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(Circulation Research. 1998;83:1069-1079.)
© 1998 American Heart Association, Inc.

Original Contributions

Nitric Oxide-20–Hydroxyeicosatetraenoic Acid Interaction in the Regulation of K⁺ Channel Activity and Vascular Tone in Renal Arterioles

Cheng-Wen Sun, Magdalena Alonso-Galicia, M. Reza Taheri, John R. Falck, David R. Harder, , Richard J. Roman

From the Department of Physiology and Cardiovascular Research Center (C.-W.S., M.A.-G., M.R.T., D.R.H., R.J.R.), Medical College of Wisconsin, Milwaukee and the Department of Molecular Genetics (J.R.F.), University of Texas Southwestern Medical Center, Dallas.

Correspondence to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail rroman{at}mcw.edu

	Abstract

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Abstract—The present study examined whether inhibition of P4504A enzyme activity and the formation of 20-HETE contributes to the activation of K⁺ channels and vasodilator effects of nitric oxide (NO) in renal arterioles. Addition of an NO donor to the P4504A2 enzyme that produces 20-HETE increased visible light absorbance at 440 nm indicating that NO binds to heme in this enzyme. NO donors also dose-dependently inhibited the formation of 20-HETE in microsomes prepared from renal arterioles. In patch-clamp experiments, NO donors increased the open-state probability of a voltage-sensitive, large-conductance (195±9 pS) K⁺ channel recorded with cell-attached patches on renal arteriolar smooth muscle cells. Blockade of guanylyl cyclase with [1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one] (ODQ, 10 µmol/L), or cGMP-dependent kinase with 8R,9S,11S-(-)-9-methoxycarbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)-trinden-1-one (KT-5823) (1 µmol/L) did not alter the effects of NO on this channel. In contrast, inhibition of the formation of 20-HETE with 17-octadecynoic acid (1 µmol/L) activated this channel and masked the response to NO. Preventing the NO-induced reduction in intracellular 20-HETE levels also blocked the effects of NO on this channel. Sodium nitroprusside (SNP) increased the diameter of renal interlobular arteries preconstricted with phenylephrine to 80±4% of control. Blockade of guanylyl cyclase with ODQ (10 µmol/L) attenuated the response to SNP by 26±2%; however, fixing 20-HETE levels at 100 nmol/L reduced the response by 67±8%. Blockade of both pathways eliminated the response to SNP. These results indicate that inhibition of the formation of 20-HETE contributes to the activation of K⁺ channels and the vasodilator effects of NO in the renal microcirculation.

Key Words: arachidonic acid • cytochrome P450 • renal circulation • vascular smooth muscle

	Introduction

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Recent studies have indicated that the tonic release of nitric oxide (NO) plays a central modulatory role in the regulation of renal tubular and vascular function and the long-term control of arterial pressure.^{1 2 3} Despite the importance of NO in the control of renal vascular tone, little is known about its mechanism of action. It is generally assumed that the effects of NO in the renal circulation are secondary to stimulation of guanylyl cyclase and the formation of cGMP as has been reported in other vascular beds.^{4 5} In this regard, NO and cGMP have been shown to activate a large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel in vascular smooth muscle (VSM) cells isolated from the trachea,⁶ aorta,⁷ coronary,^{8 9 10} mesenteric,¹¹ cerebral,¹² and pulmonary¹³ arteries. Activation of this channel hyperpolarizes VSM and limits Ca²⁺ influx through voltage-sensitive channels. Previous findings that inhibitors of guanylyl cyclase and cGMP-dependent protein kinase (PKG) attenuate the vasodilator response to NO support a primary role for cGMP in this response.^{4 5 11 14} On the other hand, recent findings that NO can still dilate some vessels depolarized with a high K⁺ medium or treated with K⁺ channel blockers indicate that other mechanisms may also be involved.^{6 10 11 13 15} In this regard, activation of PKG has been reported to contribute to the vasodilator response to NO by increasing the reuptake of Ca²⁺ into the sarcoplasmic reticulum^{16 17 18} and by decreasing the sensitivity of the contractile mechanism to Ca²⁺.^{5 19}

There is also evidence that the vasodilator response to NO in some vascular beds is cGMP-independent.^{7 11 20 21 22 23} For example, Carrier et al¹¹ reported that inhibition of PKG only blocked about half of the vasodilator effect of NO in small mesenteric arteries. Other investigators have reported that the vasodilator response to NO in bronchial smooth muscle,²² aortic rings,⁷ and cerebral²¹ arteries cannot be blocked by inhibitors of guanylyl cyclase. This has lead to a search for alternative pathways by which NO might promote vasodilation. One such mechanism has recently been identified by Bolotina et al.⁷ These authors reported that NO can directly activate a K_Ca channel in VSM cells isolated from rabbit aorta and that this may mediate the cGMP-independent actions of NO.

The purpose of the present study was to evaluate the relative contribution of cGMP versus cGMP-independent pathways in mediating the vasodilator response NO in the renal microcirculation and to determine the role of K⁺ channels in this response. Given recent observations that NO inhibits a variety of heme-containing enzymes, including NO synthase²⁴ and P450 enzymes of the 1A, 2B1, and 3C families,^{25 26} we also examined whether NO inhibits the cytochrome P4504A enzyme responsible for the formation of 20-HETE in renal VSM cells. Inhibition of this enzyme potentially could contribute to cGMP-independent actions of NO, because 20-HETE is a potent endogenous inhibitor of K_Ca channels in both renal and cerebral arteries.^{27 28}

	Materials and Methods

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General
Experiments were performed on 10- to 12-week-old male Sprague-Dawley rats purchased from Harlan Sprague-Dawley Laboratories (Indianapolis, IN). The rats were housed in an animal care facility at the Medical College of Wisconsin that is approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols received prior approval from the Animal Care Committee of the Medical College of Wisconsin.

