraluminally with the PLC inhibitor U73122 (1 µmol/L, 30 minutes). U73122 prevented (P<0.05) Ach-induced NO-dependent dilation (in the presence of 25 mmol/L KCl-PSS). In contrast, U73122 failed to alter L-NNA–resistant dilation (Figure 7). Because PE-induced contraction was not altered after U73122 (Table), blockade of the PLC was most likely limited to the endothelial cells. Furthermore, external application of U73122 (1 µmol/L, 30 minutes) limited the peak PE contraction to 40% of its normal amplitude, and the contraction was no longer sustained (data not shown). The inactive form of the PLC inhibitor U73343 had no influence on Ach (10 µmol/L)-induced NO-dependent dilation (45±3% compared with 52±5% in absence of U73343; n=6).

Involvement of Intracellular Ca²⁺ in NO-Mediated Dilation
Pressurized arteries were treated intraluminally with the Ca²⁺ chelator BAPTA-AM (10 µmol/L). Ach-induced dilation was maintained in the presence of BAPTA-AM (87±5% versus control, 72±11%; n=5 per group). The NO-dependent component of Ach-induced dilation (in the presence of 25 mmol/L KCl-PSS) was reduced (P<0.05) by BAPTA-AM (23±10% versus 55±7% in the absence of BAPTA-AM; n=6 per group). In contrast, endothelial incorporation of BAPTA-AM did not reduce Ach-induced KCl-sensitive dilation (after NO synthase blockade) but in fact caused a significant (P<0.05) increase from 59±5% in control to 86±8% after BAPTA-AM (n=5 per group). PE-induced contraction was not affected by BAPTA-AM in the presence of L-NNA and 25 mmol/L KCl-PSS (63±10% and 59±6%, respectively) when compared with control responses (Table). SNP (1 µmol/L)-induced maximal dilation was not altered by BAPTA-AM (data not shown).

Involvement of Hsp90 in NO-Mediated Dilation
Incorporation of anti–Hsp90 Abs reduced NO-mediated dilation (22±5%, P<0.05, n=7) induced by Ach (10 µmol/L), as compared with vehicle in the presence of 25 mmol/L KCl-PSS. EDHF-mediated dilation was not altered (92±2%, n=7). The preconstriction induced by PE was not altered by Hsp90 Abs in the presence of L-NNA (70±4%) and 25 mmol/L KCl (72±5%), as compared with vehicle (Table).

	Discussion

Top Abstract Introduction Material and Methods Results Discussion References

 
In the present study, we have demonstrated that receptor-operated NO-dependent and NO-independent dilation of pressurized resistance arteries involved distinct pathways. NO-dependent dilation was prevented by selectively targeting the G protein ß-subunit with neutralizing Abs. Our data further demonstrate that PLC activation accounts for Ca²⁺-dependent NO production. In contrast, NO-independent but KCl- and apamine-sensitive (presumably EDHF) dilation^20,26–29 was resistant to blockade of G protein ß-subunits, independent of PLC activity and a change in intracellular Ca²⁺ levels. Presumably, G protein -subunits accounted for NO-independent dilation.

Both NO and EDHF are involved in the endothelium-dependent dilator responses to ₂-AR and muscarinic receptor agonists.^31,32 Conceivably, the differential production of NO and EDHF could reflect selective activation of - and ß-subunits of the G protein. To address that issue, we used neutralizing antibodies selectively internalized into the endothelium of pressurized vessels. Our data indicate that this approach can adequately target endothelial cells (Figure 1) and avoid the confounding effect of smooth muscle cell Ab internalization as demonstrated by the stability of the contraction induced by PE (Table). Although this approach has been successfully used by others to target G protein subunits in cultured endothelial and smooth muscle cells,^10,18 to our best knowledge this study is the first to use G protein subunit Abs to specifically target endothelial cells in pressurized vessels. This allowed us to link the effect of endothelial G protein subunit inhibition to overall vascular responses. As a consequence, neutralization of the endothelial ₂-AR pathway partly revealed the smooth muscle contractile response to Oxy (Figure 4),²⁴ which represents another demonstration of the selective endothelial incorporation of the antibodies. Ach, on the other hand, has no effect in denuded mesenteric vessels (data not shown), which render the endothelium the unique functional target of the muscarinic agonist.

Consistent with earlier studies, our data demonstrate the specificity of the coupling of ₂-ARs to G_i proteins^32,33 and substance P³² and muscarinic receptors to G_q proteins.^34,35 As expected, anti–G_q-subunit Abs selectively abolished substance P- and Ach-induced endothelium-dependent dilation, whereas anti–G_i-subunit Abs selectively abolished ₂-AR–mediated endothelium-dependent dilation. Thus, -subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries. Apparently, -subunit activation by receptor stimulation is an essential initial step in NO and EDHF production.

