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Am J Physiol Heart Circ Physiol 275: H1322-H1328, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 4, H1322-H1328, October 1998

Rabbit polymorphonuclear leukocytes release a factor that causes constriction of the coronary vasculature

Joanne L. Hart-Favaloro and Owen L. Woodman

Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Polymorphonuclear leukocytes (PMN) have been shown to have numerous vasoactive effects, particularly in large artery bioassays. This study shows that rabbit PMN passively release a contractile factor that constricts the coronary vasculature of isolated, Langendorff-perfused rabbit hearts. The mechanism of action of this factor does not involve inhibition of nitric oxide (NO), production of cyclooxygenase metabolites, 5-hydroxytryptamine, or endothelin, or the activation of alpha -adrenoceptors but is a Ca2+-dependent process, because the constriction is inhibited by the Ca2+-channel blocker amlodipine. The activity of this factor is significantly inhibited if it is pretreated with trypsin or heated to 90°C for 10 min, and the active factor is concentrated in the retentate of 100-kDa cutoff centrifuge filters, indicating that the factor is a protein >100 kDa in size. This study shows that rabbit PMN spontaneously release a protein factor that causes constriction of isolated, perfused rabbit hearts by a NO-independent but Ca2+-dependent mechanism.

neutrophil; vasoactive protein

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A NUMBER OF REPORTS describe the release of vasoactive, particularly contractile, agents from polymorphonuclear leukocytes (PMN) in a variety of isolated vessels and vascular beds. These constrictor responses are mediated by numerous agents including reactive oxygen species (7, 20) and leukotrienes (8, 19) as well as a number of unknown factors (17, 29, 31-33). The mediators of the constrictor responses in isolated hearts include oxygen radical species such as superoxide anions (14, 20) and the hydroxyl radical (7), which inhibit nitric oxide (NO)-mediated relaxation. Other studies report that vascular constriction in the isolated, perfused rabbit heart is caused by PMN-derived leukotriene synthesis (8, 19, 24). We previously described (9, 10, 32) the release by PMN of a factor that causes endothelium-dependent contraction of large arteries by inhibiting the effects of endothelium-derived NO. This factor is released by PMN and does not require an interaction between the PMN and the endothelium. In addition, we showed that the effect of this factor is increased in atherosclerosis (10, 33). We now extend our studies to examine the effects of rabbit PMN-derived products on the microvasculature of the rabbit heart.

A common consequence of coronary vessel atherosclerosis is myocardial ischemia that results in an inflammatory response leading to infiltration of PMN on reperfusion of the tissue. These PMN are associated with damage to the myocardium and the coronary vessels through release of radical oxygen species, arachidonic acid metabolites, platelet-activating factor (PAF), and lysosomal enzymes (13). They also decrease perfusion of the coronary microvasculature by plugging capillaries (5), and this has been suggested to be the cause of the "no-reflow" phenomenon (4). Because several reports show that PMN have the ability to release contractile mediators and there is evidence that PMN accumulate under the conditions of myocardial ischemia, it is possible that PMN-derived vasoconstrictor factor(s) may exacerbate the impaired perfusion of the myocardium by releasing contractile mediators.

The aim of this study was to examine the mechanism of action of a vasoconstrictor factor that is released by PMN in the rabbit isolated coronary circulation, with a view to elucidating the identity of this factor.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

General Procedures

New Zealand White rabbits of either sex (2.4 ± 0.1 kg, n = 37) were anesthetized by intravenous administration of Saffan (0.9% alfaxolone and 0.3% alfadolone). The carotid artery was cannulated, and blood was collected into a syringe containing sodium citrate (3.8% wt/vol, final concn). When all the blood was removed (~100 ml) the thorax was opened, and the heart was removed and placed in Krebs-bicarbonate solution (composition in mM: 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4 · 7H2O, 5.0 D-glucose, 25.0 NaHCO3, and 2.5 CaCl2 · 2H2O).

