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Cardiovascular Research 2000 45(4):1001-1009; doi:10.1016/S0008-6363(99)00414-9
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Reversal of glibenclamide-induced coronary vasoconstriction by enhanced perfusion pulsatility: possible role for nitric oxide

Pasquale Pagliaro, Nazareno Paolocci, Takayoshi Isoda, Walter F Saavedra, Genshiro Sunagawa and David A Kass*

The Division of Cardiology, Department of Medicine, and Department of Bioengineering, The Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA

* Corresponding author. Present address: Halsted 500, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21287, USA. Tel.: +1-410-955-7153; fax: +1-410-955-0852 dkass{at}bme.jhu.edu

Received 3 August 1999; accepted 19 November 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: ATP-sensitive potassium channels (K+ATP) prominently contribute to basal coronary tone; however, flow reserve during exercise remains unchanged despite channel blockade with glibenclamide (GLI). We hypothesized that increasing perfusion pulsatility, as accompanies exercise, offsets vasoconstriction from K+ATP-channel blockade, and that this effect is blunted by nitric oxide synthase (NOS) inhibition. Methods: In 31 anaesthetized dogs the left anterior descending artery was blood-perfused by computer-controlled servo-pump, with real-time arterial perfusion pulse pressure (PP) varied from 40 and 100 mm Hg at a constant mean pressure and cardiac workload. Results: At control PP (40 mm Hg), GLI (50 µg/min/kg, i.c.) lowered mean regional coronary flow from 37±5 to 25±4 ml/min (P<0.001). However, this was not observed at 100 mm Hg PP (41±2 vs. 45±4). NOS inhibition by NG-monomethyl-L-arginine (L-NMMA) did not alter basal flow at 40 mm Hg PP, but modestly lowered flow (–5%, P<0.001) at higher PP (100 mm Hg), reducing PP-flow augmentation by –36%, and acetylcholine (ACh) induced flow elevation by –39%. Co-infusion of L-NMMA with GLI resulted in net vasoconstriction at both PP levels (–60% and –40% at 40 and 100 mm Hg PP, respectively). Unlike GLI, vasoconstriction by vasopressin (–43±3% flow reduction at 40 mm Hg PP) or quinacrine (–23±7%) was not offset at higher pulsatility (–44±4 and –23±6%, respectively). Neither of the latter agents inhibited ACh- or PP-induced flow responses, nor did they modify the effect of L-NMMA on these responses. Conclusions: Increased coronary flow pulsatility offsets vasoconstriction from K+ATP blockade by likely enhancing NO release. This mechanism may assist exercise-mediated dilation in settings where K+ATP opening is partially compromised.

KEYWORDS Coronary circulation; Vasoconstriction/vasodilation; Nitric oxide; K-ATP channels; Blood pressure


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
ATP-sensitive K+ channels (K+ATP) play an important role in the regulation of basal coronary tone [1–3] and contribute to vasodilation under conditions of increased metabolic demand [4] or ischemia [5]. In conscious dogs, inhibition of these channels by glibenclamide (GLI) lowers resting coronary flow by 30–50% [6,7]. Glibenclamide also abolishes vasodilation associated with

β1-adrenoceptor stimulation [8,9] and incremental cardiac pacing [10], supporting the role of K+ATP channels in vasodilator reserve when metabolic stimuli are enhanced. Intriguingly, however, blockade of K+ATP channels by GLI does not inhibit exercise-induced coronary flow reserve [6,7,11], due in large part to increases or an enhanced role of adenosine and nitric oxide (NO)-dependent dilation. This has led to the concept of redundancy of dilator-reserve mechanisms, with one or another pathway compensating for the reduced effectiveness of another [12].

