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Circulation Research. 1997;80:1-10

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(Circulation Research. 1997;80:1-10.)
© 1997 American Heart Association, Inc.

Articles

Mechanisms of Coronary Microvascular Dilation Induced by the Activation of Pertussis Toxin–Sensitive G Proteins Are Vessel-Size Dependent

Heterogeneous Involvement of Nitric Oxide Pathway and ATP-Sensitive K+ Channels

Tatsuya Komaru, Toshinori Tanikawa, Akihiko Sugimura, Toshinobu Kumagai, Kouichi Sato, Hiroshi Kanatsuka, Kunio Shirato

The First Department of Internal Medicine, Tohoku University, School of Medicine, Sendai, Japan.

Correspondence to Tatsuya Komaru, MD, The First Department of Internal Medicine, Tohoku University, School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, 980 Japan. E-mail komaru@int1.med.tohoku.ac.jp


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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G proteins are critically important mediators of many signal transduction systems. In the present study, we investigated the effect of direct activation of pertussis toxin (PTX)–sensitive G protein (GPTX) on coronary arterial microvascular tone in 37 open-chest anesthetized dogs in vivo. Coronary arterial microvessels on the surface of the beating left ventricle were visualized by performing fluorescence coronary microangiography using an intravital microscope with a floating objective system. Microvessels were divided into two groups, small microvessels (inner diameter, <=130 µm) and large microvessels (inner diameter, >130 µm). Topically applied mastoparan (G protein activator, 10, 30, and 100 µmol/L) produced homogeneous microvascular dilation in a concentration-dependent manner (10 µmol/L, 7.9±2.0%; 30 µmol/L, 10.3±2.4%; and 100 µmol/L, 16.7±4.5% in small microvessels; 10 µmol/L, 5.3±1.2%; 30 µmol/L, 9.8±2.5%; and 100 µmol/L, 15.5±3.9% in large microvessels). These dilations were reversed to constriction by pretreatment with PTX (300 ng/mL, 2 hours) in both microvessel groups. Blockade of nitric oxide production by N{omega}-nitro-L-arginine (LNNA, 300 µmol/L) offset the mastoparan-induced dilation in large microvessels but not in small microvessels. Cosuperfusion of glibenclamide (10 µmol/L) with LNNA produced constriction of all sizes of microvessels in response to mastoparan, whereas charybdotoxin (10 nmol/L) did not affect the mastoparan effect. Pretreatment with glibenclamide alone reversed mastoparan dilation to constriction in small microvessels, whereas it only offset the dilation without producing constriction in large microvessels. We conclude that the activation of GPTX produces homogeneous coronary arterial microvascular dilation and that the underlining mechanisms of the dilation are vessel size dependent. The L-arginine–nitric oxide pathway mediates the dilation only in large microvessels, whereas ATP-sensitive K+ channel activation plays a central role in the dilation of small microvessels when GPTX is directly activated. ATP-sensitive K+ channels are also involved in the dilation of large microvessels in a synergistic fashion with nitric oxide production.


Key Words: coronary circulation • guanine nucleotide regulatory protein • arterioles • microcirculation • vasodilation


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Heterotrimeric GTP-binding regulatory proteins (G proteins) play central roles in transducing various biological signals from the outside to the inside of the cell.1 2 3 4 5 6 Many endogenous substances bind their specific receptors on the surface of the cell membrane, activate specific G proteins, and modulate the activity of effectors such as ion channels and enzymes. These receptor-dependent signal transduction systems are important pathways that mediate various cellular functions. Recent evidence has raised the possibility that G proteins also transduce signals in a receptor-independent manner.7 8 9 10

We have previously shown that GPTX mediates the coronary microvascular dilation during a reduction in perfusion pressure in vivo.11 The mechanism of G protein activation during such microvascular responses is not known. Several investigators, including the authors, have shown that adenosine does not play a major role in this microvascular control during coronary autoregulation and ischemia.12 13 14 One possibility is that some physical stimuli other than agonists activate the G protein. To date, it has not been directly shown whether activation of GPTX causes coronary microvascular dilation in vivo. Activation of GPTX may cause vasodilation via the L-arginine–nitric oxide pathway or via hyperpolarization by K+ channel activation. On the other hand, it may cause vasoconstriction via an inhibitory effect on adenylyl cyclase or via phospholipase C activation in vascular smooth muscle.2 3 5

