Coronary Arteriolar Dilation to Acidosis
Role of ATP-Sensitive Potassium Channels and Pertussis Toxin–Sensitive G Proteins
Background—We previously demonstrated that coronary arteriolar dilation in response to acidosis is mediated by the opening of ATP-sensitive potassium (KATP) channels. However, the signal transduction involved in the KATP-channel activation during acidosis has not been elucidated. A recent study in cardiac myocytes implied that pertussis toxin (PTX)–sensitive G proteins may be involved in the signal transduction for KATP-channel activation. However, it remains unclear whether this transduction process also occurs in the vascular tissue and, in particular, whether it exerts functional dilation in response to acidosis.
Methods and Results—To examine the signaling pathway for acidosis-induced dilation, porcine coronary arterioles were isolated, cannulated, and pressurized for in vitro study. The GTPase activity in reconstituted G proteins was examined at different levels of pH. Extravascular acidosis (pH 7.3 to 7.0) produced a graded dilation of coronary arterioles. This dilation was not affected by removal of endothelium but was significantly attenuated after inhibition of KATP channels and G proteins by glibenclamide and PTX, respectively. Glibenclamide and PTX attenuated the acidosis-induced arteriolar dilation to the same extent, and combined administration of both inhibitors did not further inhibit the vasodilation. These results indicated that both inhibitors act on the same vasodilatory pathway. Furthermore, vasodilation of coronary arterioles to the KATP-channel opener pinacidil and to the endothelium-independent vasodilator sodium nitroprusside was not affected by PTX. Because PTX inhibited acidosis-induced vasodilation without inhibiting KATP-channel function, it is suggested that PTX inhibits the vasodilatory pathway upstream from KATP channels. GTPase activity in reconstituted G proteins was significantly enhanced by a reduction in pH, indicating that G proteins were directly activated by acidosis.
Conclusions—On the basis of these findings, we conclude that acidosis-induced coronary arteriolar dilation is mediated by the opening of smooth muscle KATP channels through the activation of PTX-sensitive G proteins.
ATP-sensitive potassium (KATP) channels have been shown to be involved in the regulation of membrane potential of vascular smooth muscle cells in various tissues,1 including coronary arteries.2 Activation of this potassium channel contributes to the regulation of coronary blood flow during reduction of perfusion pressure3 or under metabolic stresses such as hypoxia,4 ischemia,3 and increased metabolic demand,5 conditions generally associated with tissue acidosis. Recent in vitro studies have indicated that acidosis produces vascular relaxation/dilation via hyperpolarization,6 and we subsequently demonstrated that the opening of KATP channels is responsible for the dilation of coronary arterioles to acidosis.7 However, the signal transduction pathway involved in this vasodilation has not been elucidated. Because the majority of coronary resistance (>60%) resides in arterioles <150 μm in diameter8 and these vessels are responsible for the regulation of coronary blood flow, it is important to understand the basic vasomotor control mechanism of these arterioles in response to acidic stress.
Guanine nucleotide–binding proteins (G proteins) are known to be an important component of signal transduction pathways that carry information received at the cell surface to the appropriate cellular response.9 Pertussis toxin (PTX) inactivates Gi and Go proteins by means of ADP ribosylation of the carboxyl termini of α-subunits, the putative site of receptor–G protein interaction. The holo–G protein (αβγ) is required for ADP ribosylation.10 There are 2 ways that PTX inhibits G protein–mediated signaling. On the one hand, PTX blocks receptor–G protein interactions11 ; on the other, ADP ribosylation by PTX can also prevent the dissociation of the Gα and Gβγ subunits and thus inhibit effector function.12 It has been shown that inhibition of G protein signal transduction by PTX inactivates KATP channels of pancreatic cells13 and cardiac myocytes,14 suggesting that activation of KATP channels may be through G proteins. However, it remains unclear whether this transduction process also occurs in the vascular tissue and, in particular, whether it exerts functional dilation in response to acidosis. In addition, the direct effect of acidosis on G protein activity has not been characterized. In the present study, we tested the hypothesis that the opening of KATP channels through activation of PTX-sensitive G proteins is responsible for the signal transduction initiating vasodilation in response to acidosis. To achieve this goal without interference from neurohumoral and hemodynamic factors, the experiments were performed in pressurized, isolated coronary arterioles 50 to 100 μm in diameter and in G protein–reconstituted liposomes.