An important signalling mechanism that could play a role in this behaviour is the rise in coronary perfusion pulsatility. During exercise, the arterial pulse pressure (PP) typically doubles [13–15],and greater flow pulsatility can itself modestly increase coronary flow by an NO-dependent mechanism [16]. This effect is considerably amplified by low-levels of adenosine [17]. It has been reported that both shear-stress stimulated [1] and agonist-evoked [5,19] NO release are unaffected by glibenclamide, and that K+ATP-channel blockade effects are principally confined to the distal microvascular bed [20–22]. Thus, it is feasible that enhanced pulsatility of coronary flow might trigger NO-mediated vasodilator responses, offsetting distal resistance rise from K+ATP blockade. The present study tested this hypothesis, employing an in vivo model in which the pulsatility of coronary flow was directly and independently controlled by a servo-pump, either in the presence of GLI alone or in combination with NO-inhibition. To test the specificity of vasoconstriction/pulsatility interactions, further studies were performed with arginine-vasopressin (AVP) or quinacrine (QUI). Both agents were chosen as they induce diffuse vasoconstriction without inhibiting receptor-mediated NO release (i.e. by acetylcholine [23,24]).


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
The protocol was reviewed and approved by the Animal Care and Use Committee of the Johns Hopkins University and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication N. 85–23, revised 1985). The servo-system used to regulate in situ coronary pulsatile perfusion has been previously reported in the detail [16]. Briefly, adult mongrel dogs (n=31; weight 28–36 kg) were anaesthetized with pentobarbital (30 mg/kg i.v.) and fentanyl (50 mg/kg i.v.) and ventilated with enhanced inspired oxygen. Ventilation was adjusted to yield physiological arterial pO2, pCO2, and pH. Left external jugular and femoral veins were cannulated for fluid administration and anaesthesia maintenance. Indomethacin (50 mg i.m.) was then administered. Both femoral, left carotid, and left subclavian artery, were cannulated for withdrawal of oxygenated blood into the servo-pump, and for blood sampling and pressure monitoring in the aortic root and LV cavity.

After heparinization (8000 IU bolus, 1000 IU/h), the LAD was cannulated and left carotid arterial blood diverted through this cannula to maintain flow while the servo-pump was primed.

Arterial blood was withdrawn by roller-pump from a femoral artery and pressurized at a constant 100 mm Hg mean by passage through a distensible reservoir submerged under pressurized water.

Blood then passed through a filter, heat exchanger, and one-way valve, and into a chamber with a movable floor that was linked to a linear motor (Ling Apparatus, CT). Digital real-time feedback was used to position the motor and generate the desired pulse pressure. Blood then exited the chamber to perfuse the LAD cannula. Pressure within the perfusion line was measured by micromanometer (SPC 350, Millar Inst., TX), and used to calculate the feedback error signal for servo-control. The servo-command signal was generated from the central aortic pressure wave.

This waveform was digitally stored to memory, modified to achieve the desired pulse amplitude, and then "played back" while maintaining synchrony with LV contraction. We have previously shown that alterations of regional (or global) coronary PP does not itself alter myocardial wall function or energetics [16,17,25]. Chamber function was minimally changed, as hearts ejected into the native vasculature under all conditions. Blood temperature in the servo-system was maintained at 37°C, with blood gases matching those of the systemic circulation.

2.1 Pharmaceuticals
NG-monomethyl-L-arginine (L-NMMA; CalBiochem, La Jolla CA), arginine-vasopressin, quinacrine, acetylcholine (Sigma Chemical Co., St. Louis, MO), and adenosine (Adenocard, Fujisawa), were each dissolved in isotonic saline. GLI [Sigma] was prepared in deionized water with pH adjusted to 8–8.5 with 0.1 N NaOH and 0.1 N HCl. Pinacidil (RBI, Natick, MA) was dissolved in dimethylsulphoxide (DMSO) and isotonic saline, with a final concentration of DMSO of <0.01%. Vehicle was tested independently and had no effect on coronary flow. All the drugs were administered intracoronary through a side-port in the servo-perfusion line.

2.2 Experimental protocols
After servo-perfusion was established in the isolated coronary bed, the pulse pressure was set to 40 mm Hg, and conditions stabilized for 15–20 min. Data were then obtained at steady-state with the servo-pump generating PPs of either 40 or 100 mm Hg. Data were recorded 4–5 min following a change in PP, with repeat recordings made at each PP to confirm reproducibility. Baseline data were obtained in all animals. PP-flow responses during co-infusion of various agents were obtained in subsets of these animals.

