Cardiovascular Research

Cardiovascular Research 2000 46(3):547-556; doi:10.1016/S0008-6363(00)00040-7
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Endothelium-dependent hyperpolarization and relaxation resistance to N^G-nitro-L-arginine and indomethacin in coronary circulation

Zhi-Dong Ge^a, Xiao-Hui Zhang^b, Peter Chin-Wan Fung^b and Guo-Wei He^a,c,*

^aCardiovascular Research Laboratory, Grantham Hospital, Department of Surgery, The University of Hong Kong, Hong Kong SAR, China
^bDivision of Medical Physics, Department of Medicine, The University of Hong Kong, Hong Kong SAR, China
^cCardiovascular Research, Albert Starr Academic Center for Cardiac Surgery, St. Vincent Hospital, Portland, Oregon, USA

^* Corresponding author. Professor G.-W. He, Chair of Cardiothoracic Surgery, University of Hong Kong, Grantham Hospital, 125 Wong Chuk Hang Road, Aberdeen, Hong Kong. Tel.: +852-2518-2631; fax: +852-2814-8635 gwhe{at}hkucc.hku.hk

Received 13 July 1999; accepted 10 January 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: It is controversial whether endothelium-dependent relaxation resistance to inhibitors of nitric oxide (NO) and prostacyclin synthases is completely attributed to endothelium-derived hyperpolarizing factor (EDHF). This study examined NO release and K⁺ channels involved in endothelium-dependent relaxation and hyperpolarization resistance to N^G-nitro-L-arginine (L-NNA) and indomethacin in coronary arteries with emphasis on the microarteries. Methods: NO release, isometric force, and membrane potential of porcine coronary arteries were measured using a NO-specific electrode, wire myograph, and microelectrode, respectively. Results: In large arteries pretreated with indomethacin, bradykinin (BK) evoked a rise in [NO] from 5.5±2.4 nM to 105.0±19.6 nM and hyperpolarization. L-NNA treatment significantly reduced the BK-stimulated rise in [NO] to 32.1±11.3 nM but did not affect the hyperpolarization. In the presence of indomethacin and L-NNA, U₄₆₆₁₉ contracted and depolarized (from –51±3 mV to –30±4 mV) vascular smooth muscle in microarteries. The addition of BK produced dose-dependent relaxation (maximal: 70.2±5.7%) and repolarization (membrane potential: –50±4 mV). Oxyhemoglobin eliminated indomethacin and L-NNA-resistance rise in [NO] but not relaxation (42.3±4.4%) and repolarization (-40±2 mV) by BK. Tetraethylammonium, charybdotoxin, and iberiotoxin partially decreased the BK-induced responses. Apamin alone did not affect the relaxation by BK; however, in combination with charybdotoxin it almost completely abolished the BK-induced relaxation and hyperpolarization. Conclusions: In porcine coronary arteries, both EDHF and NO contribute to BK-induced relaxation resistance to indomethacin and L-NNA. Large conductance Ca²⁺-activated K⁺ channels (BK_Ca) may play an important role in mediating the BK-induced responses and small conductance Ca²⁺-activated K⁺ channels might function as ‘backup’ mechanisms when BK_Ca is curtailed.

KEYWORDS Coronary circulation; Endothelial factors; K-ATP channel; K-channel; Membrane potential; Nitric oxide

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This article is referred to in the Editorial by B. Vanheel and J. Van de Voorde (pages 370–375) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The endothelium relaxes vascular smooth muscle cells by at least three factors: nitric oxide (NO) [1], prostacyclin (PGI₂) [2], and endothelium-derived hyperpolarizing factor (EDHF) [3]. After activation by shear stress or stimuli, endothelial nitric oxide synthase (NOS) converts L-arginine to nitric oxide (NO). NO causes relaxation by activating soluble guanylate cyclase in smooth muscle cells to increase intracellular cyclic GMP [4,5] or by the activation of K⁺ channels in some vasculature [5]. PGI₂ is produced by the action of prostacyclin synthase on endoperoxides, the production of which are induced by cyclooxygenase (COX). This factor evokes vasodilation via the activation of adenylate cyclase. In contrast, the enzyme responsible for EDHF production is unknown. However, it is putatively believed that EDHF hyperpolarizes vascular smooth muscle cells by activating K⁺ channels, which results in the closure of voltage-dependent Ca²⁺ channels, the reduction of intracellular free Ca²⁺, and subsequently the relaxation of blood vessels [6,7].