Isolation of Rabbit PMN

PMN were isolated from whole blood under sterile conditions using cell culture grade, endotoxin-tested reagents and solutions. Briefly, blood was taken from the rabbits and the plasma was removed by centrifugation (15 min, 150 g). Most of the erythrocytes were then removed by sedimentation with Dextran T500 (5 ml, 6% wt/vol). The leukocyte-rich top layer was then removed and centrifuged. The resulting pellet was resuspended in 10 ml of a 4:1 mixture of isotonic saline:platelet-poor plasma and then underlayered with 3 ml of Lymphoprep solution and centrifuged again to remove the mononuclear cells. Remaining erythrocytes were lysed by resuspending the pellet in HEPES buffer containing isotonic ammonium chloride (composition 10 mM HEPES and 150 mM NH4Cl, pH 7.4) for 10 min. After erythrocyte lysis the PMN were sedimented (5 min, 1,000 g) and washed twice in HEPES-buffered Tyrode solution [composition in mM: 10 HEPES, 137 NaCl, 11.9 NaHCO3, 0.4 NaH2PO4, 2.7 KCl, 0.26 MgCl2, and 11 D(+)-glucose and 0.25% (wt/vol) BSA, pH 7.4]. Samples of some PMN suspensions were stained with gentian violet and trypan blue for counting and assessment of viability, respectively. In some experiments a further sample was applied to a microscope slide using a cytospin centrifuge. These slides were fixed with ethanol and then stained with Giemsa stain, and the PMN cell types were counted. The PMN were resuspended at a final concentration of 5 × 107 cells/ml in Tyrode solution and incubated for 30 min. After the incubation period the supernatant of the PMN suspension, containing products released by PMN, was collected by centrifugation (5 min, 1,000 g). This supernatant was either used immediately or stored for up to 4 mo at -20°C.

Assays to Assess Activation Status of PMN

Myeloperoxidase assay. Myeloperoxidase (MPO) was assayed as an indication of the extent of degranulation the PMN had undergone. The assay used was based on the method of Suzuki et al. (36). The reaction mixture consisted of 80 mM sodium phosphate buffer (pH 5.4), 1.6 mM 3,3',5,5'-tetramethylbenzidine (TMB), and 0.3 mM H2O2 in 200 µl of phosphate-buffered saline and 20 µl of Tyrode solution containing 0.25% BSA. This solution was incubated for 2 min at 37°C, and then the reaction was started by adding 30 µl of the PMN supernatant for the test samples or 30 µl of Tyrode solution for the blank. The reaction mixture was incubated for 3 min at 37°C and then stopped by adding 1.75 ml of 200 mM sodium acetate buffer (pH 3.0), and the solution was placed on ice. The absorbance of the samples was read at 655 nm within 10 min of the addition of the sodium acetate. Units of activity of MPO are expressed as the change (Delta ) in absorbance (A) per 106 PMN per minute.

Superoxide anion assay. The production of superoxide anions by PMN was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c. The cell suspension (500 µl, 4 × 106 cells/ml) was mixed with 500 µl of cytochrome c (2 mg/ml in Tyrode solution) and incubated at 37°C for 5 min. The cells were then removed from the solution by centrifugation, and the absorbance was read, to calculate basal superoxide anion release, in a quartz cuvette at 550 nm (A550). The spectrophotometer (Ultraspec 2000, Pharmacia Biotech) was zeroed against a reaction mixture containing the cell suspension and cytochrome c (as above) and 30 U SOD. A reaction mixture of cell suspension and cytochrome c (as above) with 1 µM N-formylmethionyl-leucyl-phenylalanine (FMLP) was used as a positive control. With this method the amount of superoxide generated per milliliter can be calculated by [superoxide anions] (nmol) = 47.7 × A550, because the absorbance peak at 550 nm is caused by superoxide-dependent cytochrome c reduction (15). Results are expressed as nanomoles per 106 cells per 5 min.

Isolated Langendorff-Perfused Rabbit Heart Preparation

The heart was dissected free from any extraneous tissue, cannulated at the base of the aortic arch, and connected to a roller pump (Minipuls 3, Gilson). The heart was retrogradely perfused with Krebs-bicarbonate solution (composition described in General Procedures) at a constant rate to give a perfusion pressure (PP) of ~50 mmHg. The nonrecirculating solution was pumped through a glass condenser to deliver the solution at 37°C, and the preparation was surrounded by a heated water jacket to maintain this temperature. A hook was placed through the apex of the spontaneously beating heart and connected via a pulley to a force transducer (model FT03, Grass Medical Instruments) to measure the force of contraction and to trigger heart rate measurement. The pressure in the system (PP) was measured via a pressure transducer (model CDX-111, Cobe). PP, developed tension, and rate of contraction were all recorded on a Maclab recording system (MacLab 4e, Apple Computer). The preparation was allowed to equilibrate for at least 15 min, during which time the passive force was adjusted to 4 g. Drugs were introduced to the heart with a microsyringe via a rubber injection point in the perfusion circuit.

Experimental Protocols

General protocol. The response to the constrictors ANG II (100 pmol) or the thromboxane mimetic 9,11-dideoxy-11alpha ,9alpha -epoxymethanoprostaglandin F2alpha (U-46619, 1 nmol), the endothelium-dependent dilator bradykinin (BK, 1 nmol) and the endothelium-independent dilator sodium nitroprusside (SNP, 100 nmol) were examined at the beginning of each experiment to establish that the microvessels of the preparation were capable of constriction and endothelium-dependent and endothelium-independent dilatation.

Dose-response curves to PMN supernatant. After the confirmation of functional responses to BK, SNP, and ANG II or U-46619, a dose-response curve to a sample of PMN supernatant was derived by randomly adding volumes of 10, 30, 100, and 300 µl.