To test whether enhanced flow pulsatility offset constrictor effects of K+ATP blockade, GLI (n=12, 50 µg/min/kg, i.c.) was first infused and then PP increased. The efficacy of K+ATP channel blockade in vivo was additionally confirmed by co-infusion of pinacidil (5 mg/min i.c., n=2) or adenosine [26] 5–25 mg/min i.c., n=5)-induced flow elevation. Pinacidil and adenosine flow responses declined by 80% and 70%, respectively. To test the role of NO-stimulation, GLI infusion was stopped and L-NMMA infused (n=5; 5 mg/minx20 min, i.c.). Data were then measured at both PPs, GLI was re-infused, and PP again varied in the presence of both agents. In two additional subsets, the protocol was repeated using either AVP (0.5–2 µg/min) or QUI (6.7 µg/kg/minx20 min; both n=8). Each agent was infused intracoronary, and titrated to induce a mean flow reduction at PP=40 mm Hg similar to that with GLI. Both agents act by multiple mechanisms to generate diffuse vasoconstriction. AVP constricts by blocking voltage-sensitive Ca2+ channels and receptor mediated intracellular Ca2+ release [27,28]; while K+ATP inhibition is less prominent [24]. AVP can also modestly reduce ventricular contractility [29].

Quinacrine inhibits Ca2+ and ATP sensitive K+ channels [30,31] and PLA2 [32], the latter reducing generation of arachidonic acid metabolites that are linked with smooth muscle hyperpolarization and relaxation [33,34]. Thus both agents induce more diffuse constriction than GLI.

To confirm that neither AVP nor QUI acted by direct inhibition of NO release, we determined the vasodilator response to ACh (300 µg i.c.) in animals before and after AVP and QUI (n=5). In these same animals, we tested whether the effects of L-NMMA on inhibiting pulse-mediated and ACh-induced vasorelaxation were altered by co-treatment with AVP. If so, this would suggest a primary NO-inhibition effect of AVP itself. Data were obtained at PP=40 or 100 mm Hg with and without AVP, both before and after L-NMMA.

2.3 Data analysis and statistics
Data were digitized at 200 Hz and analysed off-line with custom software. Mean and pulsatile coronary flow, systolic, diastolic, and mean coronary pressure, LV pressure and aortic flow were measured in each study. For comparison of paired samples in each group and between groups, Student's t test for paired and unpaired data were used, with a Bonferroni correction for multiple comparisons. Data are presented as mean±S.E.M.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Chamber hemodynamics and pulsatility
Left ventricular systolic pressure averaged 127±4 mm Hg in control, and was similar with all interventions (130±3, 126±6, 123±8, 122±13 for i.c. GLI, glibenclamide+L-NMMA, AVP, and QUI, respectively). Heart rates generally ranged between 110 and 120/min. Importantly, there were no significant changes in chamber hemodynamics associated with alterations in perfusion PP for any of the conditions. We have previously reported regional function is similarly unaltered by this manoeuver, and that PP-flow responses are not influenced by even moderate changes in regional or global contractile state [16,17].

3.2 Glibenclamide and perfusion pulsatility
Fig. 1A displays phasic coronary pressure and flow tracings measured under basal conditions and following administration of GLI. Data for control (40 mm Hg) and enhanced (100 mm Hg) pulsatile pressures are superimposed, and portions where net flow was enhanced by higher perfusion pulsatility are denoted by shading. At baseline, elevating PP generated higher flow during systolic ejection, but little net if not a slight decline in flow during diastole.