The chemical identity of EDHF is controversial, although epoxyeicosatrienoic acids, the cytochrome P-450 metabolites of arachidonic acid, have been proposed as possible candidate for EDHF in porcine and bovine coronary arteries [8,9]. Therefore, a routine way to study EDHF at present is to use COX and NOS inhibitors to inhibit the production of both PGI₂ and NO. However, it has been recently found that oxyhemoglobin (HbO), a potent scavenger of NO, was capable of inhibiting endothelium-dependent relaxation and decreasing the NO concentration in the presence of N^G-nitro-L-arginine (L-NNA) [10]. This finding suggests that the NOS inhibitors could not completely block the production of EDNO and that HbO–NO is not a relaxing factor. It is even claimed that in the presence of high concentrations of NOS inhibitors, there is still sufficient NO (in the rabbit carotid artery sample) to account fully for the endothelium-dependent relaxation and hyperpolarization, as the relaxing and hyperpolarizing factor [11]. Therefore, the existence of another relaxing and hyperpolarizing factor is questionable, at least in some vasculature.

Based on selective blockers, at least five types of K⁺ channels have been identified involving the EDHF-mediated relaxation in different blood vessels. These are large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels, small-conductance Ca²⁺-activated K⁺ (SK_Ca) channels, ATP-sensitive K⁺ (K_ATP) channels, voltage-dependent inward rectifier K⁺ channels, and voltage-dependent delayed rectifier K⁺ channels [6,12,13]. In porcine coronary conduit arteries, several previous studies, including some by us, investigated the contribution of K⁺ channels to bradykinin (BK)-induced relaxation and hyperpolarization resistance to COX- and NOS-inhibitors [13–15]. However, the results are not uniform, they even conflict with each other. Furthermore, to our knowledge, the involvement of K⁺ channel types in the BK-induced, EDHF-mediated relaxation and hyperpolarization in the porcine coronary resistance arteries has not been studied.

Thus, the present study examined nitric oxide release and K⁺ channel types involved in BK-induced relaxation and hyperpolarization of porcine coronary circulation in the presence of indomethacin (Indo, a potent inhibitor of COX) and L-NNA (an inhibitor of NOS) or ±HbO with emphasis on coronary microarteries. NO concentration, isometric force, and membrane potential of arterial rings were measured using a NO-specific electrode and meter, wire myograph, and glass microelectrode, respectively.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Fresh porcine hearts collected from a local slaughterhouse were placed in a container filled with cold Krebs’ solution and immediately transferred to the laboratory. Upon receipt of the heart, large epicardial and intramyocardial microcoronary arteries (usually the tertiary branches of the left anterior descending artery) were carefully dissected, and carefully removed to protect the endothelium. The vessel was cleaned of fat and connective tissue and cut into cylindrical rings of 4-mm (for epicardial arteries) or 2-mm (for intramyocardial arteries) length under a microscope. The Krebs’ solution was pre-aerated with a gas mixture of 95% O₂–5% CO₂ and had the following composition (in mM): 144 Na⁺, 5.9 K⁺, 2.5 Ca²⁺, 1.2 Mg²⁺, 128.7 Cl^–, 25 HCO₃^–, 1.2 SO₄^2–, 1.2 H₂PO₄^–, and 11.0 glucose.

2.1 Measurement of NO and membrane potential in large arteries
Epicardial arterial rings were cut open along the longitudinal axis and pinned, with the endothelium side upward, on the bottom of a 2.5-ml organ bath. NO released from endothelial cells was measured using a NO-specific electrode and meter (ISO-NO, World Precision Instruments, Sarasota, FL, USA). The design of the NO-specific electrode has been described previously in detail [16,17]. In short, NO was allowed to diffuse through a semipermeable membrane followed by oxidation at a working platinum electrode resulting in the presence of a small electric current. This redox signal was directly proportional to the NO concentration. The tip of the NO electrode was slowly inserted vertically into the endothelial cell surface until a current was detected. From that location the electrode was then retracted for 2 microns form the cell surface with the micromanipulator.