NO Binding Studies
These experiments were performed with microsomes prepared from Sf9 insect cells transfected with a full-length P4504A2 cDNA ligated into the SmaI restriction site of the PVL 1393 baculovirus expression vector. We recently published the details regarding the cloning, expression, and characterization of the catalytic activity of this protein.²⁹ Microsomes prepared from Sf9 cells transfected with the 4A2 viral construct, or the vector alone, were resuspended in a 1-mL cuvette at a final protein concentration of 0.5 mg/mL in a 50 mmol/L sodium phosphate buffer (pH=7.4) containing 80 mmol/L NaCl. Similar studies were performed with microsomes (1.5 mg/mL) prepared from renal microvessels and the liver of rats. The cytochrome P450 content of the diluted P4504A2 and microsomal proteins were determined by measuring the reduced CO difference spectrum according to the method of Omura and Sato.³⁰ The effects of NO on the visible light absorption spectrum of the 4A2 protein and microsomal preparations were recorded with a DU 680 scanning spectrophotometer (Beckman Instruments), 1 minute after adding various concentrations of the NO donor 1-Hexanamine, 6-(2-hydroxyl-1-methyl-2-nitrosohydrazino)-N-methyl- (Mahma NONOate) (10^-7 to 10^-3 mol/L) to the cuvette. Experiments were performed with both the oxidized and the sodium dithionite-reduced forms of these proteins.

The concentration of NO in the samples was determined by the oxyhemoglobin-trapping technique.³¹ Various concentrations of Mahma NONOate were added to a cuvette containing 1 mL of 3 µmol/L oxyhemoglobin dissolved in the same buffer used in the NO binding studies. One minute after the addition of the donor, a difference spectrum was recorded over the range of 380 to 500 nm and the concentration of NO estimated from the change of absorbance at 401 and 419 nm with an extinction coefficient of 100 mmol/L^-1cm^-1.

Biochemical Studies
Isolation of Renal Microvessels
Rats were anesthetized with pentobarbital (50 mg/kg body weight), and the aorta below the renal arteries was cannulated. The kidneys were flushed with 10 mL of Tyrode's solution (in mmol/L) NaCl 145, KCl 6, NaHCO₃ 4.2, MgCl₂ 1, CaCl₂ 0.05, HEPES 10, and glucose 10 to which albumin (60 g/L) and Evans blue (10 g/L) were added. The kidneys were removed and pushed through a 180-µm sieve with the barrel of a 30-mL glass syringe. Intact vascular trees collected from this sieve were incubated for 30 minutes at 37°C in 5 mL of the Tyrode's solution containing collagenase (196 U/mL), soybean trypsin inhibitor (10 000 U/mL), DTT (1 mg/mL), and albumin (1 mg/mL) to remove tubular tissue. This digest was filtered onto a 75-µm nylon mesh and rinsed with 20 mL of fresh Tyrode's solution. Preglomerular arteries filled with Evans blue were identified with a stereomicroscope and collected by microdissection.

Preparation of Microsomes
Renal microvessels were homogenized in a 2 mL of 10 mmol/L potassium phosphate buffer (pH 7.7) containing 250 mmol/L sucrose, 1 mmol/L EDTA, 10 mmol/L MgCl₂, 0.1 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/mL aprotinin, 2 µmol/L leupeptin, and 1 µmol/L pepstatin. The homogenates were centrifuged at 3000g for 5 minutes, 15 000g for 20 minutes, and 100 000g for 60 minutes. The microsomal pellet was resuspended in 100 mmol/L potassium phosphate buffer (pH 7.4) containing: 30% glycerol, 1 mmol/L EDTA, 1 mmol/L DTT, and 0.1 mmol/L phenylmethyl sulfonyl fluoride. The microsomes were frozen in liquid nitrogen and stored at -80°C until used in an assay.

Metabolism of Arachidonic Acid
Microsomes (0.5 mg/mL) were incubated for 30 minutes at 37°C with [1-C¹⁴]arachidonic acid (AA) (0.1 µCi/mL; 10 µmol/L) in 1 mL of a 100 mmol/L potassium phosphate buffer (pH 7.4) containing 5 mmol/L MgCl₂, 1 mmol/L EDTA, 1 mmol/L NADPH, and an NADPH-regenerating system (10 mmol/L sodium isocitrate and 0.4 U/mL isocitrate dehydrogenase). The samples were incubated in closed glass test tubes. A stream of O₂ gas was gently blown over the surface of the reaction to maintain a PO₂ at 100 Torr, because the production of P450 metabolites of AA is oxygen dependent.³² Reactions were terminated by acidification to pH 4 with 1 mmol/L formic acid, and the reactions were extracted with ethyl acetate. The metabolites formed were separated with a 2x250 mm, C18-reverse phase, HPLC column and a linear elution gradient ranging from acetonitrile/water/acetic acid (50/50/0.1) to acetonitrile/acetic acid (100/0.1) for 40 minutes. The radioactive products were monitored with a radioactive flow detector (Model 120, Radiomatic Instrument Co).

Patch-Clamp Studies
Isolation of Renal VSM Cells
VSM cells were isolated from interlobular arteries (<100 µm) microdissected from the kidneys of rats. The vessels were incubated for 15 minutes at 37°C in 1 mL of the low Ca²⁺ Tyrode's solution containing 1.5 mg/mL papain (14 U/mg) and 1 mg/mL DTT, followed by an incubation for 15 minutes at 37°C in 2 mL of the Tyrode's solution containing 2 mg/mL collagenase (196 U/mL), 0.5 mg/mL elastase (90 U/mL), and 1 mg/mL soybean trypsin inhibitor (10 000 U/mL). The supernatant was collected and the cells spun down at 500g for 5 minutes, resuspended in fresh low Ca²⁺ Tyrode's solution, and stored at 4°C. Patch-clamp experiments were completed within 4 hours after the cells were isolated.