Accordingly, the purpose of the present study was (1) to test the hypothesis that the activation of GPTX produces coronary microvascular dilation and (2) to elucidate the underlining mechanisms of the microvascular responses during the activation of the G protein. We applied mastoparan to activate GPTX, because Higashijima and colleagues15 16 have shown that it forms an amphiphilic {alpha} helix upon binding to the cell membrane and activates GPTX in a receptor-independent way. We examined the functional link of GPTX to the L-arginine–nitric oxide pathway and to K+ATP channels in coronary microcirculation in vivo.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
General Preparation
Mongrel dogs of either sex (n=37; body weight, 4.4 to 11.4 kg [7.0±0.3 kg]) were premedicated with ketamine (50 mg IM) and anesthetized with an intravenous injection of {alpha}-chloralose (60 mg/kg, Wako Chemicals). Additional doses of the anesthetics were given as necessary. The animals were intubated with a cuffed endotracheal tube and mechanically ventilated with a respirator (model NSH-34RH, Harvard Apparatus). A positive end-expiratory pressure of 3 to 5 cm H2O was applied to prevent lung atelectasis. Arterial blood gases were kept within physiological ranges by adjusting the volume or frequency of ventilation and/or by application of supplemental oxygen. Intravenous infusion of sodium bicarbonate was applied, when necessary, to maintain the physiological pH of the blood. Body temperature was kept at 37°C with a heat blanket. The right jugular vein was cannulated for administration of anesthetics and fluid infusion. A catheter was introduced into the right carotid artery, and its tip was placed in the ascending aorta for the measurement of aortic pressure with a strain-gauge transducer (model MPU 0.5, Toyo Sokki).

A left thoracotomy was performed in the fifth intercostal space, and the heart was suspended on the pericardial cradle. A plastic wrap was used to separate the lung from the heart. A catheter was introduced into the left atrium for injection of fluorescein isothiocyanate dextran (molecular weight, 150 000; Sigma Chemical Co), which was used as a contrast media for fluorescence coronary microangiography. The heart rate was kept constant at 140 bpm by left atrial pacing after suppression of the sinus node with a local injection of 10% formaldehyde (0.5 mL) into the sinoatrial nodal area. The heart surface was kept moist throughout the experiment by continuously dripping warmed Krebs' solution (mmol/L: NaCl 118.2, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, calcium disodium EDTA 0.026, and glucose 5.5, maintained at 37°C and pH 7.40) at a rate of 60 mL/h except in the case of superfusion with PTX or mastoparan.

To reduce excessive cardiac movement, two 24-gauge stainless steel needles were inserted horizontally (5 to 7 mm apart) into the midmyocardium of the left ventricle. Both ends of each needle were fixed to a needle holder held with coil springs. This apparatus allows the heart to move perpendicularly but limits excessive horizontal movement, thereby maintaining the area of interest in the microscopic field of view.

Image Acquisition System
For direct visualization of coronary microvessels in beating left ventricles, an intravital microscope system equipped with a floating objective developed in our laboratory was used. Details of this system have been previously reported.17 18 Briefly, the floating objective consists of a pair of convex lenses that transmit the real image to a standard microscope without any change in magnification. The lower part of the objective facing the heart surface can move perpendicularly in unison with the cardiac motion. The movable portion is lifted by an objective lifter that prevents its direct contact with the heart surface. The upper part of the floating objective was fixed to the stage of a standard microscope. The transmitted real images of the coronary microvessels in the epimyocardial layer were then observed with the standard microscope. The microscope objective used for this study was a Leitz model PL-fl (x10; numerical aperture, 0.30).

Epi-illuminated fluorescence coronary microangiography was performed to obtain microvascular images and measure microvascular diameters. The surface of the left ventricle was epi-illuminated by incident light from a mercury lamp (HBO-100EW/2, Nikon Inc). The maximal wavelength of the illuminating light was 495 nm, obtained by using a B2 excitation filter (Nikon). Intermittent exposure to ultraviolet light occurred no more than eight times in one experiment, and the exposure period for taking one motion picture was <10 seconds. An earlier study has shown that intermittent exposure to ultraviolet light for short periods does not lead to microvascular injury.19 The emitted light was then passed through a 510-nm filter. A highly sensitive TV camera (C 1000-12, Hamamatsu Photonics), a high-resolution TV monitor (C 1846-01, Hamamatsu Photonics), and a videocassette recorder (AG-7350, Panasonic) were used to monitor and to store the enhanced vascular images. The spatial resolution of this system was {approx}2 µm. Video images were recorded at 60 frames per second.