The membrane potential of the vascular smooth muscle cells was measured using a conventional glass microelectrode. As previously described by us [13], the microelectrode filled with 3 M KCl (tip resistance 100–120 M) was advanced using a pipette holder mounted on a three-dimensional vernier-type hydraulic micromanipulator and inserted into a single smooth muscle cell from the endothelial side. The following criteria were used to assess the validity of a successful impalement: (1) a sudden negative shift in voltage; followed by (2) a stable negative voltage for more than 2 min; and (3) an instantaneous return to the previous voltage level on dislodgement of the microelectrode.

2.1.1 Protocol
All arterial strips were equilibrated for 60 min at 37±0.1°C before and after the NO-specific electrode and membrane potential microelectrode were inserted towards the endothelial cell surface. The NO instruments were calibrated prior to daily experiment with the standard NO generated from the reaction of liquid nitrite (NaNO₂) and a solution containing H₂SO_4, K₂SO₄, and KI. Stable baseline values were obtained for at least 15 min before stimulation. BK (0.1 µM)-caused change in NO concentration and membrane potential was studied in the presence of Indo 7 µM or plus L-NNA 300 µM and HbO 20 µM. Only one NO concentration or membrane potential value was calculated for each vascular ring.

2.2 Isometric force studies in microarteries
A microarterial ring was guided onto a suitable length of stainless-steel wires (40 µm in diameter) through its lumen under the microscope. The wire was fixed tightly on a jaw in a two-channel Mulvany myograph and another wire was passed lightly through the vascular lumen and then anchored to the other jaw of the same chamber. These two wires were attached to a force transducer or to a micrometer, respectively. A previously described method [18] was used to normalize vascular rings under a condition simulating the transmural pressure in vivo encountered by the coronary microartery. Briefly, the arterial rings were progressively stretched until the passive transmural pressure reached 100 mmHg. The internal circumference was then set to a normalized value, estimated to be equivalent to 90% of the circumference at a passive transmural pressure of 100 mmHg.

2.2.1 Protocol
One of the two rings taken from the same artery in the myograph was incubated with Indo 7 µM and L-NNA 300 µM as control, and the other with Indo and L-NNA plus HbO 20 µM or K⁺ channel blockers. These blockers and their concentrations were tetraethylammonium (TEA, a non-specific blocker of K⁺ channels) 1 mM, charybdotoxin (ChTX, a blocker of BK_Ca and voltage-dependent K⁺ channels) 0.1 µM, iberiotoxin (IbTX, a specific blocker of BK_Ca channels) 0.1 µM, apamin (a specific blocker of SK_Ca channels) 0.5 µM, and glibenclamide (GBC, a blocker of K_ATP channels) 3 µM. Thirty minutes later, thromboxane A₂ mimetic U₄₆₆₁₉ 3 µM was applied to contract the vessels. When stable contractions to U₄₆₆₁₉ were obtained, BK was cumulatively applied to establish concentration–response curves. Only one concentration–relaxation curve was obtained from each coronary ring. From a number of rings (usually 6–11 rings) in each group, a mean concentration–relaxation curve was established.

2.3 Simultaneous measurement of membrane potential and isometric force in microarteries
The myograph was mounted within a metal-screened cage. The membrane potential of vascular smooth muscle cells was measured using the glass microelectrode. This setting allows simultaneous measurement of isometric force and the membrane potential contributing to the production of this force.

2.3.1 Protocol
Isometric force and membrane potential were continuously measured under the following situations: (1) in the presence of Indo 7 µM and L-NNA 300 µM (resting membrane potential); (2) at a stable contraction caused by U₄₆₆₁₉ (3 nM); and (3) under the effect of BK (0.1 nM–0.1 mM). Only one membrane potential and isometric force were obtained for each arterial ring. For each group, a mean membrane potential was calculated from 6 to 11 rings.

2.4 Data analysis
NO concentration was expressed in nM calculated by comparison with the standard curve determined each day prior to experiment. Relaxation was expressed as the percentage decrease in isometric force induced by U₄₆₆₁₉. The effective concentration of BK that caused 50% of maximal relaxation was defined as EC₅₀. The EC₅₀ was determined from each concentration–relaxation curve using a logistic curve-fitting equation: E=MA^P/(A^P+K^P), where E is response, M is maximal relaxation, A is concentration, K is EC₅₀ concentration, and P is the slope parameter [13]. From this fitted equation, the mean EC₅₀ value plus or minus the standard error of the mean (S.E.M.) was calculated for each group.