Effect of specific antagonists on response to PMN supernatant. In experiments in which an inhibitor or antagonist was used, the responses to BK, SNP, ANG II or U-46619, and a 300-µl sample of PMN supernatant were obtained. This protocol was then repeated in the presence of phentolamine (10 µM), amlodipine (10 µM), indomethacin (3 µM), bosentan (10 µM), or hemoglobin (10 µM), which were infused for at least 15 min before and then throughout retesting of the functional responses to the agonists and the PMN supernatant samples. Responses to phenylephrine (100 nmol), U-46619 (1 nmol), arachidonic acid (10 µg), endothelin (100 pmol), and BK (1 nmol) were also tested where appropriate to confirm antagonism of the relevant receptor or inhibition of the target enzyme. Appropriate time control experiments were also carried out.

Experiments to determine whether factor is a protein. Samples of PMN supernatant were split into three groups, a control group, a group subjected to 90°C for 10 min (heated), and a group incubated with glass beads to which the proteolytic enzyme trypsin was attached (166 U/ml for 5 h). After this incubation period, the trypsin beads were then removed by centrifugation so that no trypsin was left in the sample. Each of these samples (300 µl) were then assayed in the isolated heart preparation.

Molecular size estimation using centrifuge filters. PMN supernatant samples were separated by size exclusion through centrifuge filters (Centriprep CP100 filters, Amicon) into fractions containing molecules greater than or less than 100 kDa.

Drugs and Reagents Used

Arachidonic acid, ANG II acetate salt, BK acetate salt, indomethacin, L-phenylephrine hydrochloride, 5-hydroxytryptamine creatinine sulfate, SNP, trypsin bound to DITC glass, U-46619, and bovine hemoglobin were all obtained from Sigma Chemical. Phentolamine mesylate was obtained from Ciba-Geigy, endothelin-1 from Auspep, amlodipine from Pfizer, and bosentan sodium salt from Roche. Hemoglobin, SNP, phentolamine, endothelin, bosentan, phenylephrine, and 5-hydroxytryptamine (5-HT) were dissolved in water and further diluted in Krebs solution. BK and ANG II were dissolved in ethanol, diluted to a stock solution in 0.25 M NaHCO3, and then further diluted in 0.9% NaCl. Arachidonic acid was dissolved in hexane and then diluted in 0.1 M Na2CO3. Indomethacin was dissolved and diluted in 0.1 M Na2CO3, and amlodipine was dissolved in ethanol and diluted in Krebs solution. Saffan was obtained from Pitman-Moore and diluted twofold with sterile 0.9% saline. The chemicals used for the PMN isolation were obtained from the following companies: trisodium citrate, ammonium chloride, and Giemsa stain (BDH Chemicals); Dextran T500 (Pharmacia); Lymphoprep (Nycomed); HEPES (BDH); and Tyrode salts, albumin bovine fraction V, trypan blue, and crystal violet (Sigma Chemical). For the PMN activity assays cytochrome c, FMLP, SOD, cytochalasin B, and TMB were obtained from Sigma Chemical, and H2O2 was purchased as a 30% (wt/vol) solution from BDH Chemicals. Cytochalasin B and FMLP were prepared in dimethyl sulfoxide, SOD in H2O and TMB in N,N-dimethylformamide.

Data Presentation and Statistical Analysis

The results are expressed as means ± SE. PMN product-induced volume-response curves were compared by ANOVA. Responses pre- and postantagonist (or inhibitor) treatment were compared using Student's paired t-test. Statistical significance was accepted when P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Composition and Activation Status of PMN Suspension Used to Obtain PMN Supernatant

The PMN suspension contained 84% neutrophils, 1% basophils, 3% eosinophils, 1% monocytes, and 12% lymphocytes (n = 40). MPO activity in the PMN supernatant was significantly enhanced if the PMN were treated with FMLP and cytochalasin B [MPO activity (Delta A · 106 cells-1 · min-1): control 0.024 ± 0.008, FMLP + cytochalasin B treated 0.184 ± 0.056; n = 4]. Similarly, FMLP-stimulated superoxide anion production was observed both before and after the 30-min incubation period [superoxide anion release (nmol · 106 cells-1 · 5 min-1): preincubation, basal 0.73 ± 0.28, FMLP-stimulated 3.30 ± 0.80; postincubation, basal 0.46 ± 0.06, FMLP-stimulated 2.40 ± 0.76; n = 3]. These results indicate that the cells had not undergone degranulation or activation during the preparation or incubation procedures, and therefore PMN supernatant samples were prepared from quiescent PMN.

