Vol. 83, Issue 5, 1617-1622, 1997

Flow-induced responses in cat isolated pulmonary arteries

Larissa A. Shimoda¹, Nan A. Norins², and Jane A. Madden^3,4

¹ Department of Biomedical Engineering, Marquette University, Milwaukee 53233; ² Departments of Pediatrics and ³ Neurology, The Medical College of Wisconsin, Milwaukee 53226; and ⁴ Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Shimoda, Larissa A., Nan A. Norins, and Jane A. Madden. Flow-induced responses in cat isolated pulmonary arteries. J. Appl. Physiol. 83(5): 1617-1622, 1997.Isolated, cannulated, endothelium-intact cat pulmonary arteries, averaging 692 ± 104 µm in diameter, were set at a transmural pressure of 10 mmHg and monitored with a video system. Intraluminal flow was increased in steps from 0 to 1.6 ml/min by using a syringe pump. An electronic system held pressure constant by changing outflow resistance. Flow-diameter curves were generated in physiological saline solution. At constant transmural pressure, the arteries constricted in response to increased intraluminal flow. Constriction was not affected by removing extracellular Ca²⁺ but was abolished after treatment with ryanodine to deplete intracellular Ca²⁺ stores, with the endothelin-1 synthesis inhibitor phosphoramidon, with the endothelin A-receptor antagonist BQ-123, with the protein kinase C inhibitor staurosporine, or with glutaraldehyde to reduce endothelial cell deformability. The results indicate that isolated pulmonary arteries can constrict in response to intraluminal flow and suggest that constriction is mediated by endothelin-1 and depends on intracellular Ca²⁺ release and protein kinase C activation.

endothelin-1; BQ-123; shear stress

INTRODUCTION

IN THE SYSTEMIC CIRCULATION, changes in the mechanical forces exerted on the blood vessel wall can actively influence vascular tone either through the release of endothelium-derived factors (5, 6, 16) or by direct activation of smooth muscle cells (11). In contrast, in the pulmonary circulation, increased pulmonary arterial pressure or blood flow is normally accompanied by a decrease in pulmonary vascular resistance due to passive distension and/or recruitment (1, 9, 23, 24). However, active pulmonary vasomotor responses have been evoked by changes in transmural pressure (17, 20, 26). These responses may have important implications for the normal and pathophysiological functions of the pulmonary vasculature.

Although the vasoactive effect of luminal flow has been studied in vessels from several vascular beds, including the coronary (18), cerebral (11, 25), mesentery (28), skeletal (14), and femoral (27), it has not been investigated in pulmonary arteries. The purpose of this study was to determine the responses of isolated cannulated small-diameter pulmonary arteries to changes in luminal flow at constant transmural pressure. In arteries from other organs, reports vary as to whether flow-induced responses depend on the endothelium and intracellular and/or extracellular Ca²⁺. Therefore, we also investigated some of the possible endothelium-mediated and signal-transduction pathways by which flow might modulate pulmonary vascular tone. These studies were conducted by using a system that controls both transmural pressure and intraluminal flow independently in isolated cannulated vessels (25). Thus relevant forces can be controlled, and arterial reactivity is not influenced by the state of the surrounding tissue.

METHODS

Vessel preparation. This study was approved by the Animal Care and Use Committee of the Zablocki Veterans Affairs Medical Center. Fourteen adult mongrel cats (2.5-4.0 kg) of either sex were premedicated with ketamine hydrochloride (15 mg/kg) and anesthetized with pentobarbital sodium (30 mg/kg ip). The animals were exsanguinated by severing the carotid artery, the thorax was opened, and the lungs were removed. Thirty-three intrapulmonary artery segments averaging 692 ± 104 µm outside unstressed diameter were dissected from the apical portions of both lung lobes. Arteries were identified by wall thickness and orientation to the bronchioles. The dissected arteries were placed in cold (4°C) physiological saline solution (PSS) until use.

The system used to study cannulated arteries consisted of a water-jacketed plastic chamber in which proximal (inflow) and distal (outflow) cannulas are mounted. The cannulas were glass micropipettes tapered with a pipette puller (Brown-Flaming P-77, Sutter Instrument), with the tips cut so that the inflow and outflow cannula-tip diameters were equal.