Data are expressed as mean±1 S.E.M. for n observations, where n equals the number of coronary arterial rings. For the isometric force studies, two rings cut from the same artery were randomly allocated into two groups, and the data were evaluated using paired Student's t-tests. For membrane potential and NO measurement, unpaired Student's t-tests were used to calculate the difference. P<0.05 was considered to be statistically significant.

2.5 Drugs
The drugs used and their sources were as follows: BK, A₂₃₁₈₇, L-NNA, Indo, TEA, ChTX, apamin, GBC, and hemoglobin were from Sigma Chemical, St. Louis, MO, USA. U₄₆₆₁₉ was from Cayman Chemical, Ann Arbor, MI, USA. IbTX was from Calbiochem, La Jolla, CA, USA. L-NNA (dissolved in distilled water) and Indo (dissolved in ethanol) were stored at 4°C. The solution of U₄₆₆₁₉ was held frozen until required.

Commercial bovine hemoglobin was dissolved in 0.9% NaCl to make up a 3-ml stock solution. The stock solution was subsequently reduced to HbO by addition of a small amount (<0.3 g) of sodium dithionite. Excessive sodium dithionite was extracted by running the solution through an Econo-Pac 10DG column (Bio-Rad) equilibrated with 0.9% NaCl. The HbO solutions were frozen in aliquots at –20°C and stored for up to 14 days.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effects of L-NNA and HbO on NO release and membrane potential in large arteries
NO released from the endothelial cells of coronary conduit arteries was 5.5±2.4 nM (n=7) at the resting status. In the presence of Indo 7 µM, addition of BK 0.1 µM caused a rapid and marked rise (generally within 1.5 min) in NO, followed by a sustained elevation generally lasting for at least 8 min. The peak concentration of NO usually appeared within 2.0–2.5 min after the addition of BK and reached 105.0±19.6 nM [n=7, Fig. 1(a)]. After treatment with Indo 7 µM and L-NNA 300 µM, the kinetics of NO release from coronary endothelial cells and amplitude altered significantly [Fig. 1(b)]. The concentration of NO release by BK was significantly diminished in 1–2.5 min. The maximal concentration was reduced to 32.1±1.3 nM (n=8, P=0.02). HbO 20 µM rapidly scavenged the NO in the incubation media. No NO signal could be detected during the HbO incubation of 30 min. Addition of BK 0.1 µM caused a minimal increase in [NO] generally below 8 nM [Fig. 1(b)].

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Bradykinin (BK)-stimulated nitric oxide release and membrane hyperpolarization in coronary conduit arteries and the effects of NG-nitro-L-arginine and oxyhemoglobin. (a) Mean time (min)–NO concentration (nM) curves in response to BK. , Indo (indomethacin); , Indo+L-NNA, the combination of indomethacin and NG-nitro-L-arginine; , Indo+L-NNA+HbO, the combination of indomethacin, NG-nitro-L-arginine, and oxyhemoglobin. *, P<0.05; *, P<0.01 vs. the control. (b) Original tracings of NO release. BK was applied as indicated by arrows. (c) BK-induced membrane hyperpolarization.

 
In the presence of Indo, the resting membrane potential of coronary conduit arteries was –57±3 mV (n=28). Addition of BK 0.1 µM evoked a marked membrane hyperpolarization (16±1 mV) of vascular smooth muscle cells. The membrane potential was hyperpolarized to –73±3 mV (n=22). The combination of Indo and L-NNA did not significantly alter the resting membrane potential (–56±3 mV, n=26, P=0.9) and BK-induced hyperpolarization (14±1 mV, n=19, P=0.2). The membrane potential was hyperpolarized to –71±3 mV (P=0.7) by BK. After the pretreatment of arteries with Indo, L-NNA, and HbO, the resting membrane potential of vascular smooth muscle cells was –55±3 mV (n=24, P=0.6 vs. the Indo). BK-induced hyperpolarization was significantly reduced to 13±1 mV (n=17, P=0.03, Fig. 1(c); BK-induced membrane potential: –67±3 mV).