Current Recordings
K⁺ currents were recorded with the cell-attached, patch-clamp technique at room temperature as we described previously.^{28 33 34} The pipettes were constructed from 1.5-mm borosilicate glass capillaries and had tip resistances of 8 to 10 megohms. A List EPC-7 patch-clamp amplifier (List Biological Laboratories, Inc) was used to record single-channel currents. The amplifier output was digitized at a rate of 10 kHz and filtered at 2 kHz. Data analysis was performed with pClamp software (version 6.03, Axon Instruments). Open-state probability (NP_o) was calculated using the following equation:

where T_j is the sum of the open time at a given conductance level, j represents multiples of a given conductance, and T is the total recording time.

The effects of NO donors on K⁺ channel activity were initially characterized with cell-attached patches on renal arteriolar VSM cells bathed in a normal bath solution (140 mmol/L Na⁺, 5 mmol/L K⁺, and 1.2 mmol/L Ca²⁺) at resting membrane potential (0 mV pipette potential). All other experiments were performed with a pipette potential of -40 mV and with the cells bathed in a high KCl (145 mmol/L) and a low Ca²⁺ (100 nmol/L) solution to null resting membrane potential and to minimize Ca²⁺ gradients and flux across the cell.

Solutions
The pipette solution contained (in mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, and HEPES 5 (pH 7.4). The composition of the physiological bath solution contained (in mmol/L) NaCl 140, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.1, glucose 10, and HEPES 5 (pH 7.4). The high K⁺, low Ca²⁺ bath solution contained (in mmol/L) KCl 145, CaCl₂ 0.37, MgCl₂ 1.1, glucose 10, HEPES 10, and EGTA 1 (pH 7.4). The concentration of free Ca²⁺ in this solution (10^-7 mol/L) was calculated by a computer program.

Effects of NO Donors on K⁺ Currents
Single-channel K⁺ currents were recorded from cells bathed in the normal physiological bath solution during a 2-minute control period at resting membrane potential. The bath was exchanged with a solution containing sodium nitroprusside (SNP, 10^-4 mol/L) or Mahma NONOate (10^-4 mol/L), and K⁺ currents were recorded again after a 5-minute equilibration period. Similar experiments were performed on cells bathed with a high K⁺, low Ca²⁺ solution with a pipette potential of -40 mV.

Characterization of Type of K⁺ Channels Activated by NO Donors
The type of K⁺ channels activated by NO were characterized by constructing a current-voltage relationship and determining the voltage-dependence of NP_o across a range of potentials from -40 to 40 mV. These experiments were performed with the cell-attached mode and the bath solution contained 145 mmol/L K⁺ to null resting membrane potential. Single-channel conductances were calculated from the slopes of the current-voltage relationships.

The effects of selective blockade of K_Ca channels with iberiotoxin (100 nmol/L) and tetraethylammonium chloride (TEA) (0.3 mmol/L),^{35 36 37 38 39} small conductance, Ca²⁺-activated, K⁺ channels with apamin (100 nmol/L),³³ and delayed rectifier channels with 4-aminopyridine (5 mmol/L)^{28 35 36} on the response to SNP were also determined. The concentrations of the blockers used were based on the results of previous studies, which characterized the effects of these compounds on K⁺ channels in renal VSM cells.^{28 33 35 36} A diffusion block technique was used in these experiments.⁴⁰ The tip of the pipette was filled with the normal pipette solution while the remainder of the pipette was back-filled with a solution containing the blocker. A high-resistance seal was formed, and baseline channel activity was recorded at a pipette potential of -40 mV. The effects of the channel blockers became apparent 10 minutes later, and K⁺ currents were again recorded during a 2-minute experimental period. The bath was then exchanged with a solution containing 10^-4 mol/L SNP and K⁺ channel activity was redetermined 5 minutes later.

Effect of Blockade of cGMP Pathway on Response to NO Donors
Baseline K⁺ currents were recorded during a control period with the cell-attached mode and a pipette potential of -40 mV. The bath was exchanged with a solution containing a soluble guanylyl cyclase inhibitor, [1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one] (ODQ, 10 µmol/L, Alexis Co),^{14 41} or vehicle (0.1% ethanol). After a 10-minute equilibration period, the effects of ODQ on K⁺ channel activity were determined. Then, SNP (10^-4 mol/L) was added to the bath, and K⁺ currents were recorded after a 5-minute equilibration period.

The effects of blocking PKG with 8R,9S,11S-(-)-9-methoxycarbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)-trinden-1-one (KT-5823, Biomol Research Laboratories, Inc)¹¹ on the activation of K⁺ channels produced by SNP were studied. Baseline K⁺ channel activity was recorded with a pipette potential of -40 mV during a 2-minute control period. The bath was exchanged with a solution containing 1 µmol/L KT-5823 or vehicle (0.1% ethanol), and K⁺ currents were recorded after a 10-minute equilibration period. The effectiveness of the blockade of PKG was tested by studying the effects of 10 µmol/L 8-Bromo-cGMP on K⁺ channel activity in cells treated with vehicle or KT-5823. Finally, SNP (10^-4 mol/L) was added to the bath, and K⁺ currents were recorded after a 5-minute equilibration period.

Effect of Blockade of 20-HETE Pathway on Response to NO Donors
These experiments examined the effects of preventing the NO-induced fall in intracellular 20-HETE levels by adding exogenous 20-HETE (100 nmol/L) to the bath. We assumed that adding 20-HETE to the bath would clamp intracellular levels during the experiment, because 20-HETE is lipid soluble and rapidly equilibrates between the intracellular and extracellular compartments. Baseline K⁺ channel activity was recorded during a 2-minute control period with a pipette potential of -40 mV. Vehicle (0.1% ethanol) or 20-HETE (100 nmol/L) was added to the bath and K⁺ channel activity was recorded during a 2-minute experimental period. SNP (10^-4 mol/L) was added to the bath, and K⁺ channel activity was recorded after a 5-minute equilibration period.