Microvascular Diameter Measurement
The microvascular images including the plasma layer were enhanced by fluorescein isothiocyanate dextran (30 mg/mL in saline, 0.2 mL), which was injected via the catheter placed in the left atrium each time the fluorescent images were obtained. The inner diameter of arterial microvessels was measured on a high-resolution monitor screen (C1846-01, Hamamatsu Photonics) using a videomanipulator (C 2117, Hamamatsu Photonics). On the monitor screen, one cursor was set on the vessel wall of interest, and another cursor was set on the nearest point of the other side of the vessel wall. The distance between the two cursors was automatically calculated by the videomanipulator, thereby yielding the microvascular diameters. Arterial microvessels could be easily discriminated from venous ones by the sequential enhancement of microvessels from artery to capillary and vein. Microvascular diameters were measured at least three times at the end-diastolic phase. To compare diameters before and after each intervention, diameters were measured at the same place, with vascular branchings or other vessels used as reference points.

Experimental Protocols
Experiments were performed {approx}30 minutes after the surgical preparation and instrumentation, when all monitored variables had become stable.

A pilot study (3 dogs, seven vessels) showed that mastoparan superfusion (100 µmol/L) produced maximal dilation in 5 minutes and that, thereafter, the microvascular diameter gradually decreased. Based on these observations, all data concerning mastoparan were collected in 5 minutes by use of the following protocols.

Group 1
In 11 dogs (group 1), the concentration-response relationship of mastoparan was constructed. After control diameter measurement, three concentrations of mastoparan (10, 30, and 100 µmol/L) were sequentially superfused. Five minutes after the onset of superfusion of each concentration of mastoparan, the microvascular image was obtained. At the end of this protocol, 100 µmol/L of sodium nitroprusside was superfused.

Group 2
In 4 dogs (group 2), the effect of PTX on mastoparan-induced responses was investigated. After the control measurement, PTX (300 ng/mL) was superfused for 2 hours. Microvascular diameters were measured every 30 minutes during the 2-hour pretreatment with PTX. Thereafter, three concentrations of mastoparan were sequentially applied as described above. In this protocol, additional doses of {alpha}-chloralose ({approx}10 mg/kg) were usually injected every half hour to maintain an adequate anesthetic level during the superfusion of PTX. Microvascular diameters, hemodynamic variables, and blood gas data were measured >20 minutes after the administration of anesthetics.

Group 3
In 7 dogs (group 3), the effect of LNNA (nitric oxide inhibitor) on mastoparan-induced responses was tested. After the control measurement, LNNA (300 µmol/L) was topically superfused onto the heart surface until the end of the experiment. Twenty minutes after beginning the LNNA superfusion, three concentrations of mastoparan were applied.

Group 4
In 5 dogs (group 4), the effect of cosuperfusion of LNNA and glibenclamide (K+ATP channel inhibitor) on mastoparan-induced responses was investigated. After the control measurement, LNNA (300 µmol/L) and glibenclamide (10 µmol/L) were superfused onto the heart surface until the end of experiment. Twenty minutes after the onset of the cosuperfusion, three concentrations of mastoparan were applied.

Group 5
In 4 dogs (group 5), the effect of cosuperfusion of LNNA and charybdotoxin (large-conductance K+Ca2+ channel inhibitor) on mastoparan-induced microvascular responses was investigated. The protocol was the same as in group 4, but glibenclamide was replaced with charybdotoxin (10 nmol/L).

Group 6
In 6 dogs (group 6), the effect of glibenclamide alone on the mastoparan responses was investigated. The protocol was the same as in group 4, but LNNA was not applied.

Blood pressure was continuously monitored, and blood samples for analyses of blood gases and blood pH were obtained at the start and end of each experiment.

Drugs
Mastoparan (1 mg, Bachem California) was freshly dissolved in 0.2 mL of distilled water at first and then diluted to the target concentration with Krebs' solution on each experimental day. Lyophilized PTX (100 µg, Seikagaku Co) was dissolved with 10 mL of distilled water and stored at 4°C. The PTX solution was freshly diluted with Krebs' solution to 300 ng/mL on each experimental day. LNNA (Sigma) and sodium nitroprusside (Wako Chemicals) were freshly dissolved with Krebs' solution. Glibenclamide (Sigma) was dissolved with dimethyl sulfoxide at first, and then the target concentration was obtained by diluting with Krebs' solution. The final concentration of dimethyl sulfoxide was 0.01 vol%. Charybdotoxin (100 µg, Peptide Institute) was dissolved with 230 µL of distilled water, and the aliquots of charybdotoxin (20 µL, 100 µmol/L) were stored at -20°C until use. An aliquot was freshly dissolved with Krebs' solution on the day of the experiment.