Pigs (8 to 12 weeks old, of either sex) were sedated with telazol 4.4 mg/kg IM and xylazine 2.2 mg/kg IM, then anesthetized and heparinized with pentobarbital sodium 20 mg/kg IV and heparin 1000 U/kg IV, respectively, through the marginal ear vein. Pigs were intubated and ventilated with room air. After a left thoracotomy was performed, the heart was electrically fibrillated, excised, and immediately placed in cold (5°C) saline solution.
Isolation and Cannulation of Microvessels
The techniques for isolation of porcine coronary arterioles were described previously.15 In brief, a mixture of india ink and gelatin in physiological salt solution (PSS) containing (in mmol/L) NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS 3.0 was perfused into the left anterior descending artery and the circumflex artery to allow visualization of the coronary microvessels. The subepicardial coronary arterioles (50 to 100 μm in ID) were dissected from surrounding cardiac tissue under cold (5°C) PSS containing albumin (1%; Amersham) at pH 7.4. Each isolated microvessel was then transferred for cannulation to a Lucite vessel chamber containing albumin-PSS (pH 7.4) equilibrated with room air at ambient temperature. Both ends of the vessel were cannulated with micropipettes filled with albumin-PSS. The cannulated vessel was securely tied to the micropipettes with 11-0 ophthalmic suture (Alcon).
After a vessel was cannulated, the preparation was transferred to the stage of an inverted microscope (Diaphot 300, Nikon) coupled to a CCD camera (TM-34KC, Pulnix) and video micrometer (Microcirculation Research Institute, Texas A&M University Health Science Center). The micropipettes were connected to independent pressure reservoir systems,16 and intraluminal pressures were measured through side arms of the 2 reservoir lines by low-volume-displacement strain-gauge transducers (Statham P23 Db, Gould). The isolated vessels were pressurized without flow by setting both reservoirs at the same hydrostatic level (60 cm H2O). IDs of the vessel were measured throughout the experiment by video microscopic techniques incorporated with the MacLab (ADInstruments) data acquisition system.16
Experimental Protocols for Isolated Vessel Studies
Each cannulated vessel was bathed in albumin-PSS and equilibrated with room air, and the temperature was maintained at 36°C to 37°C. The vessel was set to its in situ length and allowed to develop basal tone at 60 cm H2O intraluminal pressure. After a stabilization period of 60 minutes, acidosis-induced vasodilation was studied by incremental addition of HCl (0.05N) to the vessel bath to reduce extravascular pH.7 The bath Pco2 was not altered by addition of HCl. Vessel diameter was measured at a pH of 7.4, 7.3, 7.2, 7.1, and 7.0 before and after disruption of endothelium by perfusion of a nonionic detergent (CHAPS; 0.4%) to the vessel. Denudation of the endothelium was verified by the absence of vasodilation to the endothelium-dependent vasodilator bradykinin 100 nmol/L as described previously.7 The contribution of KATP channels to acidosis-induced dilation was examined by a specific KATP-channel inhibitor glibenclamide (5 μmol/L, 15 minutes of incubation). The role of PTX-sensitive G proteins in acidosis-induced vasodilation was examined after incubation of the vessels with PTX 100 ng/mL for 60 minutes. The KATP-channel opener pinacidil 0.03 to 3 μmol/L and the endothelium-independent vasodilator sodium nitroprusside 1 nmol/L to 10 μmol/L were used to examine KATP-channel and vascular smooth muscle function, respectively. All chemicals were administered to the vessel bath. At the end of each experiment, each vessel was relaxed completely with sodium nitroprusside 100 μmol/L to obtain the maximum diameter at 60 cm H2O intraluminal pressure.