The effects of 17-octadecynoic acid (17-ODYA), an inhibitor of the formation of 20-HETE,⁴² on the activation of K⁺ channels produced by SNP was also studied. Baseline K⁺ currents were recorded with a pipette potential of -40 mV during a 2-minute control period. 17-ODYA (1 µmol/L) or vehicle (0.1% ethanol) were added to the bath and K⁺ channel activity was recorded after a 10-minute period. SNP (10^-4 mol/L) was then added to the bath and K⁺ currents were redetermined after a 5-minute equilibration period.

Isolated Vessel Studies
Rats were anesthetized with pentobarbital (50 mg/kg body weight), the aorta was cannulated, and the kidneys were flushed with 20 mL of ice-cold Tyrode's solution. The left kidney was removed and small interlobular arteries (50 to 100 µm) were isolated by microdissection. The vessels were mounted on glass micropipettes and intraluminal pressure was maintained at 90 mm Hg. After a 30-minute equilibration period, the effects of agonists and inhibitors on the inner diameter of the vessel were determined with a video system composed of a stereomicroscope (Carl Zeiss Inc), a television camera (KP-130 AV, Hitachi), a videocassette recorder (A6 to 7330, Panasonic), and a measuring system (VIA-100, Boeckeler Instrument Co). The vessels were perfused and bathed with a physiological salt solution (PSS) containing (in mmol/L) NaCl 119, CaCl₂ 1.6, NaHCO₃ 12, MgSO₄ 1.2, NaH₂PO₄ 1.2, KCl 4.7, glucose 10, and EDTA 0.026 that was equilibrated with a 95% O₂–5% CO₂ gas mixture to maintain pH at 7.4. Indomethacin (5 µmol/L) and baicalein (0.5 µmol/L) were added to the bath solution to block the endogenous metabolism of AA via the cyclooxygenase and lipoxygenase pathways.⁴³ The specificity of these inhibitors was tested by studying their effects on the metabolism of AA by isolated glomeruli. Glomeruli were used for these studies because they avidly metabolize AA by cyclooxygenase, lipoxygenase, and cytochrome P450 pathways. In this system, baicalein (1 to 2 µmol/L) specifically inhibited the formation of 12-HETE and had no effect on the formation of 6-keto-PGF₁, epoxyeicosatrienoic acids (EETs), or 20-HETE. Indomethacin at concentrations of 1 to 5 µmol/L completely inhibited the formation of 6-keto-PGF₁ without affecting the formation of EETs, 20-HETE, or 12-HETE. However, at higher concentrations (10 µmol/L), indomethacin also inhibited the formation of 20-HETE and EETs by about 50%.

Effect of Blockade of cGMP and 20-HETE Pathways on Vasodilator Response to SNP
The contribution of cGMP and 20-HETE to the vasodilator effect of NO was evaluated by determining the response to SNP (10^-7 to 10^-3 mol/L) in vessels preconstricted with phenylephrine (1 µmol/L) under control conditions, and after guanylyl cyclase was inhibited with ODQ (10 µmol/L) or intracellular 20-HETE levels were fixed at a high level by adding 20-HETE (100 nmol/L) to the bath. We also determined the effects of blocking both pathways simultaneously. In other experiments, the role of K⁺ channels in the vasodilator response to NO was evaluated by studying the response to SNP (10^-7 to 10^-3 mol/L) in vessels preconstricted with phenylephrine (1 µmol/L) after a depolarizing concentration of KCl (50 mmol/L) or the selective K_Ca channel blocker,³⁸ iberiotoxin (100 nmol/L), was added to the bath.

Effect of ODQ and 20-HETE on cGMP Production in Renal Microvessels
The effectiveness of ODQ to block guanylyl cyclase activity was assessed by measuring its effects on cGMP levels in renal microvessels stimulated with 10^-3 mol/L SNP. Renal microvessels (5 to 10 mg protein) were preincubated for 30 minutes at 37°C in PSS. The microvessels were transferred to vials containing 1 mL of PSS, 0.5 mmol/L of 3-isobutyl-1-methylxanthine, to prevent breakdown of cGMP and SNP (10^-3 mol/L). Vehicle (0.1% ethanol), ODQ (10 µmol/L), or 20-HETE (100 nmol/L) were then added and the samples were incubated for 10 minutes at 37°C. The reactions were terminated by adding trichloroacetic acid to a final concentration of 6%. The samples were homogenized and centrifuged, and the supernatant was extracted with water-saturated ether. The aqueous phase was collected and dried under nitrogen. The pellet was resuspended in 1 mL of 1 mol/L NaOH and the protein content was determined by the Bradford method (Bio-Rad) for normalization of cGMP levels per mg of protein. The dried samples were reconstituted, and cGMP levels were determined with a commercially available radioimmunoassay kit (PerSeptives Inc). The range of the standard curve was 2 to 128 µmol/tube with a B_o of 39.6% and a B₅₀ of 9 µmol/tube. Repeated analysis of a pooled vessel sample averaged 1610±80 µmol/tube with a coefficient of variation of <5%.

Statistics
Values presented are mean±SEM. The significance of the differences in mean values between and within groups was examined with an analysis of variance for repeated measures followed by a Duncan's multiple-range test.⁴⁴ Single-channel conductances were fitted using least-squares linear regression. A P value <0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NO Binding Studies
Representative tracings comparing the effects of NO on the visible light absorption difference spectra of microsomes prepared from renal microvessels and the liver of the rat and Sf9 insect cells transfected with a P4504A2-baculovirus construct are presented in Figure 1. The cytochrome P450 content of the liver, renal microvessel, and P4504A2 transfected cell microsomal preparations averaged 0.3, 0.1, and 0.1 nmol/mg protein, respectively. Addition of Mahma NONOate to either oxidized or dithionate-reduced microsomes prepared from the liver or the renal microvessels (Figure 1A and 1B) increased absorption at 440 nm which is characteristic of the formation of ferric- and ferrous-nitrosyl complexes at the heme binding site of P450 enzymes.⁴⁵ Similarly, oxidized microsomes prepared from insect cells expressing the P4504A2 enzyme also exhibited an increase in absorbance at 440 nm after addition of the NO donor (Figure 1C). However, if the P4504A2 protein was first reduced with dithionate, the NO donor did not increase absorption at this wavelength. In additional experiments, we found that the concentration of Mahma NONOate that produced a maximal increase in absorption was 10^-5 mol/L and that this produced a measured NO concentration of 10^-6 mol/L, 1 minute after addition of the compound to the cuvette.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of Mahma NONOate (10-5 mol/L) on the visible light spectrum of both oxidized and reduced microsomes prepared from the liver (A) and renal vessels (B) of rats and Sf9 insect cells transfected with a baculovirus-P4504A construct expressing P4504A2 protein (C). Samples were reduced with a few grains of sodium dithionite. The absorption difference spectra were obtained 1 minute after addition of the NO donor to the cuvette.