Effect of PMN Supernatant on Isolated, Perfused Rabbit Heart

PMN supernatant caused a dose-dependent increase in PP that was not caused by the vehicle (Tyrode solution with 0.25% BSA). A reproduction of an original trace is shown in Fig. 1, and the group data are presented in Fig. 2. The vehicle had no effect on either the force or rate of cardiac contraction; however, the PMN supernatant caused a decrease in force of contraction but no change in heart rate (Fig. 1).


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  		Fig. 1.   Experimental record showing effect of polymorphonuclear leukocyte (PMN) supernatant (10-300 µl) on isolated, perfused rabbit hearts. PMN supernatant causes a dose-dependent constriction of coronary circulation. Response occurs quickly, and baseline perfusion pressure returns to control levels within 5 min. There is no effect of PMN supernatant on heart rate, although developed force is reduced when there are increases in perfusion pressure.


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  		Fig. 2.   PMN supernatant (bullet , n = 16) causes a dose-dependent increase in perfusion pressure; however the vehicle, Tyrode solution (, n = 6), has no effect. * P < 0.05 compared with vehicle, ANOVA. Delta , Change.

Effect of Specific Antagonists on Response to PMN Supernatant

NO-binding agent hemoglobin. The response to 300 µl of PMN supernatant was unaffected by the presence of hemoglobin (10 µM) [Delta PP (mmHg): control 21 ± 7, hemoglobin treated 27 ± 7; n = 6], but the response to BK (100 pmol) was significantly inhibited by this concentration of hemoglobin [Delta PP (% of baseline): control -6.1 ± 0.5, hemoglobin treated -0.3 ± 1.7; n = 3, P < 0.05, Student's paired t-test].

Cyclooxygenase inhibitor indomethacin. The response to 300 µl of PMN supernatant was unaffected by the presence of indomethacin (3 µM) (Delta PP: control 23 ± 5, indomethacin treated 33 ± 13 mmHg; n = 6), but the response to arachidonic acid (10 µg) was significantly enhanced by this concentration of indomethacin (Delta PP: control 5 ± 2, indomethacin treated 15 ± 3 mmHg, n = 6; P < 0.05, Student's paired t-test), because in the presence of cyclooxygenase inhibition the arachidonic acid is metabolized by lipoxygenase to contractile leukotrienes.

Nonselective alpha -adrenoceptor antagonist phentolamine. The response to 300 µl of PMN supernatant was not affected by the presence of phentolamine (10 µM) (Delta PP: control 28 ± 4, phentolamine 48 ± 10 mmHg; n = 8), but the response to the alpha 1-adrenoceptor agonist phenylephrine (100 nmol) was significantly inhibited by this concentration of phentolamine (Delta PP: control 7 ± 4, phentolamine treated 0.3 ± 0.8 mmHg, n = 8; P < 0.05, Student's paired t-test).

Nonselective endothelin antagonist bosentan. The response to 300 µl of PMN supernatant was unaffected by the presence of bosentan (10 µM) (Delta PP: control 15 ± 3, bosentan 10 ± 4 mmHg, n = 7), but the response to endothelin-1 (100 pmol) was significantly inhibited by this concentration of bosentan (Delta PP: control 35 ± 10, bosentan 2 ± 2 mmHg; n = 8, P < 0.01).

Ca2+-channel antagonist amlodipine. The responses to PMN supernatant (300 µl) was significantly inhibited by amlodipine (10 µM) (Fig. 3). The response to U-46619 (1 nmol) was also significantly inhibited by this concentration of amlodipine (Delta PP: control 9 ± 1, amlodipine treated 3 ± 1 mmHg; n = 5, P < 0.05, Student's paired t-test).


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  		Fig. 3.   Increases in perfusion pressure caused by PMN supernatant (300 µl) in absence (control) and presence of L-type Ca2+-channel blocker amlodipine. Response to PMN supernatant (300 µl) is significantly inhibited by amlodipine (10 µM); n = 5. * P < 0.05 compared with control, Student's paired t-test.

Time control experiments. The response to PMN supernatant was not different after a 30-min time period (Delta PP: control 52 ± 17, after 30 min 35 ± 9 mmHg; n = 5). The baseline PP was higher after the time period, but the responses to bradykinin, arachidonic acid, phenylephrine, endothelin, and U-46619 were all unaffected by the 30-min time period (data not shown).

Determination of Chemical Nature and Molecular Size of PMN-Derived Contractile Factor

Both heating (90°C, 10 min) and treating the PMN supernatant with trypsin (166 U/ml, 37°C for 5 h) significantly inhibited the activity of the PMN factor (Fig. 4). Estimation of the molecular size of the protein using centrifuge filters showed that the factor was concentrated in the retentate of the 100-kDa cutoff filter (Fig. 5).


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  		Fig. 4.   Trypsin treatment (166 U/ml for 5 h) and heating PMN supernatant to 90°C for 10 min caused significant reductions in response to 300-µl samples of PMN supernatant; n = 10. * P < 0.05, ** P < 0.001 compared with control, ANOVA and Newman-Keuls.