Figure 1
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  		Fig. 1 (A) Top: phasic tracings of coronary perfusion pressure waveforms generated by servo-control system for 40 (solid) and 100 (dashed) mm Hg pulse pressures. Middle: Corresponding phasic coronary flow waveforms under basal conditions. Higher perfusion pulsatility resulted in a net rise in flow measured during the systolic period and early diastolic period (shaded regions). Net flow generally rose by 10–15%. Lower: Similar phasic coronary flow tracings after administration of glibenclamide. Enhanced flow at higher pulsatility was amplified during both systole and diastole. (B) Summary data for coronary flow measured during systolic and diastolic periods under baseline conditions and following glibenclamide. There was greater flow enhancement during systole and a significant rise in diastolic flow at higher PP after glibenclamide infusion. * P<0.01 vs. 40 mm Hg pulse pressure, # P<0.01 vs. PP response in controls. (C) Net mean coronary flow under both conditions. While glibenclamide significantly reduced mean flow at a pulse pressure of 40 mm Hg, flow was unchanged from control at higher pulsatility.

 
However, in the presence of GLI, higher pulsatility enhanced both systolic and diastolic flow, with the net result being a much larger increase in mean coronary flow at an identical mean perfusion pressure. Fig. 1B provides mean data. Increasing pulsatility under basal conditions resulted in a 66±9% increase in flow during the systolic-period, but an –11±2% decline in diastolic-flow. After GLI, enhanced pulsatility increased flow during both systole (166±26%) and diastole (+28±5%) (both P<0.02 vs. control). Importantly, both systolic and diastolic perfusion periods were unchanged since only the pulse amplitude was altered by this manoeuver.

Net effects on mean coronary flow are shown in Fig. 1C. At baseline pulsatility (40 mm Hg), flow was 37±3 ml/min, and this fell significantly to 25±4 ml/min after GLI administration. Mean flow was slightly higher (41±2 ml/min) with enhanced perfusion pulsatility, and remained unaltered despite GLI (45±4 ml/min, P=NS). Thus, elevating coronary perfusion pulsatility reversed the vasoconstrictive effect of glibenclamide.

3.3 Effect of L-NMMA and GLI+L-NMMA on pulsatile perfusion response
A potential mechanism for reversal of GLI-induced vasoconstriction by enhanced pulsatility was a greater role of mechanically-stimulated NO-release. GLI effects dominate in distal resistance beds following the distribution of K+ATP channels, which could potentially maintain more proximal NO-responsiveness to enhanced pulsatility. To test this hypothesis, L-NMMA was first infused alone, then co-infused with GLI, and perfusion pulsatility varied. NOS-inhibition alone had no significant effect on mean coronary flow at a PP of 40 mm Hg, but modestly lowered mean flow at PP=100 mm Hg (–5±1%, P<0.001). Flow augmentation due to PP declined from +14±2% to +9%±1.5 after L-NMMA, (–36%; P<0.001). Flow responses to ACh infusion (300 µg i.c.) were also reduced by 39±3.8%, consistent with prior results [16].

In the presence of L-NMMA, increasing perfusion pulsatility no longer offset vasoconstriction induced by GLI. Fig. 2A displays phasic coronary flow waveforms at the two pulse pressures. Unlike the results with GLI alone, combined GLI and L-NMMA no longer resulted in enhanced mean diastolic flow, but converted to a net decline of –8%±2% (P<0.02).


Figure 2
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  		Fig. 2 Effect of combined L-NMMA and glibenclamide on phasic coronary flow. (A) Unlike glibenclamide alone (Fig. 1A, lower panel), combined glibenclamide and L-NMMA led to a net decline in flow during diastole (arrow) at higher PP, with flow enhancement only measured during the systolic period. (B) Mean coronary flow at both PPs for control and combined L-NMMA+GLI conditions, showing persistent vasoconstriction at higher PP with this combination. # P<0.01 vs. control.

 
Mean coronary flow (Fig. 2B) at a PP of 40 mmHg fell from 36±3 to 13±2 ml/min (–60±8%; P<0.01) in the presence of GLI+L-NMMA, and similar constriction was now also observed at higher pulsatility (39±4 vs. 23±9 ml/min, –40±6%; P<0.001).