Biochemical Studies
P4504A enzyme activity was assayed under control conditions and after addition of various concentrations of SNP (10^-5, 10^-4, 10^-3 mol/L) or the noncyanide-releasing NO donor 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) (10^-5, 10^-4, 10^-3 mol/L) to the incubations. Renal vascular microsomes produced 14,15-, 11,12-, and 8,9-dihydroxyeicosatrienoic acids and 20-HETE when incubated with AA under control conditions (Figure 2). Addition of SNP and PAPA NONOate inhibited the formation of 20-HETE in a concentration-dependent manner. At a concentration of 10^-3 mol/L, both compounds completely inhibited the formation of 20-HETE.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of SNP and PAPA NONOate on the production of 20-HETE in microsomes prepared from preglomerular arterioles isolated from the kidneys of rats. The microsomes were incubated with [14C] AA (0.5 µCi/mL, 10 µmol/L) in the presence of vehicle, SNP, or PAPA NONOate. Results are expressed as a percentage of the control production of 20-HETE which averaged 6.8±0.3 pmol/min per milligram protein. Numbers in parentheses indicate the number of vessel isolations and incubations performed with each concentration of donor. *Significant difference (P<0.05) vs control.

Patch-Clamp Studies
A representative record illustrating the effects of SNP (10^-4 mmol/L) on K⁺ channel activity in renal arteriolar VSM cells bathed with a normal physiological bath solution at resting membrane potential is presented in Figure 3. SNP enhanced the opening of a large-conductance K⁺ channel. NP_o increased from 0.002±0.0002 to 0.009±0.001 after the addition of SNP (n=6 cells). This was caused by an increase in the number of channel openings (from 6±1 to 21±4 events every 2 minutes). Mean open time was not significantly altered (from 0.49±0.11 to 0.64±0.16 ms). SNP also had no significant effect on the single-channel conductance (5.13±0.05 pA before and 5.39±0.20 pA after SNP).

View larger version (25K): [in this window] [in a new window]   Figure 3. Representative tracing depicting the effects of SNP (100 µmol/L) on the activity of K+ channels recorded from cell-attached patches of rat renal VSM cells bathed with a normal physiological salt solution. Currents were recorded at room temperature at resting membrane potential (pipette potential, 0 mV). The control and SNP tracings presented are gap-free and are presented in consecutive order.

Additional studies were performed on 8 cells bathed in a high K⁺ solution so that membrane potential could be controlled and the types of K⁺ channels activated by SNP could be better characterized. At least three types of K⁺ channels with a large (8.0±0.3 pA), intermediate (6.5±0.1 pA), and small conductance (2.8±0.1 pA) were typically recorded from cell-attached patches at a pipette potential of -40 mV (Figure 4, upper panel). The opening of the large-conductance K⁺ channel gradually increased and reached a stable plateau value 5 minutes after addition of SNP (10^-4 mol/L) to the bath. The increase in the activity of this channel was sustained for at least 15 minutes (the longest period monitored) and channel activity returned toward control after SNP was removed from the bath. On average, SNP (10^-4 mol/L) increased the NP_o of the large-conductance K⁺ channel 10-fold (Figure 4, lower panel). Mean open time increased from 1.8±0.2 to 4.0±0.3 ms after SNP, and the number of channel openings increased from 81±24 to 654±50 events every 2 minutes. SNP had no effect on the unitary current of this channel (7.8±0.1 pA before and 8.6±0.3 pA after SNP). SNP also had no significant effect on the NP_o or the unitary current of the small or intermediate conductance K⁺ channels (Figure 4, lower panel). The effects of SNP were concentration dependent. In 5 cells, SNP at concentrations of 10^-6, 10^-5, and 10^-4 mol/L increased the NP_o of the large-conductance K⁺ channel by 126±70%, 338±60%, and 1343±444%, respectively. Similar effects were seen in five cells after the addition of Mahma NONOate to the bath. In these experiments, Mahma NONOate (10^-4 mol/L) selectively increased the NP_o of the large-conductance K⁺ channel 10-fold from 0.4±0.2% to 4.8±1.9% of recording time (n=5), whereas it had no significant effect on the NP_o of the intermediate- or small-conductance channels.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of SNP (100 µmol/L) on the activity of K+ channels recorded from cell-attached patches of rat renal VSM cells. Currents were recorded at room temperature with a pipette potential of -40 mV. The cells were bathed with a high K+ (145 mmol/L), low Ca2+ (100 nmol/L) solution to null membrane potential. A, B, and C, Examples of an opening of the small (2.8 pA)- intermediate (6.5 pA)- and large (8.0 pA)- conductance K+ channel. *Significant difference (P<0.05) vs control. Values presented are mean±SE recorded from 8 cells before and after SNP.