Administration of Drugs
As mentioned above, Krebs' solution (37°C) was continuously superfused onto the observation area throughout the experiment at the rate of 60 mL/h, unless otherwise stated. To apply all agents except mastoparan and PTX, a 10 times higher concentration of each agent was superfused via the side port of the Krebs' solution superfusion line at a rate of 6 mL/h, and the rate of superfusion of Krebs' solution was reduced to 54 mL/h. With this method, the target concentration of each agent was obtained at the heart surface. When mastoparan or PTX was applied, the superfusion of Krebs' solution was stopped, and the target concentration of these agents was superfused onto the heart surface at a rate of 10 mL/h with a syringe pump (STC 521, Terumo). The entire superfusion line was continuously warmed by a warm-water circuit using a thermostat water bath to keep the superfusate temperature at 37°C on the heart surface. In groups 3, 4, 5, and 6, the mastoparan solution also contained the target concentration of blockers (LNNA, glibenclamide, charybdotoxin, or a combination of these) to maintain the blockade of nitric oxide production, K+ATP channels, or K+Ca2+ channels throughout the experiment.

Data Analysis
Aortic pressure was recorded on a Rectigraph (model 8K, San-Ei Sokki). All variables were described as mean±SEM. To evaluate the microvascular responses caused by the agents, the percent change in diameter from baseline was calculated. When microvessels were pretreated with blocker(s), the diameter after the end of each pretreatment was considered as the baseline. Regression analysis (polynomial) was performed to assess the relationships between the microvascular responses caused by mastoparan and the vessel sizes in each protocol. The regression analysis revealed the size dependence of mastoparan-induced dilation in the presence of LNNA. The x intercept of the regression line was between microvascular diameters of 100 and 150 µm. On the basis of this observation, arterial microvessels were divided into two groups according to their control diameters: small microvessels (inner diameter, <=130 µm) and large microvessels (inner diameter, >130 µm). Changes in aortic pressure and microvascular diameters were statistically analyzed using one-way ANOVA for repeated measures and Student's t test for paired samples, modified by the Bonferroni multicomparison method to detect significant changes.20 To compare the percent changes in microvascular diameters produced by mastoparan between the groups, Student's t test for unpaired samples was applied. The differences were accepted as significant at P<.05.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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The TableDown shows the hemodynamic and blood gas data during the experiments. The aortic pressure did not change in any of the groups during mastoparan superfusion; however, nitroprusside superfusion decreased the aortic pressure in group 1. Blood gases and blood pH were within normal limits throughout the experiment.


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  		Table 1. Aortic Pressure, Blood pH, and Blood Gas Data During Each Protocol

As shown in Figure 1Down, mastoparan superfusion produced vasodilation of the coronary arterial microvessels. Regression analysis did not demonstrate any significant correlation between the control diameter and the microvascular responses by mastoparan at any concentration of mastoparan. The dilation was concentration dependent in both large (>130-µm) and small (<=130-µm) microvessels. In large vessels (11 vessels; mean diameter, 180±12 µm), the magnitude of the vascular responses for each concentration was as follows: 10 µmol/L, 5.3±1.2%; 30 µmol/L, 9.8±2.5%; and 100 µmol/L, 15.5±3.9% (P<.05 versus control diameter in every concentration). In small vessels (12 vessels; mean diameter, 87±7 µm ), the magnitude of the dilation was as follows: 10 µmol/L, 7.9±2.0%; 30 µmol/L, 10.3±2.4%; and 100 µmol/L, 16.7±4.5% (P<.05 versus control diameter at every concentration). There was no statistical difference in mastoparan-induced vasodilation between the large and small vessels at any concentration of mastoparan. Nitroprusside (100 µmol/L) produced further dilation in both large (26.8±3.9%) and small (44.6±6.1%) microvessels.





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  		Figure 1. Scatterplots showing coronary arterial microvascular responses caused by mastoparan (10 µmol/L [A], 30 µmol/L [B], and 100 µmol/L [C]) in vivo. Mastoparan was superfused onto the heart surface. Mastoparan produced homogeneous vasodilation in a concentration-dependent manner. Regression analysis showed no significant correlation between control microvascular diameter and vascular responses.

PTX superfusion per se did not produce any change in baseline diameter in either large vessels (7 vessels; mean diameter, 188±16 µm; 30 minutes, 2.3±1.4%; 60 minutes, 0.4±0.9%; 90 minutes, -1.4±0.9%; and 120 minutes, -0.3±0.3%) or small vessels (7 vessels; mean diameter, 101±9 µm; 30 minutes, 3.0±2.3%; 60 minutes, 3.1±1.2%; 90 minutes, 0.5±1.2%; and 120 minutes, 0.4±1.5%). When microvessels were pretreated with PTX, mastoparan-induced vasodilation was abolished regardless of the control diameter (Fig 2Down). Mastoparan actually produced vasoconstriction at higher concentrations. The constriction was statistically significant at 100 µmol/L in small vessels and at 30 and 100 µmol/L in large vessels. These data indicate that the dilatory component of coronary microvascular responses to mastoparan is mainly mediated by the activation of GPTX and that mastoparan constricts microvessels in a GPTX-independent manner.