Preparation of Reconstituted G Proteins
To directly examine the effect of acidosis on G protein activity, heterotrimeric G proteins were purified from bovine brain by previously described procedures.17 The protein purity and activity were monitored by SDS-PAGE/immunoblotting and [35S]GTP-γ-S binding.17 The homogeneous G protein preparation was reconstituted into liposomes by use of detergent-mediated methods, and the reconstitution efficiency was measured in each preparation as described previously.17
Effect of pH on G Protein Activity
G protein–reconstituted vesicles were resuspended with reconstitution buffer into aliquots (pH preadjusted to 7.0, 7.1, 7.2, 7.3, and 7.4 with HCl) and were equilibrated for 3 hours on ice. The γ-32P–labeled GTP (10 μmol/L final concentration, 10 μL) was added in appropriate pH buffer to the vesicles (40 μL) and incubated at 37°C for 1 minute (each treatment was done in triplicate). Steady-state GTP hydrolysis as a function of GTPase activity was measured by the charcoal adsorption method previously described.18 Briefly, 5% charcoal in 20 mmol/L ice-cold phosphoric acid was added to the reaction mixture, and the samples were moved onto ice. Samples were mixed thoroughly and centrifuged at 20 000g for 10 minutes at 4°C to pellet the charcoal. Radioactivity was measured in the supernatants for released Pi from GTP: Pi released=(nmol GTP added)(sample cpm)/total cpm added.
Experiments with liposomes containing zero protein were performed simultaneously, and the values were subtracted for nonspecific activity. GTP hydrolysis by pure G proteins (without phospholipid) was used as a control. All values were normalized for protein concentration and time.
Drugs and chemicals were obtained from Sigma Chemical Co except as specifically stated. Phospholipids were from Avanti Polar Lipids, Inc. [γ-32P]GTP was from Amersham. PTX and sodium nitroprusside were dissolved in PSS. Glibenclamide and pinacidil were dissolved in dimethyl sulfoxide (DMSO), then diluted in PSS to obtain the desired final concentration. The concentration of DMSO in the vessel bath was <0.03%. A vehicle control study indicated that this concentration of DMSO influenced neither resting diameter nor arteriolar responses to acidosis, as reported previously.7
All diameter changes were normalized to the maximum dilation in the presence of sodium nitroprusside 100 μmol/L and expressed as percentage of maximum dilation. G protein activity was expressed as Pi released from GTP. All data are presented as mean±SEM. Differences in baseline diameter before and after pharmacological interventions were compared by Student’s paired t test. Statistical comparisons of acidosis-induced vasodilation and the alteration of G protein activity under different treatments were performed with 1-way or 2-way ANOVA and tested with Fisher’s protected least significant difference multiple range test when appropriate. Significance was accepted at P<0.05.
Acidosis-Induced Coronary Arteriolar Dilation
All isolated arterioles developed a similar level of basal tone (Table⇓) within 40 minutes at 36°C to 37°C bath temperature with 60 cm H2O intraluminal pressure. Acidosis produced dilation of coronary arterioles in a hydrogen ion concentration–dependent manner, ie, 16±1%, 28±2%, 54±3%, and 73±3% of maximum dilation at pH 7.3, 7.2, 7.1, and 7.0, respectively (Figure 1⇓).
Effects of Endothelial Denudation, Glibenclamide, and PTX on Acidosis-Induced Dilation
Disruption of endothelium abolished coronary arteriolar dilation to 100 nmol/L bradykinin, ie, diameter increased from 77±13 to 101±20 μm and from 75±14 to 76±13 μm before and after denudation, respectively. However, coronary arteriolar dilation to acidosis (pH 7.3 to 7.0) was not altered in these denuded vessels (Figure 1⇑), indicating that this vasodilatory response was not mediated by the endothelium. Incubation of the vessels with glibenclamide 5 μmol/L or PTX 100 ng/mL did not affect resting vascular tone (Table⇑), but acidosis-induced vasodilation was significantly attenuated (Figure 1⇑). The efficacy of glibenclamide and PTX in inhibiting vasodilation to acidosis was not different.