The voltage dependence of the large-conductance K⁺ channel activated by NO is depicted in Figure 5A. The NP_o of this channel increased at depolarized potentials and the unitary current varied linearly with pipette potential. The slope conductance determined over the range of pipette potentials from 40 to -40 mV averaged 195±9 pS (Figure 5B), which is lower than the value that we have previously reported for this channel in excised membrane patches (250 pS).^{28 33 34} A decreased conductance of the K_Ca channel recorded with cell-attached patches has been reported previously. It is thought to be caused by effects of intracellular Mg²⁺ and Na⁺ which lower the conductance of this channel when it is studied in intact cells.⁴⁰

View larger version (12K): [in this window] [in a new window]   Figure 5. Characterization of the K+ channel activated by SNP in rat renal VSM cells. A, Voltage dependence of the opening of the large-conductance K+ channel activated by SNP (100 µmol/L). B, Current-voltage relation of the large-conductance K+ channel activated by SNP (100 µmol/L). Currents were recorded with cell-attached patches at the pipette potentials indicated. The cells were bathed with a high K+ (145 mmol/L), low Ca2+ (100 nmol/L) solution to null membrane potential. Each point represents a mean value±SE obtained from 5 to 9 cells.

The effects of the selective blockade of K_Ca channels with TEA (0.3 mmol/L)³⁹ and iberiotoxin (100 nmol/L)³⁸ on the response to NO are presented in Figure 6. Inclusion of TEA in the pipette solution reduced the conductance of the large-conductance K⁺ channel by 37% from 7.8±0.4 to 4.9±0.5 pA (n=4) and induced a characteristic "flickery-type" blockade. This is consistent with the rapid on/off kinetics of the interaction of TEA with the K_Ca channel.³⁹ Addition of SNP (10^-4 mol/L) still increased the number and duration of openings of the partially blocked K_Ca channel in the presence of TEA (Figure 6A). The NP_o increased from 0.03±0.02 to 0.34±0.05 after addition of 10^-4 mol/L SNP. In contrast, the opening of the large-conductance K⁺ channel in renal VSM cells was blocked nearly completely by inclusion of 100 nmol/L iberiotoxin in the pipette solution. Baseline NP_o fell from 0.36±0.10 to 0.03±0.01 (Figure 6B), and iberiotoxin prevented the increase in NP_o after SNP (10^-4 mol/L). These findings are consistent with the reported mode of action of iberiotoxin that binds with high affinity and a low dissociation rate constant to a receptor on the external surface of the K_Ca channel and prevents movement of K⁺ ions through the pore.^{38 46}

View larger version (18K): [in this window] [in a new window]   Figure 6. Representative tracings depicting the effects of TEA (A) and iberiotoxin (IBTX) (B) on the large-conductance K+ channel activated by SNP (100 µmol/L). Currents were recorded at room temperature with cell-attached patches and a pipette potential of -40 mV. The cells were bathed with a high K+ (145 mmol/L), low Ca2+ (100 nmol/L) solution to null membrane potential. TEA (0.3 mmol/L) and iberiotoxin (100 nmol/L) were added to the pipette solution by the diffusion block technique described in Materials and Methods.

The effects of ODQ on K⁺ currents recorded from cell-attached patches of renal arteriolar VSM cells are presented in Figure 7A. ODQ significantly decreased the NP_o of the large-conductance K⁺ channel from 0.10±0.01 to 0.05±0.01 (n=6 cells). It had no effect, however, on the activation of the channel produced by SNP. SNP still increased the NP_o of the large-conductance K⁺ channel from 0.05±0.01 to 0.60±0.01 after ODQ, and there was no difference in the response to SNP in cells treated with vehicle or ODQ.

View larger version (25K): [in this window] [in a new window]   Figure 7. Effects of blockade of the cGMP pathway on the activation of the K+ channel produced by SNP (100 µmol/L). A, Effects of the soluble guanylyl cyclase inhibitor ODQ (10 µmol/L) on the response to SNP. B, Effects of the cGMP-dependent protein kinase inhibitor KT-5823 (1 µmol/L) on the response to 8-Bromo-cGMP and SNP. Currents were recorded with cell-attached patches and a pipette potential of -40 mV. The cells were bathed with a high K+ (145 mmol/L) solution to null membrane potential. *Significant difference (P<0.05) vs control.

Similar experiments depicting the effects of blockade of the cGMP pathway with KT-5823 (1 µmol/L) are presented in Figure 7B. KT-5823 reduced baseline NP_o of the K_Ca channel from 0.15±0.04 to 0.07±0.01 (n=7). 8-Bromo-cGMP (10 µmol/L) markedly increased the NP_o of this channel in cells treated with vehicle, but its effects were completely blocked in cells treated with KT-5823. In contrast, KT-5823 had no effect on the response of these same cells to SNP (10^-4 mol/L). SNP increased the NP_o of the K_Ca channel from 0.11±0.01% to 1.8±0.70% of recording time in the presence of KT-5823. There was no significant difference in the response to SNP in cells treated with vehicle or KT-5823.

The effects of blockade of the 20-HETE pathway on the K⁺ channel response to SNP are presented in Figure 8. In vehicle-treated cells, addition of SNP produced a 10-fold increase in the NP_o of the K_Ca channel. Blockade of the production of 20-HETE with 17-ODYA (1 µmol/L) mimicked the effects of SNP and increased the baseline NP_o of the K_Ca channel from 0.34±0.10 to 0.83±0.20 (Figure 8A). In the presence of 17-ODYA, SNP had no further effect to increase the NP_o of this channel. Addition of 20-HETE (100 nmol/L) reduced baseline NP_o and completely abolished the effect of SNP to activate the K_Ca channel (Figure 8B).

View larger version (23K): [in this window] [in a new window]   Figure 8. Effects of 17-ODYA (1 µmol/L) and 20-HETE (100 nmol/L) on the activation of the KCa produced by SNP (100 µmol/L) recorded from rat renal VSM cells. Currents were recorded at room temperature with cell-attached patches and a pipette potential of -40 mV. The cells were bathed with a solution containing a high concentration of KCl (145 mmol/L) to null membrane potential. A, Summary of the effects of 17-ODYA on the activation of KCa channels produced by SNP. B, Effects of 20-HETE on the activation of KCa channel produced by SNP. Values are mean±SEM. Numbers in parentheses indicate the number of cells studied. *Significant difference (P<0.05) vs control.