An arterial segment was tied in place on the proximal cannula with 22-µm nylon suture, and the lumen was flushed with PSS. The distal end of the artery was then tied onto the distal cannula. The exterior of the vessel was suffused with PSS from a reservoir at 37°C and aerated with a gas mixture containing O₂, CO₂, and N₂, giving a PO₂ of 130-150 Torr, PCO₂ of 37-40 Torr, and pH 7.37. The artery was filled with PSS aerated with the same gas mixture as the reservoir, and all side branches were tied off. A micrometer connected to the proximal cannula was used to take out the slack in the artery. The artery was then pressurized to 10 mmHg and allowed to stabilize for 60-90 min without flow before study.

Flow control system. The system used to study flow effects in isolated cannulated blood vessels has been described previously (25). Briefly, a syringe pump (Harvard model 976) connected to the inflow cannula can be set to a constant flow. A pulse damper is used to minimize oscillations in flow caused by the syringe pump. An electronically driven micromanipulator (Oriel A18008 Encoder Mike, Oriel, Stratford, CT) is incorporated into a specially designed feedback-control circuit. The micromanipulator is used to adjust the resistance of the tubing connected to the outflow cannula. The motion of the micromanipulator is determined by the difference between the pressure measured by the inflow and outflow pressure transducers and the pressure set by the user. Pressure and flow are independently controlled: the flow by the syringe pump and the pressure by the micromanipulator servomechanism.

The accuracy of the pressure measured by the inflow and outflow transducers and displayed on the controller as the mean value was verified by measuring the actual luminal pressure. A 5- to 10-µm-diameter, beveled-tip micropipette was inserted through the artery wall, and the luminal pressure was measured by using a micropressure measuring system (Instruments for Physiology and Medicine, model 5A, San Diego, CA). In the five arteries in which this was done, the transmural pressure value displayed on the controller agreed within ±1 mmHg of the luminal pressure recorded with the micropuncture system. Step changes in flow caused <5 mmHg transient change in transmural pressure (measured on the controller and with the micropipette), which stabilized within 10 ± 0.4 s.

Vessel diameter measurements. A color video camera (Panasonic Digital 5000) mounted on a stereomicroscope (Olympus SZ-STB1) above the vessel chamber projected the artery image on a video monitor (Sony PVM-1390), and the external arterial diameter (±1.5 µm) was measured by using a video scaler (FORA IV-550). The external diameter was always measured at the same point on the arterial wall, as judged by the presence of various distinguishing features such as adhering connective tissue or side branches located near the site. The video image of the artery could also be recorded on a videocassette recorder (Panasonic AG-1730) for later analysis or review. Diameters were measured immediately after mounting the artery, after equilibration, and throughout the protocols described below.

Experimental protocols. All arteries were tested for viability by measuring the contractile response induced by 30 mM KCl. The vessels were tested for a functionally intact endothelium by adding 10⁶ M norepinephrine (NE) followed by 10⁶ M acetylcholine (ACh) at the peak of the NE-induced constriction. Arteries that did not contract by at least 20% to KCl and/or dilate to ACh were discarded.

The effects of flows from 0.108 to 1.6 ml/min on arterial diameter were studied while transmural pressure was held constant at 10 mmHg. This range of flows was chosen to encompass values reported in the literature for similarly sized in situ pulmonary arteries at resting cardiac output (0.3-0.5 ml/min; Refs. 15, 24). Flow was maintained for 3 min after each step change, at which time the external diameter measurement was stable.

The following protocols were performed to determine whether the endothelium-derived constricting factor endothelin-1 (ET-1) was participating in the flow-induced response. Flow vs. diameter (F/D) curves were performed before and after exposure to the ET-1 synthesis inhibitor phosphoramidon (10⁵ M) and the endothelin A (ET_A) -receptor antagonist BQ-123 (10⁶ M). The inhibitory effect of BQ-123 was verified by comparing the response to exogenous ET-1 (10⁹ M) in the absence and presence of BQ-123. F/D curves were also performed before and after treating the arteries with the protein kinase C (PKC) inhibitor staurosporine (10⁹ M) after both the exterior and the luminal PSS were replaced with a Ca²⁺-free solution and after sarcoplasmic reticulum Ca²⁺ stores were depleted with ryanodine (RYN; 10⁶ M). This dose of RYN has been shown to abolish caffeine-induced increases in intracellular Ca²⁺ in vascular smooth muscle (29).