Biochemical studies have demonstrated that - and ß-subunits from a single G protein subtype could influence distinct intracellular processes,^3,4 such as NO and EDHF production. To directly address that issue, we have created experimental conditions to separate NO-mediated (in the presence of indomethacin and high-external K⁺) and EDHF-mediated (in the presence of indomethacin and L-NNA) agonist-induced dilations as described by others.^26–29 Neutralization of G protein ß-subunits prevented NO-dependent dilation induced by Ach and Oxy (Figure 5 and online Figure 2). The process subtending EDHF-dependent dilation clearly differs from that of NO-dependent dilation and was independent of Gß-subunit activation (Figure 5 and online Figure 2). Conceivably, in addition to being the initial step in receptor-mediated G protein activation, -subunit activation may then selectively trigger EDHF production.

It has been shown that on G protein activation, ß-subunits activate PLC and increase intracellular free Ca²⁺.⁸ To ensure that our experimental approach did not disrupt the pathway considered to normally account for NO production,^30,36–38 we have examined the effects of PLC inhibition, Ca²⁺ buffering, and neutralization of Hsp90 on NO-dependent dilation. Inhibition of PLC (Figure 7) prevented NO- but not EDHF-dependent Ach-mediated dilation. Consistent with this finding, BAPTA-AM reduced NO-dependent dilation but failed to alter EDHF-dependent dilation. This partial inhibition of NO-mediated dilation with BAPTA-AM is consistent with data obtained in similar experimental conditions³⁰ and may be owing to the lack of total Ca²⁺ clamp, despite the sharp reduction in intracellular Ca²⁺ previously reported.³⁰ Alternatively, this may reflect the Ca²⁺-independent, Hsp90-mediated sustained activation of NO synthase.³⁹ Reduction in NO- but not EDHF-dependent dilation by endothelial incorporation of Hsp90 Abs further confirms the specificity and the high degree of compartmentalization of the NO pathway. Thus, Ca²⁺-dependent NO production most likely involved Gß-subunit-dependent PLC activation.^18,40 EDHF production clearly differed from NO production, as it was insensitive to Hsp90 neutralization and PLC blockade and was Ca²⁺-independent.

In contrast to earlier studies performed in conductance arteries,^23,41,42 the present data indicate that the NO-independent dilation of resistance vessels was insensitive to Ca²⁺ buffering with BAPTA-AM. One mechanism by which activation of muscarinic receptors can lead to NO- and Ca²⁺-independent dilation is through an increase K⁺ efflux from endothelial cells,⁴³ which can induce hyperpolarization.⁴⁴ Because of the direct contact between endothelial and smooth muscle cells in resistance arteries, hyperpolarization could be transmitted from endothelial to smooth muscle cells by myoendothelial gap junctions.^30,45,46 In that eventuality, the endothelium-dependent hyperpolarization would not require the Ca²⁺-dependent synthesis of an EDHF.⁴⁵ In support of this hypothesis, agonist-induced endothelium-dependent vasodilation of resistance arteries in endothelial NO synthase knockout mice is mediated by an EDHF-like principle that requires functional gap junctions but not NO, prostacyclin, or a cytochrome P450 metabolite production.^29,45 In conductance arteries, myoendothelial junctions are lacking. This may explain why the production of an EDHF by the endothelium is required for endothelium-dependent hyperpolarization of smooth muscle cells.⁴⁷

The responses observed after G protein -subunit Ab internalization should be interpreted with caution because neutralization of endothelial G protein -subunits prevented receptor/G protein coupling. This is an inherent problem of targeting G protein -subunits. What our data convincingly show is the close association between NO-dependent responses and G protein ß-subunit activation. Consequently, the residual dilatory response resistant to ß-subunit blockade most likely reflected -subunit activation. Such a model has been previously described with neutralizing Abs in patch-clamp studies using the inside-out configuration.¹⁷ These authors demonstrated that 11,12-epoxyeicosatrienoic acid–induced Ca²⁺-sensitive K⁺ channel activation was mediated by G_s-subunits but not ß-subunits in isolated smooth muscle cells. Thus, it is conceivable that in endothelial cells of pressurized resistance arteries a direct coupling between muscarinic receptors, G_q-subunits, and K⁺ channels may be involved and lead to NO-independent dilation.

In conclusion, mobilization of the G protein ß-subunit is pivotal to NO-dependent dilation triggered through endothelial muscarinic and ₂-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to ß-subunit Abs. Although not directly activating the NO pathway, -subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.

	Acknowledgments

 
This work was supported by the Canadian Institutes of Health Research (MOP 14496), the Heart and Stroke Foundation of Québec, and the Montreal Heart Institute Research Foundation. Philippe Véquaud is a postdoctoral fellow of the Fonds de Recherche en Santé du Québec, and Eric Thorin is a Research Scholar of the Heart and Stroke Foundation of Canada. The authors thank Drs Michel Lavallée and Nathalie Trescases for their helpful comments.

Received June 11, 2001; revision received August 16, 2001; accepted August 16, 2001.

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