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  		Figure 2. Scatterplots showing microvascular responses caused by mastoparan (10 µmol/L [A], 30 µmol/L [B], and 100 µmol/L [C]) in vivo under pretreatment with PTX (300 ng/mL) for 2 hours. PTX totally abolished mastoparan-induced microvascular dilation. Higher concentrations of mastoparan produced microvascular constriction regardless of vessel size.

LNNA superfusion (300 µmol/L) did not affect the diameters in large vessels (8 vessels; mean diameter, 171±18 µm; -5.3±3.3%) or small vessels (11 vessels; mean diameter, 84±8 µm; -0.6±2.8%). Fig 3Down shows the individual microvascular responses to the three concentrations of mastoparan in the presence of LNNA. Regression analysis demonstrated a significant correlation between control diameters and microvascular responses at the two higher concentrations of mastoparan (Fig 3B and 3CDownDown). That is, LNNA inhibited the mastoparan-induced dilation only in the larger microvessels; it failed to inhibit the dilation in smaller vessels.





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  		Figure 3. Scatterplots showing microvascular responses caused by mastoparan (10 µmol/L [A], 30 µmol/L [B], and 100 µmol/L [C]) in vivo in the presence of LNNA (300 µmol/L). Regression analysis showed a significant correlation between control microvascular diameters and vascular responses at 30 and 100 µmol/L of mastoparan. The regression lines were y=60.77-0.764x+0.0213x2 at 30 µmol/L of mastoparan, and y=90.13-0.966x+0.0240x2 at 100 µmol/L of mastoparan.

Cosuperfusion of glibenclamide and LNNA tended to decrease the baseline diameter in both large vessels (7 vessels; mean diameter, 180±17 µm; -5.6±2.9%) and small vessels (7 vessels; mean diameter, 86±11 µm; -4.1±3.6%), but these changes did not attain statistical significance. As shown in Fig 4Down (open symbols), mastoparan caused microvascular constriction in the presence of glibenclamide and LNNA in both large and small microvessels. When glibenclamide was replaced with charybdotoxin (small microvessels: 8 vessels; mean diameter, 69±5 µm; large microvessels: 6 vessels; mean diameter, 184±15 µm), mastoparan-induced dilation still occurred in small microvessels (Fig 4Down, closed symbols).





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  		Figure 4. Scatterplots showing microvascular responses caused by mastoparan (10 µmol/L [A], 30 µmol/L [B], and 100 µmol/L [C]) in vivo in the presence of glibenclamide (10 µmol/L, {circ}) or charybdotoxin (10 nmol/L, {bullet}) in addition to LNNA (300 µmol/L). The addition of glibenclamide to LNNA abolished the mastoparan-induced microvascular dilation in all sizes of coronary microvessels. The dilation of small microvessels still occurred when glibenclamide was replaced with charybdotoxin.

The effect of glibenclamide alone on the mastoparan responses was investigated (Fig 5Down). Glibenclamide did not change the baseline diameter in either small vessels (10 vessels; mean diameter, 80±9 µm; -1.2±1.6%) or large vessels (7 vessels; mean diameter, 191±17 µm; -1.8±1.4%). Glibenclamide blocked the mastoparan-induced dilation. As vasoconstriction occurred in small microvessels, the regression analysis revealed a weak but significant correlation between the control diameters and mastoparan-induced responses at 10 and 100 µmol/L of mastoparan (Fig 5A and 5CDownDown).





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  		Figure 5. Scatterplots showing microvascular responses caused by mastoparan (10 µmol/L [A], 30 µmol/L [B], and 100 µmol/L [C]) in vivo in the presence of glibenclamide (10 µmol/L) alone. Glibenclamide significantly inhibited mastoparan-induced microvascular responses in all sizes of microvessels. Regression analysis showed a significant correlation between control microvascular diameters and vascular responses at 10 and 100 µmol/L of mastoparan. The regression lines were as follows: y=-11.86+0.114x-0.00024x2 at 10 µmol/L of mastoparan, and y=-15.38+0.121x-0.00017x2 at 100 µmol/L of mastoparan.