3.4 Interaction of AVP and quinacrine and perfusion pulsatility
Unlike GLI, vasoconstriction induced by AVP or QUI was not offset by enhancing perfusion pulsatility (Fig. 3A, upper panel). At 40 mm Hg basal PP, AVP lowered mean flow by –43±3%, (P<0.01), matching changes induced by GLI. However, a very similar –44±4% reduction in mean flow was also observed at higher PP (P=NS vs. PP–40 mm Hg). Flow during systole rose with higher PP similar to responses with GLI; however diastolic flow declined >30% offsetting this change (Fig. 3B, upper). Analogous data were obtained with QUI (Fig. 3, lower panels) with flow reduction at 40 mm Hg PP being similar to that observed at higher PP (–23±6.5% vs. –23±5.9%, P=NS).


Figure 3
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  		Fig. 3 (A) Mean coronary flow at 40 and 100 mmHg pulse pressure during concomitant i.c. arginine vasopressin (AVP) or quinacrine infusion. Substantial vasoconstriction with reduced flow was observed at both levels of PP. (B) Mean coronary systolic and diastolic flow at both PPs measured with both agents. Systolic flow rose at higher PP, whereas in contrast to GLI results, diastolic flow declined. This explained the net decline in total mean flow. * P<0.05 vs. 40 mm Hg pulse pressure, # P<0.01 baseline vs. post-drug.

 
To test whether the disparity between GLI versus the alternative constrictors was due to inhibition of NO-release by AVP or QUI, the vasodilator response to ACh (300 µg i.c.) was compared before and after infusion of each agent; with and without co-treatment with L-NMMA. Acetylcholine increased mean flow similarly with or without AVP (Fig. 4A, left). Furthermore, while the ACh-flow response was substantially blunted by L-NMMA alone (P<0.001), the addition of AVP did not modify this response (Fig. 4A, right, P>0.4 for interaction). Similar results were obtained with QUI (data not shown).


Figure 4
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  		Fig. 4 (A) Increase in mean coronary flow from i.c. acetylcholine before and after AVP, and with or without concomitant treatment with L-NMMA. AVP did not inhibit acetylcholine-induced vasodilation, whereas L-NMMA reduced it by –40%. Residual dilation to ACh after L-NMMA was unaltered by co-administration of AVP. (B) Similar analysis of flow responses to increased pulsatility, for baseline and AVP conditions with or without concomitant L-NMMA. AVP alone did not enhance the PP-flow response. The response was reduced by L-NMMA, but this change was unaltered by co-administration of AVP. * P<0.01 for control vs. acetylcholinem (or PP 40 vs. PP 100 mm Hg); {dagger} P<0.05 for Ach (or PP) response contrasting data with or without L-NMMA co-treatment.

 
We further tested whether the NO-dependence of PP-induced flow augmentation was blunted by AVP (Fig. 4B). As previously noted, AVP alone did not significantly alter the flow response to enhanced pulsatility. In contrast, L-NMMA alone reduced PP-induced flow elevation by {approx}40% (P<0.05). AVP+L-NMMA altered this response similarly to L-NMMA alone (P>0.8 for AVP-L-NMMA interaction effect). These data show that disparities between PP-induced flow elevation among GLI, AVP, and QUI, were unlikely due to inhibition of NOS signalling by the latter two agents.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
This study provides the first demonstration that increases in the pulsatility of in vivo coronary perfusion can offset coronary distal-vascular constrictor effects of K+ATP-channel blockade by glibenclamide. The capacity of higher pulsatility to offset GLI-induced constriction was largely eliminated by L-NMMA, supporting pulse-signalling NO release in offsetting GLI-induced vasoconstriction. In contrast, identical increases in pulsatility had no effect on net constriction by AVP or QUI, agents acting diffusely within the coronary vasculature. These results suggest a novel model whereby metabolic (i.e. distal vascular) regulation of coronary tone can be altered by mechanical stimuli from pulsatile perfusion via an NO-dependent mechanism.