To examine whether glibenclamide and PTX had additive inhibitory effects on acidosis-induced vasodilation, the vascular response to acidosis was examined in the presence of glibenclamide, PTX, or combined glibenclamide and PTX. Both glibenclamide and PTX inhibited arteriolar dilation to acidosis (pH 7.2 and 7.0) in a comparable manner (Figure 2⇓), which is in agreement with the results shown in Figure 1⇑. In addition, combined administration of PTX and glibenclamide did not potentiate the inhibitory effect produced by PTX or by glibenclamide alone (Figure 2⇓).
Effect of PTX on Vasodilation to Pinacidil and Sodium Nitroprusside
To examine whether KATP channels and vasodilatory function of smooth muscle were directly inhibited by PTX and glibenclamide, the vasomotor responses of isolated coronary arterioles to a KATP-channel opener, pinacidil, 0.03 to 3 μmol/L, and to an endothelium-independent vasodilator, sodium nitroprusside, 0.001 to 10 μmol/L, were studied in the absence and presence of these inhibitors. Both pinacidil and sodium nitroprusside produced dilation of isolated coronary arterioles in a dose-dependent manner (Figure 3⇓). PTX 100 ng/mL did not influence pinacidil-induced arteriolar dilation (Figure 3A⇓), indicating that KATP-channel function was not altered by PTX. In addition, vasodilation to sodium nitroprusside was not affected by combined administration of PTX 100 ng/mL and glibenclamide 5 μmol/L (Figure 3B⇓), indicating that these 2 inhibitors did not influence vasodilatory function of vascular smooth muscle.
Effect of Acidosis on G Protein Activity
To directly assess the effect of pH on G protein activity, the release of Pi (as an index of G protein activity) from GTP in G protein–reconstituted liposomes was determined under various pH conditions (pH 7.4 to 7.0). The GTPase activity in reconstituted G proteins was inversely related to the reduction of pH, ie, a 4-fold increase in GTPase activity was observed when pH was reduced in a stepwise manner from 7.4 to 7.0 (Figure 4⇓).
In the present study, using isolated vessel preparations, we demonstrated that acidosis-induced dilation in coronary arterioles is endothelium-independent and is mediated by the activation of KATP channels and PTX-sensitive G proteins, because this dilation is inhibited by either glibenclamide or PTX in an identical manner. Combined administration of these 2 inhibitors does not produce additional inhibitory effects. Furthermore, purified G proteins in phospholipid vesicles are activated in the acidic environments. Because PTX does not inhibit KATP-channel and vascular smooth muscle function and the acidosis directly activates G proteins, it is suggested that acidosis-induced dilation of coronary arterioles is mediated by the opening of smooth muscle KATP channels through activation of PTX-sensitive G proteins. The present study is the first to show the involvement of PTX-sensitive G protein in the KATP channel–mediated vasodilation and the direct activation of G proteins by acidosis. To provide a perspective for our observations and conclusions, methodological considerations such as the inhibitory effects of glibenclamide and PTX will be addressed, and the possible signaling pathway for acidosis-induced dilation will be suggested. Finally, the physiological and pathophysiological significance of our findings will be discussed.
Glibenclamide has been shown to be a selective antagonist of KATP channels.1 We previously showed that the concentration of glibenclamide, 5 μmol/L, used in the present study does not influence the resting diameter and the vasodilatory response to sodium nitroprusside,7 indicating that the inhibitory action of glibenclamide on acidosis-induced dilation is not the result of a nonspecific loss of vasodilatory capacity or damage of vascular smooth muscle. In addition, PTX neither affected resting vascular tone (Table⇑) nor altered vasodilation to pinacidil or sodium nitroprusside (Figure 3⇑). It appears that the inhibitory action of PTX is selective for acidosis-induced vasodilation rather than a nonspecific inactivation of KATP channels or damage of vascular smooth muscle for vasodilation.