3.2 Influence of inhibitors on resting tone of microarteries
After normalization, the resting tone of coronary microarteries was not different among the groups. The incubation with Indo and L-NNA produced a minimal and slow increase in force. This rise in the force was unaffected by the combined treatment of Indo and L-NNA plus GBC or apamin. However, after the rings were incubated with Indo, L-NNA plus TEA 1 mM, or ChTX 0.1 µM, or HbO 20 µM, the increase in the force was significantly higher than the change in the force of rings treated with Indo and L-NNA (Fig. 2).

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Change of basal tone caused by inhibitors in coronary microarteries. The protocol of the inhibitors was as follows: control, indomethacin and NG-nitro-L-arginine; TEA, indomethacin, NG-nitro-L-arginine, and tetraethylammonium (TEA); ChTX, indomethacin, NG-nitro-L-arginine, and charybdotoxin; apamin, indomethacin, NG-nitro-L-arginine, and apamin; and HbO, indomethacin, NG-nitro-L-arginine, and oxyhemoglobin (HbO). *, P<0.05; *, P<0.01 vs. the control (paired t-test).

 
3.3 NO-related relaxation and hyperpolarization resistance to Indo and L-NNA and the effect of HbO in microarteries
In the presence of Indo and L-NNA, U₄₆₆₁₉ 3 µM caused a stable and rapidly developing contraction in coronary microarterial rings. Cumulative addition of BK and A₂₃₁₈₇ (0.1 nM–316 nM) produced a marked relaxation in the concentration-dependent manner. HbO treatment shifted the concentration–relaxation curve rightward and reduced the BK- and A₂₃₁₈₇-induced relaxation [Fig. 3(a) and (b)]. The maximal relaxation and EC₅₀ values for BK were changed from 72.1±6.2% to 43.0±4.3% (n=9, P=0.001) and from 23.7±6.4 nM to 47.8±6.9 nM (P=0.02), respectively. To observe whether sodium dithionite was completely removed by the Econo-Pac 10DG column, the effect of the solution passing through the column on BK and A₂₃₁₈₇-induced relaxation was examined. Compared with Krebs’ solution, the solution through the column did not significantly alter the BK- and A₂₃₁₈₇-induced relaxation. Moreover, no significant difference existed between L-NNA 300 µM and 1 mM in concentration–relaxation curves for BK [Fig. 3(c)].

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Concentration (log M)–relaxation curves for bradykinin and A23187 in coronary microarteries precontracted with U46619. (a) and (b): , Control, pretreated with Indo and L-NNA in Krebs’ solution; , HbO, with Indo L-NNA, and HbO in Krebs’ solution; () column, with Indo and L-NNA in the solution running through Econo-Pac 10DG column. (c) L-NNA 300 and 1000, NG-nitro-L-arginine 300 µM and 1000 µM. *, P<0.01; *, P<0.001 vs. the control.

 
Coronary microarteries had a mean resting membrane potential of –51±3 mV (n=11) under the transmural pressure of 55–70 mmHg in the presence of Indo and L-NNA. U₄₆₆₁₉-induced contraction force and membrane potential [Fig. 4(a)] were 8.8±1.1 mN and –30±4 mV (n=11), respectively. In the vessels precontracted by U₄₆₆₁₉, BK induced a concentration-dependent hyperpolarization and associated relaxation [Fig. 4(b)]. The maximal relaxation to BK was 70.2±5.7%, associated with a maximal hyperpolarization (repolarization) of the membrane potential to –50±4 mV (73.9±7.6% reversal of U₄₆₆₁₉-induced depolarization; n=6) (Fig. 5). The EC₅₀ values for the relaxation to BK (4.0±0.7 nM, n=6) were significantly lower than those for the hyperpolarization to BK (10.0±2.2 nM, P=0.003). Treatment of the arterial rings with HbO 20 µM slightly, but not significantly, elevated the U₄₆₆₁₉-induced depolarization (membrane potential: –28±3 mV, n=11, P=0.6) and contraction (11.0±1.1 mN, P=0.2). Cumulative addition of BK (0.1 nM–0.1 µM) evoked concentration-dependent hyperpolarization and accompanied relaxation. Compared with the rings pretreated with Indo and L-NNA, HbO significantly reduced BK-induced relaxation at a concentration of 1 nM–0.1 µM and elevated membrane potential at a concentration of 0.01–0.1 µM, respectively (Fig. 5). The maximal percentage relaxation and the degree of hyperpolarization to BK were reduced respectively to 42.3±4.4% (compared to 70.2±5.7% in control, P=0.003) and –40±2 mV (compared to –50±4 mV in control, P=0.05). In addition, HbO treatment also significantly changed the EC₅₀ for the relaxation to BK to 13.7±1.1 nM (n=6, P<0.001) but not for the hyperpolarization (12.0±0.7 nM, P=0.4), compared with the control rings treated with Indo and L-NNA only. There was no significant difference between the EC₅₀ value for the relaxation and the EC₅₀ value for the hyperpolarization to BK (P=0.3).