Both basal coronary tone [3] and vasodilation associated with increased metabolic demand [8,10] and ischemia [5] are impaired by K+ATP channel inhibition with GLI. For example, in studies employing constant perfusion pressure (i.e. no pulsatility), vasodilation from β1-adrenoceptor stimulation is nearly abolished [8], while that associated with tachycardia pacing is partly inhibited by GLI [10]. In contrast, vasodilation is not attenuated by even high doses of glibenclamide in exercising conscious dogs [6,7,11,12] nor with simulated exercise achieved by epinephrine and tachycardia pacing in anaesthetized dogs [4].

The present data, obtained with a pulse pressure magnitude analogous to what is observed with a moderate–severe exercise [35], provides a potential explanation for this disparity. Aortic pulse pressures and thus flow pulsatility increase during exercise in humans [13–15] and animals [35], as well with combined sympathetic stimulation and rapid heart rates [4]. In the presence of K+ATP channel blockade and thus higher distal coronary resistance, higher flow pulsatility would likely result in greater pulse-stretch and shear signalling throughout the vascular bed. The enhanced mechanical signalling would trigger additional NO release, offsetting the effects of K+ATP-blockade. The finding that L-NMMA has negligible effects on basal coronary flow but diminishes flow significantly at higher pulsatility was first reported by Recchia et al. [16], is confirmed in the present study, and is consistent with augmented NO release at higher PPs.

Importantly, mean diastolic flow rose nearly 30% at higher pulsatility in the presence of GLI despite a fall in mean diastolic perfusion pressure. The fact that this diastolic flow augmentation was eliminated by L-NMMA further supports the role of NO release to this behaviour.

Pulsatile stretch of endothelial cells [36,37] and vascular segments [38] as well as enhanced phasic shear [39] are potent triggers of NO release. Moreover, rapid systolic flow increases produce greater shear in more constricted vessels. Thus, one might speculate that the combination of enhanced phasic distension of more proximal arterioles and greater shear in constricted distal vessels results in sufficient NO release to offset GLI-induced flow diminution. This is supported by the finding that NO-dependent dilation associated with higher PP was greater after GLI than in control or AVP groups. Furthermore, neither agonist [5,19] nor mechanically-evoked NO release [18] are known to be altered by GLI. While no studies have directly examined pulse-stretch stimulated NO release in conductance versus distal microvessels, data support that basal NO release occurs more in the more proximal and less in the distal microvessels [20]. Importantly, with even 24% of the dose of L-NMMA employed in the present study, Nishikawa et al. [40] recently reported that ACh-triggered dilation in vessels >100 µ was abolished, whereas that in even smaller microvessels persisted but at a reduced level.

In contrast to GLI, neither AVP nor QUI-induced constrictor effects were reversed by increasing the pulsatility of coronary perfusion. While GLI-mediated constriction dominates in <150 µm arterioles [20–22], regulation of tone by AVP and QUI is more diffuse due to multiple mechanisms. AVP increases intracellular Ca2+ and thus myogenic tone by promoting extracellular Ca2+ influx via voltage-gated channels [27] and V1-receptor coupled pathways [28]. The latter receptors are widely distributed in the vasculature. AVP may also modulate K+ATP channels, although this effect remains controversial and appears species and vascular site-specific [27,41]. Although we were unable to directly measure NO release in these studies, data employing L-NMMA combined with AVP failed to support a direct inhibition of NO-dependent signalling by these agents. Results with QUI, which also has effects throughout the coronary vasculature by inhibiting Kca2+ [30] and K+ATP channels [31] and PLA2 [32–34,42], were similar to those with AVP.