A comparison of the effects of blockade of the cGMP and 20-HETE pathways on the activation of the K_Ca channel produced by SNP is presented in Figure 9. The results are expressed as a percent increase in NP_o to correct for differences in baseline activity in the various groups of cells. SNP produced a 10-fold increase in the NP_o of the K_Ca channel in control cells and cells treated with vehicle (0.1% ethanol). Blockade of guanylyl cyclase with ODQ or PKG with KT-5823 had no significant effect on the activation of K_Ca channels produced by SNP. In contrast, the effects of SNP on K_Ca channels were completely blocked after the endogenous production of 20-HETE was inhibited with 17-ODYA or intracellular 20-HETE levels were fixed at a high level by adding it to the bath.

View larger version (23K): [in this window] [in a new window]   Figure 9. Summary of the effects of blockade of the cGMP and 20-HETE pathways on the activation of KCa channels produced by SNP. Results are expressed as a percent increase in NPo recorded from rat renal VSM cells after addition of SNP (10-4 mmol/L) to the bath. Currents were recorded at room temperature with cell-attached patches and a pipette potential of -40 mV. The cells were bathed in high K+, low Ca2+ solution to null the membrane potential.

The significance of activation of K⁺ channels to the vasodilator response to SNP in renal interlobular arteries are summarized in Figure 10A. Depolarization of renal arteries with 50 mmol/L KCl or blockade of K_Ca channels with iberiotoxin markedly attenuated the vasodilator response to SNP by about 75%.

View larger version (23K): [in this window] [in a new window]   Figure 10. Effects of blockade of K+ channels and the cGMP and 20-HETE pathways on the vasodilator response to NO in rat renal interlobular arteries. A, Effects of addition of 50 mmol/L KCl and iberiotoxin (100 nmol/L) on the response to SNP. B, Response to SNP before and after the addition of ODQ or 20-HETE to the bath. Results are expressed as percent of control diameter after preconstriction with phenylephrine (1 µmol/L). Numbers in parentheses indicate the number of vessels studied in each group. *Significant difference (P<0.05) control response vs SNP.

The results of experiments performed to evaluate the relative contribution of the cGMP and 20-HETE pathways to the vasodilator response to NO are presented in Figure 10B. The baseline diameter of the vessels averaged 91±4 µm (n=20 vessels, 20 rats). Phenylephrine (1 µmol/L) reduced the diameter of these vessels by 50%. SNP (10^-7 to 10^-3 mol/L) dilated these vessels in a concentration-dependent manner to a maximum of 79±4% of control. The vasodilator response to SNP was only reduced at the highest concentration of SNP studied (10^-3 mol/L) in vessels treated with ODQ. In contrast, the response to low concentrations of SNP (10^-7 to 10^-5 mol/L) was blocked completely after 20-HETE was added to the bath. 20-HETE also reduced the vasodilator response to the highest dose of SNP by 75%. Simultaneous blockade of both pathways with ODQ and 20-HETE completely abolished the response to SNP.

The results of the experiments to determine the effectiveness of the blockade of guanylyl cyclase activity in renal arterioles after administration of ODQ are presented in Figure 11. Addition of 10^-3 mol/L SNP produced a 6-fold increase in cGMP levels in renal arterioles. ODQ (10 µmol/L) prevented the increase in cGMP levels. In contrast, 20-HETE (100 nmol/L) had no effect on cGMP levels in vessels treated with SNP.

View larger version (18K): [in this window] [in a new window]   Figure 11. Effects of ODQ (10 µmol/L) and 20-HETE (100 nmol/L) on cGMP levels in renal preglomerular arteries exposed to a maximal concentration of SNP (1 mmol/L). Values are mean±SEM obtained from 10 incubations. *Significant difference (P<0.05) control response vs SNP.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study evaluated the contribution of cGMP-dependent versus cGMP-independent pathways in mediating the effects NO on K⁺ channel activity and vascular tone in renal arterioles. The results indicate that NO selectively enhances the NP_o of a large-conductance (195±9 pS) K⁺ channel in renal VSM cells in a concentration-dependent manner. NO had no significant effect on the activity of the two smaller conductance channels in renal VSM cells that were previously identified as a small-conductance, apamin-sensitive, K_Ca channel³³ and the 4-aminopyridine-sensitive delayed rectifier channel.^{28 34 35 36} The K⁺ channel activated by NO is voltage-sensitive and is blocked by TEA and iberiotoxin which are selective inhibitors of the K_Ca channel.^{37 38 39} Thus, the present findings indicate that NO activates K_Ca channels in renal VSM cells, and they are consistent with the results of recent studies with smooth muscle cells isolated from other tissues.^{6 7 9 11 13 15}

Additional experiments were performed on cells bathed with a high K⁺, low Ca²⁺ solution to further explore the mechanism by which NO activates the K_Ca channel. In these experiments, resting membrane potential was nulled, T- and L-type Ca²⁺ channels were largely voltage-inactivated, and the Ca²⁺ gradient and influx were minimized. The finding that NO still activates the K_Ca channel under these conditions suggests that this effect is not mediated by depolarization or Ca²⁺ influx across the entire cell through voltage-sensitive Ca²⁺ channels. However, we cannot completely exclude the possibility that the activation of the K_Ca channels depended on a local increase in Ca²⁺ flux across the patch itself, if NO has an action to enhance voltage-sensitive or capacitive-leak Ca²⁺ currents since our pipette solution contained Ca²⁺. This possibility seems unlikely because NO is a vasodilator that lowers intracellular Ca²⁺ concentration, and all the available evidence indicates that NO inhibits, rather than enhances, Ca²⁺ channel activity in a variety of cell types.^{47 48 49 50}