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  		Fig. 5.   When PMN supernatant sample was separated into fractions by size-exclusion centrifugation, <100 kDa fraction displayed little constrictor effect but >100 kDa fraction caused a constriction; n = 3. * P < 0.05 compared with control, ANOVA and Newman-Keuls.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study describes a PMN-derived constrictor of coronary resistance vessels that is unlike any previously described agent. This protein factor causes a dose-dependent constriction that is not dependent on NO, although it is dependent on Ca2+ influx for contractile activity. The time course of the response is rapid, the constriction being observed immediately after the introduction of the PMN supernatant sample. PP then returned to baseline within a few minutes, and the response was reproducible over the period of the experiment, with no tachyphylaxis being apparent. The dose-dependent constriction caused a maximum increase of 23 ± 2 mmHg in PP in the group included in the dose-response curve, and this response appears to be limited only by the amount of PMN supernatant that could be injected, rather than being an actual maximum response.

The PMN supernatant had no effect on heart rate but did cause a reduction in developed force. It must be noted that the method for measuring developed force was primarily intended to enable the measurement of heart rate, and any direct inotropic effects would be better examined in other preparations. The reduction in developed force observed on the addition of the PMN supernatant is likely to be a result of the constrictor effect of the PMN supernatant, which then reduces perfusion to the muscle, and therefore reduces contraction also rather than being a direct effect on contractility. Such an effect has been named the "garden hose" phenomenon (25). In the present study U-46619 also causes this decrease in developed tension with an increase in PP, even though it is reported that U-46619 has a positive inotropic effect (23), suggesting that the garden hose effect is occurring in this preparation, and therefore whether the PMN supernatant has a direct effect on contractility cannot be confirmed by this study and would be better examined using isolated cardiac muscle where changes in vascular tone would not influence contractility.

Basal PP generally increased throughout the course of each experiment; however, at the levels attained during the experiments this increase was insufficient to cause alterations of the response to the PMN supernatant. Experiments were designed to be completed within 90 min to minimize the effect of the increase in basal PP, which is most likely to be caused by perivascular edema (4). This results in the compression of the vascular bed that increases resistance to flow and results in increased PP.

The response to the PMN products in the coronary microcirculation was not dependent on NO, as determined by the experiment in which hemoglobin was infused to bind and remove NO. To further investigate this, in some experiments NG-nitro-L-arginine (L-NNA) was infused into the isolated heart preparation in an attempt to inhibit nitric oxide synthase. However, the addition of L-NNA caused such a dramatic increase in PP that the experiments could not be completed (data not shown). However, the dose of hemoglobin used was able to inhibit endothelium-dependent responses to BK, indicating that the actions of NO were prevented by this treatment. The non-NO-dependent contractile mechanism in this preparation is in contrast to the results from previous studies from our laboratory (32), in which the PMN-derived products caused contraction of rabbit isolated thoracic aorta by interfering with basal NO, indicating that the mediator responsible for the results obtained the present study is likely to be different from the PMN-derived mediator responsible for contractile effects in the large artery.

PMN produce cyclooxygenase metabolites (16); however, in situ production of cyclooxygenase metabolites cannot account for the response because it was not inhibited by indomethacin, although the possibility that the factor is a cyclooxygenase metabolite that was formed during the PMN incubation period remains to be determined. The factor is not acting through alpha -adrenoceptors, which are known to mediate constriction of blood vessels, because the response was not inhibited by phentolamine, an antagonist at both alpha 1- and alpha 2-adrenoceptors. There is evidence that PMN can convert big endothelin to endothelin-1 (27, 28, 38), and this was a possible mechanism of contraction. However, because the nonselective endothelin antagonist bosentan had no effect on the response to the PMN supernatant but markedly reduced the response to endothelin-1, this is not the case. The only inhibitor used in this study that did reduce the response to the PMN supernatant was the L-type Ca2+-channel blocker amlodipine, indicating a Ca2+-dependent mechanism of action. This may mean that the PMN factor is acting directly on the Ca2+ channel to increase Ca2+ entry or that the Ca2+-channel opening is a secondary effect. Further investigation is required to ascertain the exact effect of the PMN factor on the Ca2+ channel and whether this effect is confined to the L-type Ca2+ channel.

Leukotrienes, particularly LTD4, are known to be the mediators of increased PP in several studies investigating the effects of isolated PMN in the isolated, perfused rabbit heart (1, 24). In those studies PMN participate in the production of peptidoleukotrienes by transcellular synthesis; however, in the present study the PMN cells were not present so it was not possible for true transcellular synthesis to occur. In addition, results obtained in this study indicate that the active factor is a protein, suggesting that the mediator is unlikely to be a leukotriene.