If the capacity for NO production was unimpeded by AVP, as suggested by these results, then the disparity between PP-related flow changes versus GLI points to regional differences in vessel tone. By generating diffuse rather than more selective distal constriction, AVP (and QUI) may limit pulse-distension signalling in NO-responsive vessels. An alternative explanation is that non-NO dependent pathways such as Ca2+-activated potassium channels are also importantly involved with the pulse-perfusion response and are more inhibited by AVP and QUI than by GLI. This hypothesis is supported by data showing that both ACh-mediated and shear/stretch mediated vasodilation elicits a non-NO dependent hyperpolarizing factor [33,34,40,41,43] that is dependent on activation of KCa2+ channels, channels that are partially inhibited by both AVP and QUI [27,30]. NO may itself activate KCa2+ channels [44], and this mechanism could also play a role in the ability of enhanced PP to offset GLI-induced constriction. This would be less prominent with AVP and QUI as both can inhibit this pathway. In contrast, more selective K+ATP blockade by GLI [6,7] might leave NO (and cGMP) activation of K+Ca2+ channels unimpeded. Regardless of the precise mechanism, the present data suggest the GLI-PP interaction is specific, and not a generic response between coronary constriction and higher perfusion pulsatility. This is analogous to the specificity of adenosine and PP interaction recently reported [17].

One feature of enhanced pulsatile perfusion shared by all conditions was the increase in mean flow measured during systolic ejection. Similar behaviour is observed in intact hearts ejecting at higher pulsatile load [23] and in isolated coronary beds perfused by enhanced pulsatile flow [16,17]. As reported by Goto et al. [45], and Recchia et al. [46], increasing PP above 40 mm Hg dilates large and small resistance vessels by an endothelium-independent mechanism due to plastic properties of the vascular wall. In intact hearts, such distension would likely enhance proximal capacitance of the vascular bed, and combined with higher systolic perfusion pressures and distal constriction, increase systolic-period flow. Importantly, this flow contributes to net perfusion as demonstrated by previous comparisons to myocardial (microsphere) flow [25].

There are several potential limitations to our study that should be considered. Reduction of coronary flow due to regional vasoconstriction could have modified contractility of the locally perfused bed, altering the ventricular systolic flow impediment. Furthermore, AVP itself has been reported to induce moderate contractile depression in the dog [29]. However, the greatest extent of flow reduction was observed with combined GLI and L-NMMA, yet this yielded less flow enhancement with increased perfusion pulsatility than observed with GLI alone. Furthermore, similar reductions in flow were achieved with GLI, QUI and AVP, yet the responses to pulse pressure were very different. Lastly, our prior studies employing verapamil or epinephrine [17,47] found minimal influences of cardiac contractility on the differential response to altering pulsatile perfusion.

Our in vivo preparation could not dissociate mechanisms due to altered vascular stretch from those due to phasic shear resulting from higher pulsatility. Given that local shear cannot be measured in real-time and varies with the size and distensibility of the vascular bed, there is no current method to servo-control arterial shear stress. More detailed separation of these factors can be performed in vitro, but such preparations would not demonstrate the present behaviour, given that it required coupling of different regions within the intact coronary vascular bed.

Finally, it would have been helpful to directly measure of NO release in this model, rather than rely on response changes to NOS inhibition. Unfortunately, this has proven technically difficult due to the sub-optimal capacity to sample venous blood purely derived from the isolated perfused coronary artery, as well as to limitations in the chemiluminescent assay techniques when examining partially hemolysed blood with elevated free plasma hemoglobin. However, prior studies [48] have reported increased NO-production in conditions where pulsatile perfusion is elevated, and that NO-inhibition limits this change.

Enhancing pulsatile perfusion can offset the distal-resistance vasoconstriction effects of the K+ATP blockade by a mechanism related to NO release. Critical to this interaction is the fact that mechanical-induced NO-release is maintained or even enhanced after K+ATP inhibition in vivo. This enhancement could be related to the distal site and selective constriction associated with glibenclamide. Such interactions help explain how blockade of K+ATP channels can have such potent effects on basal coronary flow, yet relatively little influence on flow augmentation during exercise. These data provide a novel mechanical mechanism for enhanced NO-dependent dilation during exercise in the presence of K+ATP blockade.

Time for primary review 21 days.


   Acknowledgements
 
The authors wish to express their gratitude to Mr. Richard Tunin for his excellent technical cooperation. This research was supported by a grant from the National Health Service (NHLBI: HL-47511-DAK), a grant from the Italian National Research Council-CNR-Short Term Mobility/99 (PP), and fellowship grant from University of Tokyo (TI).


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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