Fig 6Down shows the group data of microvascular responses by mastoparan in small and large microvessels in the presence of various blockers. In small microvessels (Fig 6ADown), mastoparan produced dilation in a concentration-dependent manner. The concentration-dependent dilation still occurred in the presence of LNNA, and the magnitude of dilation was not statistically different from that in the absence of LNNA at any of the three concentrations. When glibenclamide was cosuperfused with LNNA, mastoparan-induced dilation was abolished, and the percent change in diameter caused by mastoparan was statistically different from that in the absence of these pretreatments and from that in the presence of LNNA alone at all three concentrations of mastoparan. The addition of charybdotoxin to LNNA did not inhibit mastoparan dilation at any concentration. Glibenclamide alone reversed the dilation to constriction at every concentration of mastoparan. There were no significant differences in mastoparan responses among the PTX group, LNNA+glibenclamide, and glibenclamide alone groups. Collectively, in small microvessels, mastoparan produced dilation mainly via the activation of K+ATP channels.




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  		Figure 6. Bar graphs showing the effect of various blockers on mastoparan-induced microvascular responses in small microvessels (inner diameter, <=130 µm [A]) and large microvessels (inner diameter, >130 µm [B]). Open bars indicate no pretreatment; stippled bars, pretreatment with LNNA (300 µmol/L); hatched bars below abscissa, pretreatment with LNNA (300 µmol/L) and glibenclamide (10 µmol/L); hatched bars above abscissa, pretreatment with LNNA (300 µmol/L) and charybdotoxin (10 nmol/L); vertically lined bars, pretreatment with glibenclamide (10 µmol/L); and solid bars, pretreatment with PTX (300 ng/mL). *P<.05 vs no pretreatment; {dagger}P<.05 vs pretreatment with LNNA.

On the other hand, in large microvessels (Fig 6BUp), the mastoparan-induced dilation, which was again concentration dependent, was offset by LNNA. Glibenclamide alone also offset the mastoparan dilation, but neither LNNA alone nor glibenclamide alone produced mastoparan-induced constriction. In contrast, the combination of LNNA and glibenclamide reversed the dilation by mastoparan to vasoconstriction at the higher two concentrations of mastoparan. PTX pretreatment also reversed mastoparan-induced dilation to constriction at the higher two concentrations of mastoparan. The microvascular responses in large vessels were significantly different between the LNNA group and the cosuperfusion (LNNA+glibenclamide) group at the two higher concentrations (30 and 100 µmol/L). Charybdotoxin did not have an additional effect on the mastoparan responses in the presence of LNNA. Collectively, these results suggest that in large microvessels the nitric oxide pathway and K+ATP channel activation synergistically mediate the vascular responses to GPTX activation and that the large-conductance K+Ca2+ channel does not play a significant role in those microvascular responses.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrates the following new findings concerning the coronary microvascular regulation via GPTX. First, direct activation of GPTX causes dilation of coronary arterial microvessels in vivo. Second, the mechanism of the vasodilation by GPTX activation is dependent on vessel size. In small microvessels <130 µm in diameter, the activation of GPTX results in vasodilation by the activation of K+ATP channels, and the nitric oxide pathway does not play a role in this vascular response. On the other hand, in large microvessels, both the production of nitric oxide and the activation of K+ATP channels mediate the dilation. The contribution of the K+ATP channel activation is greater in the small microvessels than in the large ones. Third, large-conductance K+Ca2+ channels do not play a role in determining microvascular tone when GPTX is activated.

G-Protein Activation by Mastoparan
We used mastoparan to directly activate G protein. Mastoparan is a tetradecapeptide toxin from wasp venom and is known to activate G protein in a receptor-independent fashion.7 10 15 16 When this substance binds to a phospholipid membrane, it forms an amphiphilic {alpha} helix and enhances the GDP-GTP exchange reaction on heterotrimeric G protein, mimicking the agonist-bound receptors and resulting in G-protein activation.15 16 Higashijima and colleagues15 16 have shown that the effect of mastoparan is specific to GPTX.

We topically superfused this agent on the heart surface to avoid its systemic effects. The blood pressure, blood pH, and blood gases did not change during mastoparan superfusion. The present study actually demonstrated that the vasodilator component of the mastoparan-induced responses was concentration dependent and was PTX sensitive regardless of the vessel size. Our previous study has shown that superfusion of PTX for 2 hours effectively blocks the {alpha}2-receptor–mediated microvascular response,11 which is known to be exclusively mediated by Gi protein.21 Thus, mastoparan superfusion is a useful way to investigate the involvement of signal transduction via Gi/Go protein in epimyocardial coronary microcirculation in vivo.