Activation of K_Ca channels and/or the vasodilator response to NO was cGMP-dependent in several studies.^{9 10 11 12} On the other hand, Bolotina et al⁷ reported that NO can directly activate this channel in excised membrane patches of VSM cells isolated from the rabbit aorta. The results of the present study indicate that the effects of NO to activate the K_Ca channel in renal arteriolar VSM are cGMP-independent, because blockade of guanylyl cyclase with ODQ or PKG with KT-5823 had no significant effect on this response. Overall, the results of our patch-clamp studies are consistent with a direct effect of NO on K_Ca channels if NO can act via this mechanism in intact cells. However, we feel that this question remains to be answered because Bolontia et al⁷ found that NO-induced activation of the K_Ca channel in excised membrane patches could be blocked by the addition of a low concentration of protein to the bath. This suggests that NO released by endothelial cells may be inactivated by extra- and intracellular proteins before it can reach and interact with the intracellular sites gating the K_Ca channel in intact VSM cells. This concern led us to consider another mechanism. In this regard, we have reported that renal⁵¹ and cerebral arteries²⁷ metabolize AA to 20-HETE via a P4504A enzyme and that 20-HETE serves as a novel intracellular signal transduction pathway that modulates renal vascular tone by regulating the activity of the K_Ca channel in VSM cells.²⁸ Because NO inhibits several heme-containing proteins, including NO synthase²⁴ and a variety of P450 enzymes,^{25 26 45} we considered the hypothesis that NO might activate the K_Ca channel in renal arteriolar VSM by inhibiting the endogenous formation of 20-HETE. Our results indicate that NO does bind to heme in the P4504A2 enzyme and inhibits the production of 20-HETE in renal microvessels. The concentration of the NO donor needed to inhibit the formation of 20-HETE is similar to that needed to activate the K_Ca channel and dilate renal arterioles. We also found that SNP and PAPA NONOate at concentrations of 10^-4 and 10^-3 mol/L established steady-state NO concentrations of 60 and 250 nmol/L in the bath of our isolated vessel experiments. These concentrations are well within the physiological range measured in renal interstitial fluid by microdialysis⁵² and are similar to values recently reported in the effluent of isolated vessels dilated with acetylcholine.⁵³

The results of the patch-clamp studies further revealed that blockade of the endogenous synthesis of 20-HETE with 17-ODYA mimics the effects of NO on the K_Ca channel and that NO had no additional effects on this channel after 17-ODYA. Moreover, preventing the decline in intracellular 20-HETE levels by adding it to the bath completely blocked the activation of K_Ca channels produced by NO. These findings indicate that a decline in intracellular levels of 20-HETE contributes to the NO-induced activation of the K_Ca channel in renal arteriolar VSM. They also suggest that the NO-induced activation of the K_Ca channel in these cells is probably not mediated via a direct effect of NO, since SNP should still activate the K_Ca channel in the presence of 17-ODYA and 20-HETE if it acts via this mechanism.

The significance of activation of K_Ca channels to the vasodilator effect of NO was evaluated with isolated perfused renal interlobular arteries. Depolarization of renal arterioles with 50 mmol/L KCl or blockade of K_Ca channels with iberiotoxin diminished the vasodilator response to SNP by 75%. These results indicate that activation of the K_Ca channel and hyperpolarization of VSM cells play a major role in the vasodilator response to NO in renal arterioles probably by reducing Ca²⁺ influx via voltage-sensitive Ca²⁺ channels. The mechanism mediating the residual response to NO in the presence of KCl or iberiotoxin remains to be determined, but it may be mediated via cGMP-PKG activation of Ca²⁺ reuptake into the sarcoplasmic reticulum.^{16 17 18}

We also examined the relative contribution of the cGMP and 20-HETE pathways to the vasodilator response to NO in renal arterioles. Blockade of guanylyl cyclase with ODQ (10 µmol/L) had no significant effect on the vasodilator response to SNP in the range of concentrations from 10^-7 to 10^-4 mol/L. In contrast, preventing the decline in 20-HETE levels completely blocked the vasodilator response of these arteries to low concentrations of SNP and markedly attenuated the response to high concentrations of SNP (10^-3 mol/L). This residual response in the presence of fixed 20-HETE levels is probably cGMP-dependent because it was blocked by ODQ. These results suggest that inhibition of the formation of 20-HETE and activation of the K_Ca channels play a major role in the vasodilator response to NO in the renal microcirculation. The cGMP pathway also apparently contributes to this response, but to a lesser extent. Because the effects of NO to activate the K_Ca channel in the present study were cGMP-independent, cGMP must dilate renal arterioles by some other mechanism. Likely mechanisms include an effect of PKG on the reuptake of Ca²⁺ or the Ca²⁺ sensitivity of the contractile mechanism. Moreover, the finding that simultaneous blockade of the 20-HETE and cGMP pathways eliminates the vasodilator response to NO suggests that direct effects of NO on K_Ca channels plays little or no role in this response in the renal circulation.

Although the present results indicate that inhibition of the production of 20-HETE contributes to the vasodilator response to NO in the renal circulation, the relative importance of this mechanism as a mediator of NO action likely differs between species, vascular beds, and in small arterioles versus large vessels. In this regard, we have found that the expression of P4504A protein and the production of 20-HETE is greater in small arteries (<100 µm) than in the aorta and renal and carotid arteries. This may explain why the vasodilator response to NO has generally been reported to be cGMP-dependent in most studies with large arteries, which do not express P4504A enzymes; whereas, it appears to be largely cGMP-independent in the present study and in the only other study¹¹ that was performed using microvessels.

In summary, the present results indicate that NO binds to heme in P4504A enzymes and inhibits the formation of 20-HETE in renal arterioles. The activation of the K_Ca channel and the vasodilator response to NO in these vessels appears to be largely cGMP-independent and inhibition of the formation of 20-HETE by NO contributes to these effects. These findings suggest that changes in the vascular responsiveness to NO and endothelial dysfunction associated with diabetes and hypertension may reflect changes in the expression of P4504A enzymes and the production of 20-HETE in the microcirculation.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health HL-29587 and 36279. The authors wish to thank Lisa Henderson for her assistance with performing the HPLC analysis of P450 metabolites.

Received January 30, 1998; accepted August 7, 1998.

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