Another possible mediator of the constrictor effect is PAF, which is produced by PMN and can cause coronary vasoconstriction (11). PAF is reported to cause responses via the synthesis of leukotrienes and prostaglandins (12, 35), and, although we were not successful in blocking responses to leukotrienes, indomethacin had no effect on the response, suggesting that PAF-induced prostaglandin production in situ is not a contributing factor in our study. PAF has been reported to cause dilator and constrictor effects that are species dependent (11). In rabbit hearts 1 nmol PAF caused an increase in PP, which disappeared with repeated administration, along with a negative inotropic effect (37). PAF has also been reported to decrease cardiac contractility, prolong the P-R interval in the electrocardiogram, and decrease heart rate (21), whereas in our study there did not appear to be any tachyphylaxis or effect on heart rate, suggesting that PAF is not the mediator involved. In addition, the data indicate that the responsible mediator is a large protein, which is inconsistent with it being PAF.

Several reports of vasoconstriction in isolated, perfused hearts do suggest that reactive oxygen species are mediators of constriction, particularly OH-, because thiourea (26) and dimethylurea (7) attenuate the decrease in PP. The superoxide anion is also implicated in some studies, because SOD can attenuate the constrictor effects (7); however, SOD has little effect on constrictor responses in other studies (6). We did not test the response to PMN supernatant in the presence of oxygen radical scavengers, because it is unlikely that reactive oxygen species could survive the storage procedures. However, this does not rule out the possibility that reactive oxygen species are being generated in situ by a factor from the PMN supernatant.

There are some residual platelets (46 ± 9 × 106/ml) in the PMN suspension from which the PMN supernatant samples were prepared. These platelets may have released factors such as 5-HT and thromboxane A2, although it is unlikely that the number of platelets present could release large amounts of these substances (18). 5-HT cannot account for the responses seen, because exogenous 5-HT causes a biphasic change in PP with a concurrent biphasic response in heart rate (data not shown). Thromboxane A2 cannot be ruled out as a possible mediator at this stage, although it is quite unstable (half-life = 3 min) and unlikely to survive storage at -20°C (3), and our current data strongly suggest that the active factor is a protein.

The active factor is sensitive to heat treatment and trypsin treatment. The data also show that the PMN factor is concentrated in the retentate of the Centriprep 100 filter (>100-kDa fraction), suggesting that the molecule responsible is large or the active factor is bound to, or part of, a large molecule. These data suggest that the active factor is a protein, although further work remains to elucidate the biochemical properties and identity of the factor.

In contrast to all of the other literature reports, we describe a constrictor response to a stable factor that was released from PMN during their incubation period. This factor is stable, because it can be stored in the freezer for several months without losing activity. In this study the presence of PMN in the heart preparation was not necessary to produce a response, unlike other studies in which PMN were a requirement for the response to be produced. In addition, in many cases, PMN also had to be activated to produce the response (7, 22, 24, 30). In our study PMN were not added to the perfusate; rather, the medium from a suspension of PMN containing stable PMN products was used, and care was taken not to cause activation or degranulation of the cells. This indicates that PMN passively release a stable factor that causes vasoconstriction. The effect of activation on the factor release was not investigated in this study but would be of interest because previous studies showed that stimulation of PMN enhances vasoactive factor release (9, 31).

The finding that PMN passively release a factor that causes vasoconstriction of the coronary circulation suggests that this factor may have a role in the maintenance of vessel tone under normal physiological conditions. Under pathological conditions, such as the situation where PMN are recruited during reperfusion after ischemia, a constrictor factor such as this may play a role in the impaired perfusion known as the no-reflow phenomenon. This factor may also participate in the decreased vasodilator reserve, which has been also been documented in reperfusion injury (34), by promoting vasoconstriction. The Ca2+ antagonist amlodipine has been reported to accelerate recovery of both mechanical myocardial function and coronary blood flow in the ischemic myocardium (2). The results presented in this study suggest that part of the beneficial effects of amlodipine may involve the reduction of vasoconstriction mediated by PMN-derived factor(s).

In conclusion, the data presented here indicate that quiescent PMN release a factor into their incubation medium, which causes constriction of coronary resistance vessels. The active factor appears to be a protein, and the constrictor response could only be attenuated by the calcium antagonist amlodipine. At present, further work is required to elucidate the mechanism of action and identity of this mediator.

    ACKNOWLEDGEMENTS

The authors thank Vitina Sozzi for expert technical assistance and Roche for supplying the endothelin-receptor antagonist bosentan.

    FOOTNOTES

Address for reprint requests: O. L. Woodman, Dept. of Pharmacology, Univ. of Melbourne, Parkville, Victoria 3052, Australia.

Received 16 December 1997; accepted in final form 22 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Buccellati, C., G. Rossoni, A. Bonazzi, F. Berti, J. Maclouf, G. Folco, and A. Sala. Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart. Br. J. Pharmacol. 120: 1128-1134, 1997[Medline].