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Original tracings of U46619-induced contraction and depolarization (a) and bradykinin (BK)-induced relaxation and hyperpolarization in the coronary microarteries precontracted with U46619 (b). The vessels were pretreated with indomethacin and L-NNA. U46619 and BK were applied as indicated by the arrows.

 

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Simultaneous recording of membrane hyperpolarization (a) and relaxation (b) by bradykinin in coronary microarteries precontracted with U46619. All arteries were pretreated with Indo and L-NNA. *, P<0.05; *, P<0.01; *, P<0.001 vs. the respective control.

 
3.4 Effects of K⁺ channel blockers on BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA in microarteries
3.4.1 BK-induced relaxation
In the precontracted control rings, pretreated with Indo and L-NNA, BK (0.01 nM–316 nM) induced concentration-dependent relaxation. The treatment of TEA 1 mM, ChTX 0.1 µM, and IbTX 0.1 µM significantly reduced the relaxation to BK (Fig. 6). The maximal relaxation was reduced from 79.8±7.9% (n=14) to 43.0±8.0% (n=7, P=0.03) by TEA, and from 73.6±5.0% to 50.6±5.3% (n=7, P=0.01) and to 53.6±6.3% (n=7, P=0.03) by ChTX and IbTX, respectively. The EC₅₀ values for BK were not significantly changed (P>0.05).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Concentration (log M)–relaxation curves for bradykinin in coronary microarteries precontracted with U46619. All arteries were pretreated with Indo and L-NNA in addition to the K+ channel blockers indicated below. TEA, tetraethylammonium 1 mM; ChTX, charybdotoxin 0.1 µM; IbTX, iberiotoxin 0.1 µM. *, P<0.05 vs. the control.

 
Apamin 0.5 µM alone did not significantly affect BK-induced relaxation resistance to Indo and L-NNA and the relaxation curve is basically similar to the control curve (P>0.05 in all concentrations of BK; Fig. 7(a)). However, the combination of apamin 0.5 µM and ChTX 0.1 µM almost completely abolished the relaxation to BK [Fig. 7(b)]. The maximal relaxation and EC₅₀ value for BK were altered from 71.8±5.3% to 8.1±1.0% (n=7, P<0.001) and from 6.0±0.1 nM to 55.0±0.15 nM (P<0.001), respectively.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Concentration (log M)–relaxation curves for bradykinin in coronary microarteries precontracted with U46619. All vessels were pretreated with Indo and L-NNA in addition to the K+ channel blockers indicated below. Apamin+ChTX, apamin 0.5 µM and charybdotoxin 0.1 µM. *, P<0.01; *, P<0.001 vs. the control.

 
GBC 3 µM also partially decreased the BK-induced relaxation at concentrations of 10–316 nM but did not significantly change the EC₅₀ values [Fig. 8(a)]. The combination of GBC and TEA produced further decreases in BK-induced relaxation at concentrations of 1–316 nM [Fig. 8(b)]. To investigate whether or not there was a direct effect of GBC on U₄₆₆₁₉, GBC (10 nM–0.1 mM) was cumulatively applied to U₄₆₆₁₉-precontracted vessels. GBC caused a concentration-dependent relaxation with a maximal relaxation 90.6±4.4% and EC₅₀ values of 3.4±0.1 µM.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Concentration (log M)–relaxation curves for bradykinin in coronary microarteries precontracted with U46619. All vessels were pretreated with Indo and L-NNA in addition to the K+ channel blockers indicated below. GBC, glibenclamide 3 µM; TEA+GBC, tetraethylammonium 1 mM, and glibenclamide 3 µM. *, P<0.01; *, P<0.001 vs. the control.