GPTX activation might cause both vasodilation and vasoconstriction because it can affect several effectors.1 2 3 4 5 6 It may cause vasodilation via K+ channel activation or nitric oxide production. On the other hand, it may cause vasoconstriction via phospholipase C activation or via a decrease in cAMP by inhibiting adenylyl cyclase. The present study showed that mastoparan produces homogeneous microvascular dilation in a concentration-dependent manner, suggesting that the link between GPTX and K+ channel and/or nitric oxide synthase is so potent that the vasoconstriction by GPTX activation is masked in coronary microvessels when GPTX is directly activated.

The present study may have underestimated the vasodilation on GPTX activation, because PTX pretreatment unmasked the mastoparan-induced constriction. These constrictor responses reflect some mastoparan effects that are not sensitive to PTX. Mastoparan has been reported to affect membrane-bound enzyme activity such as Na,K-ATPase22 and protein kinase C.23 The changes in the activity of these enzymes and/or the effect on other G proteins that are PTX insensitive may explain this vasoconstriction.

Mastoparan was originally known to facilitate histamine secretion from mast cells.24 The vasodilation we observed in the present study might have resulted from the effect of the released histamine. The distribution and the number of mast cells exhibit considerable variation according to species and organs, but mast cells are abundant near surfaces exposed to environmental antigens, such as the skin, gastrointestinal tract, and respiratory system.25 Furthermore, Majeed26 reported that mast cells were rarely seen in normal dogs. Because mastoparan does not have specificity to mast cells in terms of G-protein activation, we believe that the role, if any, of histamine released from mast cells in mastoparan-induced microvascular responses is minor, although we did not test the effect of histamine blocker on these responses in the present study.

Activation of GPTX and the L-Arginine–Nitric Oxide Pathway
Nitric oxide production mediates the mastoparan-induced dilation in large microvessels because LNNA, a nitric oxide inhibitor, significantly inhibits the vascular responses in those vascular segments only. It is conceivable that superfused mastoparan stimulated endothelial G protein, leading to the production of nitric oxide. A link between GPTX and the nitric oxide pathway has been demonstrated in large conduit vessels. Earlier studies have reported that some agonists, such as serotonin and {alpha}2 agonists, activate the nitric oxide pathway via G-protein activation in a PTX-sensitive manner.27 28

We have previously shown that {alpha}2 agonist and neuropeptide Y, both of which activate Gi protein, cause significant constriction in vivo in this size of coronary microvessels.11 29 Accordingly, there must be a link between GPTX and vasoconstrictor mediators in the microvascular smooth muscle. It is likely that vasodilatory mechanisms, including nitric oxide production, become dominant and mask vasoconstriction in large coronary microvessels when GPTX is activated in a receptor-independent manner.

Activation of GPTX and K+ATP Channels
Glibenclamide significantly blocked mastoparan-induced dilation in both large and small microvessels. In a rat portal vein preparation, glibenclamide has been reported to block large-conductance K+Ca2+ channels as well as K+ATP channels.30 Earlier studies, however, have shown that in the coronary vascular bed, there is no overlap in the pharmacological effects between glibenclamide at the concentration we used in the present study and K+Ca2+ channel blockers.31 32 33 We tested the effect of charybdotoxin and found that it did not affect the microvascular responses to mastoparan. Thus, it is quite likely that the K+ channel that couples with GPTX in coronary microvessels is the K+ATP channel.

Kirsch et al34 and Ito et al35 have independently demonstrated a link between GPTX and K+ATP channels in cardiocytes. Ribalet and Eddlestone36 have reported the link in insulin-secreting cell lines. The present study is the first to demonstrate a functional link between GPTX and K+ATP channels in vascular tissue.

As shown in Fig 5A and 5CUpUp, the mastoparan effect with glibenclamide pretreatment was significantly correlated with the control diameters, demonstrating that the contribution of the K+ATP channel is greater in small than in large microvessels. We have previously shown that K+ATP channels exist in coronary arterial microvessels and function as regulators of microvascular tone.37 38 Sato et al37 have previously shown that levcromakalim, a specific K+ATP channel activator, produces greater vasodilation in small coronary microvessels than in larger ones and increases coronary flow velocity in vivo. Akai et al38 have demonstrated that nicorandil, a hybrid drug of nitrate and K+ATP channel activator, produces dilation of smaller coronary microvessels, whereas nitrates dilate only coronary microvessels >200 µm in diameter.39 Those findings are consistent with the present study.