2.  Burges, R. A. The pharmacological profile of amlodipine in relation to ischaemic heart disease. Postgrad. Med. J. 67, Suppl.: S9-15, 1991.

3.   Campbell, W. B., and P. V. Halushka. Lipid-derived autacoids: eicosanoids and platelet-activating factor. In: Goodman and Gilman's The Pharmacological Basis of Therapeutics, edited by J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon, and A. Goodman Gilman. New York: McGraw-Hill, 1996, p. 601-616.

4.   Engler, R. L., M. D. Dahlgren, M. A. Peterson, A. Dobbs, and G. Schmid-Schönbein. Accumulation of polymorphonuclear leukocytes during experimental myocardial ischemia. Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H93-H100, 1986[Abstract/Free Full Text].

5.   Engler, R. L., G. W. Schmid-Schönbein, and R. S. Pavelec. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am. J. Pathol. 111: 98-111, 1983[Abstract].

6.   Esser, E., and G. Loschen. Leukocyte superoxide and cardiac dysfunctions in isolated perfused rat hearts. Arch. Toxicol. 65: 361-365, 1991[Medline].

7.   Gillespie, M. N., S. Kojima, M. Kunitomo, and M. Jay. Coronary and myocardial effects of activated neutrophils in perfused rabbit hearts. J. Pharmacol. Exp. Ther. 239: 836-840, 1986[Abstract/Free Full Text].

8.   Gonzales, J., J. L. Mehta, D. L. Lawson, W. W. Nichols, and F. A. Nicolini. Variable effects of human and canine polymorphonuclear leukocytes on vascular smooth muscle tone. Cardiovasc. Res. 26: 751-756, 1992[Abstract/Free Full Text].

9.   Hamilton, J. R., J. L. Hart, and O. L. Woodman. Degranulation enhances release of a stable contractile factor from rabbit polymorphonuclear leukocytes. Am. J. Physiol. 274 (Heart Circ. Physiol. 43): H1545-H1551, 1998[Abstract/Free Full Text].

10.   Hart, J. L., C. G. Sobey, and O. L. Woodman. Cholesterol feeding enhances the vasoconstrictor effect of products from rabbit polymorphonuclear leukocytes. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1-H6, 1995[Abstract/Free Full Text].

11.   Hu, W. M., P. C. Choy, and R. Y. Man. Characterisation of the coronary vascular responses to platelet-activating factor in the isolated perfused heart. Lipids 26: 700-704, 1991[Medline].

12.   Hu, W. M., and R. Y. Man. Interaction of vasoactive substances released by platelet-activating factor in the rat perfused heart. Br. J. Pharmacol. 104: 933-937, 1991[Medline].

13.   Lucchesi, B. R. Leukocytes and ischaemia-induced myocardial injury. Annu. Rev. Pharmacol. Toxicol. 26: 201-224, 1986[Medline].

14.   Ma, X., P. S. Tsao, G. F. Viehman, and A. M. Lefer. Neutrophil-mediated vasoconstriction and endothelial dysfunction in low-flow perfusion-reperfused cat coronary artery. Circ. Res. 69: 95-106, 1991[Abstract/Free Full Text].

15.   Markert, M., P. Andrews, and B. Babior. Measurement of O-2 production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. In: Methods in Enzymology, edited by L. Packer. New York: Academic, 1984, p. 358-369.

16.   Mehta, J., P. Mehta, D. L. Lawson, N. Ostrowski, and L. Brigmon. Influence of selective thromboxane synthetase blocker CGS-13080 on thromboxane and prostacyclin biosynthesis in whole blood: evidence for synthesis of prostacyclin by leukocytes from platelet-derived endoperoxides. J. Lab. Clin. Med. 106: 246-252, 1985[Medline].

17.   Mehta, J. L., D. L. Lawson, W. W. Nichols, and P. Mehta. Modulation of vascular tone by neutrophils: dependence on endothelial integrity. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1315-H1320, 1989[Abstract/Free Full Text].

18.   Meyers, K. M., H. Holmsen, and C. L. Seachford. Comparative study of platelet dense granule constituents. Am. J. Physiol. 243 (Regulatory Integrative Comp. Physiol. 12): R454-R461, 1982.

19.   Nishida, M., T. Kuzuya, S. Hoshida, Y. Kim, A. Kitabatake, T. Kamada, and M. Tada. Polymorphonuclear leukocytes induced vasoconstriction in isolated canine coronary arteries. Circ. Res. 66: 253-258, 1990[Abstract/Free Full Text].

20.   Ohlstein, E. H., and A. J. Nichols. Rabbit polymorphonuclear neutrophils elicit endothelium-dependent contraction in vascular smooth muscle. Circ. Res. 65: 917-924, 1989[Abstract/Free Full Text].