 
3.4.2 Simultaneous recording of membrane potential and relaxation
Neither TEA 1 mM nor ChTX 0.1 µM affect the U₄₆₆₁₉-induced depolarizaton and contraction force (P>0.05 vs. the control) in porcine coronary microarteries. BK-induced hyperpolarization and relaxation was significantly inhibited by TEA and ChTX at a concentration of 0.1 µM and in the concentration range 0.01–0.1 µM, respectively [Fig. 9(a) and (b)]. Similar to the results in the above relaxation experiments, the combination of ChTX 0.1 µM plus apamin 0.5 µM almost completely abolished BK-induced membrane hyperpolarization and associated relaxation.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Concurrent recording of bradykinin-induced hyperpolarization (a) and relaxation (b) in the coronary microarteries precontracted with U46619. All vessels were pretreated with indomethacin and L-NNA in addition to the K+ channel blockers indicated in the legends. *, P<0.05; *, P<0.01; *, P<0.001 vs. respective control.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study is the first time the BK-stimulated NO release from coronary endothelial cells and the K⁺ channel types involved in endothelium-dependent hyperpolarization and relaxation resistance to Indo and L-NNA in the porcine coronary microarteries have been investigated. There are four novel findings from the study. Firstly, both EDHF and NO potently contribute to the BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA. Secondly, BK_Ca and SK_Ca channels mediate the BK-induced relaxation resistance to Indo and L-NNA probably in a synergistic manner. Thirdly, BK_Ca channels may play a relatively important role, whereas SK_Ca channels might play a compensatory role as a ‘backup’ mechanism when BK_Ca channels are curtailed. Finally, BK_Ca channels participate in the modulation of basal tone. In addition, our present study also suggests that combined the application of NOS inhibitor and NO scavenger is necessary in order to eliminate the effect of NOS inhibitor-insensitive NO in future investigations concerning EDHF in coronary circulation.

4.1 Potent effect of both EDHF and NO in BK-induced responses resistance to Indo and L-NNA
Endothelium-dependent relaxation resistance to COX- and NOS-inhibitors was previously considered to be attributed to EDHF [3,4,6,13]. Our current study, by direct measurement of NO, has shown that L-NNA could not completely eliminate the BK-stimulated rise in NO released from the porcine coronary endothelial cells, though it could partially inhibit the NO release. Similar results were obtained in the rat superior mesenteric artery [10] and in the rabbit carotid artery [11]. Our present study demonstrates that in porcine coronary circulation, to further eliminate the production of NO in order to study the NO-independent relaxation and hyperpolarization that are attributed to another factor (EDHF), the addition of NO scavengers is necessary. Our study also demonstrates that the amount of HbO added in our experiments completely scavenged NO and produced a further inhibitory effect on BK-induced relaxation and hyperpolarization related to the residual NO that is resistant to Indo and L-NNA. Furthermore, in the simultaneous studies of isometric force and membrane potential, there was a significant difference in the EC₅₀ value for BK between relaxation and hyperpolarization in the presence of Indo and L-NNA. These results suggest that EDHF could not completely account for the endothelium-dependent relaxation resistance to COX- and NOS-inhibitors since the residual NO relaxes the vessel mainly through the cyclic GMP pathway.

In the rabbit carotid artery and rat superior mesenteric artery, NOS inhibitors L-NNA and L-NNA methyl ester partially inhibited the acetylcholine-induced relaxation and NO formation [10,11]. It was proposed that the remaining relaxation in the presence of these inhibitors was due to an incomplete inhibition of NO synthase and NO might fully account for acetylcholine-induced response resistance to COX and NOS inhibitors [10,11]. In our present study, the combination of L-NNA and HbO completely abolished the BK-stimulated rise in NO. Under such conditions, BK still caused a significant hyperpolarization in coronary conduit arteries. Similarly, it caused relaxation and associated hyperpolarization in U₄₆₆₁₉-pretreated coronary microarteries. Moreover, no difference existed in EC₅₀ values between relaxation and hyperpolarization to BK. These results indicate that unlike in other vasculatures, in the porcine coronary arteries EDHF significantly contributes to BK-induced relaxation and hyperpolarization.