We have previously shown that nitric oxide and prostaglandin signaling pathways are not involved in the activation of KATP channels during vasodilation to acidosis.7 It appears that this vasomotor response is primarily endothelium-independent, because removal of endothelium had no effect on the vasodilatory process elicited by acidosis (Figure 1⇑). The present study further demonstrated that activation of PTX-sensitive G proteins in smooth muscle is essential for this vasodilatory event (Figure 1⇑). Recently, it was speculated that the KATP channel may be directly coupled to the receptor-associated Gi protein in vascular smooth muscle,2 as has been shown in other cell types.13 14 However, there is no functional evidence to support this idea. In the present study, the dilation of coronary arterioles to acidosis was inhibited by either glibenclamide or PTX in an identical fashion (Figure 1⇑), and combined administration of these 2 agents did not further potentiate the inhibitory effect (Figure 2⇑). These results suggested that these 2 agents inhibit the same pathway for vasodilation to acidosis. Because PTX inhibited acidosis-induced vasodilation without inhibiting KATP-channel function (Figure 3A⇑), this result indicates that PTX inhibits the vasodilatory pathway upstream from the KATP channels. Therefore, it is believed that opening of KATP channels during acidosis is through the activation of PTX-sensitive G proteins.
The involvement of G proteins in acidosis-induced dilation was also supported by the reconstituted G protein studies. A widely accepted model for the association of extrinsically bound proteins with acidic phospholipid–containing membranes is that the protein-membrane interaction induces a domain of acidic phospholipids that serve as the protein binding site.19 Acidic phospholipids are known to influence membrane-bound enzymes.20 In fact, a number of key proteins involved in signal transduction (eg, phospholipase C, protein kinase C, myristylated alanine-rice C kinase substrate, pp60src protein, and G proteins) require acidic phospholipids on the plasma membranes.21 22 In the present study, we reconstituted G proteins in liposomes containing the acidic phospholipids phosphatidyl ethanolamine and phosphatidyl serine. A decrease in pH may cause an electrostatic repulsion between the negatively charged head groups of acidic phospholipids. This, in turn, changes the overall molecular organization of the lipid bilayer, which could possibly activate the bound enzyme molecules, ie, G proteins in this case. Although activation of G proteins is generally believed to be through a receptor-dependent mechanism, a recent study demonstrated that shear stress can activate membrane-bound G proteins in the absence of protein receptors.17 It is possible that alteration of local pH may elicit a pathway that bypasses membrane receptors for G protein activation, thus leading to vascular dilation. However, the major limitation of the present reconstituted G protein study is that these proteins were isolated from brain tissue rather than from the microvessels, because of the technical difficulties in obtaining a sufficient amount of microvascular G proteins. Therefore, extrapolating these data to vascular tissue, especially in relation to physiological function, is somewhat uncertain and deserves further consideration. Nevertheless, it might implicate a potential role of G protein activation in transducing messages related to acidic stress of cells. Currently, the subcellular mechanism for G protein–associated activation of KATP channels is not available. It is possible that βγ-subunits dissociating from G proteins during acidosis may play a role in KATP-channel activation. Irrespective of these speculations, additional work is needed to address this possibility and to define the cellular and molecular basis for the G protein–associated KATP-channel function in vascular smooth muscle.
Because arteriolar dilation to acidosis is a major mechanism for the metabolic regulation of coronary blood flow, PTX-sensitive G proteins and KATP channels are believed to play an important role in this regard, especially during metabolic stress. Recent studies indicated that KATP channels3 and PTX-sensitive G proteins23 are involved in the coronary arteriolar dilation in response to hypoperfusion, ischemia, and increased metabolic demand, the conditions that are generally involved in tissue acidosis.24 Our results suggested that activation of the KATP channel through the PTX-sensitive G protein signaling pathway may contribute to the vasodilation observed during these metabolic disturbances. Under pathophysiological conditions such as hypercholesterolemia25 and certain forms of hypertension,26 the alteration of G protein function in vascular smooth muscle may have significant impact on the vasodilatory function of coronary arterioles in response to acidic stress, resulting in an inadequate flow and oxygen supply to the tissue during intense metabolic demands.
This study was supported by National Heart, Lung, and Blood Institute grants HL-48179 and HL-03693 (Research Career Development Award) to Dr Kuo. We thank Dr Travis W. Hein for his suggestions and review of the manuscript.
- Received May 15, 1998.
- Revision received September 9, 1998.
- Accepted September 25, 1998.
- Copyright © 1999 by American Heart Association
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