21.   Pugsley, M. K., H. Salari, and M. J. Walker. Actions of platelet-activating factor on isolated rat hearts. Circ. Shock 35: 207-214, 1991[Medline].

22.   Rossoni, G., A. Sala, C. Buccellati, J. Maclouf, G. C. Folco, and F. Berti. Vasoconstriction to polymorphonuclear leukocytes in the isolated, perfused rabbit heart: inhibition by prostacyclin mimetics. J. Cardiovasc. Pharmacol. 27: 680-685, 1996[Medline].

23.   Sakuma, I., S. S. Gross, and R. Levi. Positive inotropic effect of the thromboxane analog U46619 on guinea pig left atrium: mediation by specific receptors and association with increased phosphoinositide turnover. Can. J. Physiol. Pharmacol. 67: 943-949, 1989[Medline].

24.   Sala, A., G. Rossoni, C. Buccellati, F. Berti, G. Folco, and J. Maclouf. Formation of sulphidopeptide-leukotrienes by cell-cell interaction causes coronary vasoconstriction in isolated, cell-perfused heart of rabbit. Br. J. Pharmacol. 110: 1206-1212, 1993[Medline].

25.   Schrör, K., and T. Hohlfeld. Inotropic actions of eicosanoids. Basic Res. Cardiol. 87: 2-11, 1992[Medline].

26.   Semb, A. G., K. Ytrehus, J. Vaage, R. Myklebust, and O. D. Mjos. Functional impairment in isolated rat hearts by activated leukocytes: protective effect of oxygen free radical scavengers. J. Mol. Cell. Cardiol. 21: 877-887, 1989[Medline].

27.   Sessa, W. C., S. Kaw, M. Hecker, and J. R. Vane. The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes. Biochem. Biophys. Res. Commun. 174: 613-618, 1991[Medline].

28.  Sessa, W. C., S. Kaw, A. Zembowicz, E. Anggard, M. Hecker, and J. R. Vane. Human polymorphonuclear leukocytes generate and degrade endothelin-1 by two distinct neutral proteases. J. Cardiovasc. Pharmacol. 17, Suppl. 7: S34-S38, 1991.

29.   Sessa, W. C., and K. M. Mullane. Release of a neutrophil-derived vasoconstrictor agent which augments platelet-induced contractions of blood vessels in vitro. Br. J. Pharmacol. 99: 553-559, 1990[Medline].

30.   Shandelya, S. M., P. Kuppusamy, M. L. Weisfeldt, and J. L. Zweier. Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Evidence for plasma-mediated leukocyte activation. Circulation 87: 536-546, 1993[Abstract/Free Full Text].

31.   Sobey, C. G., G. J. Dusting, and A. G. Stewart. Tumour necrosis factor alpha  augments the release of an endothelium-dependent vasoconstrictor from human polymorphonuclear leukocytes. J. Cardiovasc. Pharmacol. 20: 813-819, 1992[Medline].

32.   Sobey, C. G., G. J. Dusting, A. G. Stewart, and O. L. Woodman. Rabbit polymorphonuclear leukocytes cause endothelium-dependent contraction of rabbit aorta. J. Vasc. Med. Biol. 2: 107-115, 1990.

33.   Sobey, C. G., J. L. Hart, and O. L. Woodman. Vasoconstrictor responses to polymorphonuclear leukocytes from atherosclerotic rabbits. Clin. Exp. Pharmacol. Physiol. 21: 153-156, 1994[Medline].

34.   Sobey, C. G., and O. L. Woodman. Myocardial ischaemia: what happens to the coronary arteries? Trends Pharmacol. Sci. 14: 448-453, 1993[Medline].

35.   Stewart, A. G., P. N. Dubbin, T. Harris, and G. J. Dusting. Platelet-activating factor may act as a second messenger in the release of eicosanoids and superoxide anions from leukocytes and endothelial cells. Proc. Natl. Acad. Sci. USA 87: 3215-3219, 1990[Abstract/Free Full Text].

36.   Suzuki, K., H. Ota, S. Sasagawa, T. Sakatani, and T. Fujikura. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal. Biochem. 132: 345-352, 1983[Medline].

37.   Tippins, J. R., J. W. Antoniw, M. R. Alison, B. Garvey, and A. Maseri. WEB 2086 inhibits neutrophil dependent increases in coronary resistance in blood perfused rabbit heart. Cardiovasc. Res. 26: 162-169, 1992[Abstract/Free Full Text].

38.   Uprichard, A. C. G., L. G. Chi, and B. R. Lucchesi. Functional consequence of big endothelin conversion: demonstration with isolated neutrophils, but not in a postinfarction model. Pharmacology 47: 277-285, 1993[Medline].

Am J Physiol Heart Circ Physiol 275(4):H1322-H1328
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society



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