4.2 Types of K⁺ channels involved in BK-induced response resistance to Indo- and L-NNA
In the present study, TEA, a non-specific blocker of K⁺ channels, decreased the BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA in the porcine coronary microarteries. Furthermore, either ChTX or IbTX reduced the BK-induced responses. Similar results were obtained in guinea pig coronary arteries [19] and in rabbit carotid arteries [20]. ChTX is an inhibitor of BK_Ca and voltage-dependent K⁺ channels and IbTX is a specific inhibitor of BK_Ca channels. These results show the contribution of BK_Ca channels to the BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA in the coronary circulation.

Apamin, a specific blocker of SK_Ca channels, did not inhibit the BK-induced relaxation resistance to COX- and NOS-inhibitors when it was applied alone. However, the combined application of apamin and ChTX almost completely abolished the BK-induced hyperpolarization and relaxation. Similarly, in the guinea pig coronary resistance arteries, apamin alone did not affect acetylcholine-induced relaxation and hyperpolarization resistance to COX- and NOS-inhibitors but eliminated the relaxation resistance to ChTX [21]. These results suggest that the blockade of BK_Ca channels is indispensable in the inhibitory effect of apamin. In addition, it seems that ChTX and apamin inhibit K⁺ channels in a synergistic manner in the porcine coronary microarteries.

In the present study, the BK-induced relaxation resistance to Indo and L-NNA was partially inhibited by TEA, ChTX or IbTX alone and abolished by the combination of apamin and ChTX. The Ca²⁺-activated K⁺ channels on the endothelial cells of porcine coronary arteries have been well identified [22,23]. TEA and ChTX can inhibit these K⁺ channels. It seems possible that these K⁺ channel inhibitors might interact with the K⁺ channels on the endothelial cells. Endothelial cell activation by an endothelium-dependent vasodilator involves two major phenomena: membrane hyperpolarization and cytosolic Ca²⁺ increase [22]. More recently, it has been reported that K⁺-channel blockers have only a marginal effect on cytosolic Ca²⁺ in endothelial cells [21]. Therefore, it is most likely that K⁺ channel blockers act on the K⁺ channels present on smooth muscle cells.

In the porcine coronary resistance arteries, the blockade of SK_Ca or K_ATP channels had no significant effect on the resting tone. Conversely, the blockade of K_Ca channels by either TEA or ChTX increased the basal tone. In porcine coronary conduit arteries [13] and in cerebral arteries [24], TEA and ChTX were also found to produce a slight contraction. Therefore, BK_Ca channels may play a regulating role in the basal tone in these vessels.

It has been reported that K_ATP channels plays a limited role in mediating the effect of endothelium-dependent relaxation and hyperpolarization resistance to COX- and NOS-inhibitors in coronary conduit arteries [8,13,19], as evidenced by the insensitivity to GBC in the BK-induced responses. In the current study, GBC partially inhibited the BK-induced relaxation resistance to Indo and L-NNA. However, in the coronary microarteries precontracted with U₄₆₆₁₉, the cumulative addition of GBC induced a concentration-dependent relaxation, suggesting that GBC may antagonize the U₄₆₆₁₉-induced contraction. Studies in the dog and pig coronary arteries found that GBC is an antagonist of thromboxane A₂ receptor and competitively antagonizes the action of U₄₆₆₁₉ [25,26]. Taken together, GBC may not be suitable for use in the investigation of the K⁺ channel types in U₄₆₆₁₉-precontracted vessels.

In summary, in porcine coronary arteries, the BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA are mediated by two distinct endothelium-derived factors: EDHF and NO. These endothelium-dependent responses involve two types of K⁺ channels: BK_Ca and SK_Ca channels, probably acting in a synergistic manner. BK_Ca channels may play a main role in mediating the BK-induced relaxation and hyperpolarization resistance to Indo and L-NNA, whereas SK_Ca channels may function as a ‘backup’ mechanism when BK_Ca channels are curtailed.

Time for primary review 27 days.

	Acknowledgements

 
This study was supported by the Hong Kong Research Grants Council (Grant Nos. HKU7280/97M and 7246/99M), the Committee of Research and Conferences (Grant Nos. 337/048/0018 and 335/048/0079), and the St. Vincent Medical Foundation, Portland, OR, USA.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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