We have previously demonstrated that GPTX plays a pivotal role in regulating vasodilation in small coronary arterial microvessels during a reduction in perfusion pressure.11 The present authors have also shown in a similar preparation that K+ATP channels regulate coronary microvascular tone during autoregulation and ischemia.40 The present study supports the notion that GPTX and K+ATP channels do not act on coronary microvessels independently of each other but affect microvascular tone as one sequence during the reduction in perfusion pressure. It is conceivable that the activation of GPTX results in the activation of K+ATP channels, leading to hyperpolarization and relaxation of smooth muscle in coronary microvessels. The trigger of GPTX activation during a reduction in perfusion pressure is not clear at this point. Adenosine has been known to activate Gi protein via the A1 receptor in cardiocytes.34 The authors, however, have shown that adenosine is not a likely agonist that mediates the hypoperfusion-induced microvascular dilation.12 Other laboratories also have provided evidence for a limited role of adenosine as a determinant of coronary conductance during a low perfusion state.13 14 Some other mechanisms such as physical forces or chemical factors may operate in this dilation.

Although the K+ATP channel is regulated by the intracellular ATP level,41 many other factors are known to affect the activity of the channel,42 43 44 45 among which is low pH.43 Ishizaka and Kuo46 have recently shown that acidosis-induced microvascular dilation is mediated by K+ATP channels. Although they did not investigate the involvement of G protein in this coronary microvascular response, it is interesting to speculate that the decrease in pHi during a low perfusion state caused the activation of Gi/Go protein, followed by K+ATP channel activation.

Vessel Size and the Heterogeneity of Regulatory Mechanisms of Coronary Microvascular Tone
The present study demonstrated that the mechanisms of the coronary microvascular responses to GPTX activation are vessel size dependent. Earlier studies have demonstrated that coronary microvascular responses to various physiological and pharmacological interventions are strikingly heterogeneous according to the vessel sizes.47 48 49 50 51 The dominance of K+ channel activation in small microvessels and that of the nitric oxide pathway in large microvessels may at least partly explain the heterogeneous microvascular responses. For instance, the authors have demonstrated that acetylcholine-induced coronary microvascular dilation is totally blocked by arginine analogues in large but not small microvessels.51 This phenomenon reflects the heterogeneous contribution of the L-arginine–nitric oxide pathway and hyperpolarization of vascular smooth muscle in acetylcholine-induced microvascular responses. The profound involvement of the K+ATP channel in small microvessels, which may closely interact with organ metabolism, and the greater involvement of the nitric oxide pathway in larger microvessels, which may play an important role in organ flow distribution, are purposive.

Because shear stress has been suggested to constitutively yield nitric oxide via GPTX,8 it might have been expected that treatment with PTX results in vasoconstriction. However, in the present study, the microvascular diameters did not change during superfusion with PTX in either small or large microvessels, which is consistent with our previous study.11 We speculate that other PTX-insensitive regulators may have compensated for the vasoconstrictor component.

Possible Physiological Role of Receptor-Independent G-Protein Activation
Recent evidence has shown that G-protein activation does not necessarily require receptor occupation by agonists.7 8 9 10 52 Metz et al52 have shown that in the pancreas beta cell, blockade of certain G proteins selectively reduces glucose-induced insulin secretion. Because there is no receptor for glucose, some receptor-independent activation of G protein should take place in this response. In the field of angiology, the possibility has been proposed that a physical force (shear stress) activates GPTX in the endothelium, leading to K+ channel activation followed by nitric oxide production and vasodilation.8 Several endogenous amphiphilic peptides, such as substance P, histamine, and bradykinin, exert their effects not only via a receptor-operated mechanism but also via the direct activation of G protein.10 Furthermore, Kowluru et al9 have recently proposed a novel mechanism of G-protein activation via transient phosphorylation of the ß subunit of G protein, which could be receptor independent. These studies raised the possibility that receptor-independent activation of heterotrimeric G protein may play an important physiological role.

Clinical Implication
Gi protein–mediated endothelium-dependent vasodilation is impaired in diseased conditions such as atherosclerosis and hypercholesterolemia.53 54 Recent evidence has shown that oxidized low-density lipoprotein inhibits Gi protein function in the aortic endothelial cell.55 Gawler et al56 have shown that Gi protein is downregulated in experimental diabetic animals. Analogous with these phenomena, it is possible that in some diseased conditions, Gi protein–mediated microvascular function is impaired, leading to defective physiological defense mechanisms such as coronary autoregulation.


*    Selected Abbreviations and Acronyms
 
GPTX = PTX-sensitive G protein
K+ATP channel = ATP-sensitive K+ channel
K+Ca2+ channel = Ca2+-activated K+ channel
LNNA = N{omega}-nitro-L-arginine
PTX = pertussis toxin


*    Acknowledgments
 
This study was supported by grants from the Scientific Research Fund of Ministry of Education, Science, and Culture, Tokyo, Japan (Nos. 07670745 and 08670752). We thank B. Bell for reading this manuscript.


*    Footnotes
 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996.

Received May 10, 1996; accepted October 8, 1996.


*    References
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