Endothelial cell deformation was restricted by perfusing the vessels with a 0.025% glutaraldehyde (GLA) solution for 30 s, as described by Mel'kumyants et al. (22) and as used previously in cerebral arteries (25). The arteries were retested with KCl and ACh before the F/D curves were repeated to ensure that the GLA perfusion had not altered the ability of the artery to respond to these agents.

Drugs and solutions. The composition of the PSS (in mM) was 141 Na⁺, 4.7 K⁺, 2.5 Ca²⁺, 0.72 Mg²⁺, 124 Cl, 1.7 H₂PO₄, 22.5 HCO₃, and 11 glucose. Ca²⁺-free PSS contained (in mM): 140 Na⁺, 4.7 K⁺, 117 Cl, 21.2 Mg²⁺, 24 HCO₃, 1 H₂PO₄, 1.17 SO²₄, 10 glucose, and 2 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid. ACh, GLA, NE, phosphoramidon, RYN, and staurosporine were obtained from Sigma Chemical (St. Louis, MO). BQ-123 and ET-1 were obtained from American Peptides (Sunnyvale, CA). All drugs were prepared fresh for each experiment.

Statistical analysis. All diameter measurement values are means ± SE, expressed as percent original diameter. Original diameter is defined as the stable diameter at zero flow either under control conditions or after an intervention. To determine the differences between groups, Student's paired t-test or analysis of variance with repeated measures and Fisher's least squares difference test were used as appropriate. A value of P < 0.05 was considered statistically significant.

RESULTS

During the preconditioning period, the arteries developed spontaneous tone so that at the end of the period their diameters were 93.8 ± 1.40% of their diameters at mounting. The average contractile response to 30 mM KCl was 27.8 ± 2.10% (P < 0.05). In response to NE, artery diameter decreased 8.20 ± 1.20% (P < 0.05). When ACh was applied at the peak of the NE-induced contraction, the arteries dilated to 103.6 ± 1.80% of their preconstricted diameter (P < 0.05), thus demonstrating the presence of a functional endothelium.

At constant transmural pressure, artery diameter decreased as the flow was increased (Fig. 1). The maximum decrease of 10.6% occurred at the highest flow (1.6 ml/min). Wall shear stress () ( = 4 µ / r³, where µ is viscosity in poise, is flow in ml/s, and r is artery radius in cm) ranged from 0 to 13.8 dyn/cm².

The ET-1 synthesis inhibitor phosphoramidon did not affect basal arterial diameter, but when the arteries were exposed to flow, they no longer exhibited a constrictor response (Fig. 2). In the absence of flow, arteries exposed to ET-1 constricted significantly (Fig. 3). The ET_A-receptor antagonist BQ-123 did not affect basal arterial diameter but it completely blocked the contractile response to ET-1 (Fig. 3). Arteries treated with BQ-123 and exposed to increasing flow showed no change in diameter (Fig. 4A). A similar lack of response to flow was seen in arteries treated with the PKC inhibitor staurosporine (Fig. 4B).

5

9

6

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9

Exposure to Ca²⁺-free solution did not significantly alter resting artery diameter, and the constrictor response to increased intraluminal flow was slightly but not significantly attenuated (Fig. 5A). Also treatment of the arteries with RYN did not produce any change in resting diameter. However, the RYN-treated arteries did not constrict as flow was increased (Fig. 5B).

6

Arteries perfused with GLA retained the ability to constrict to KCl and NE and dilate to ACh, and these responses were not significantly different from those under control conditions (Fig. 6). However, when the treated arteries were exposed to flow, their diameter remained constant (Fig. 7).

DISCUSSION

The major finding of this study was that at constant transmural pressure small-diameter pulmonary arteries isolated from cat lungs constricted as intraluminal flow increased. This flow-induced constriction may be mediated at least in part by ET-1, since it was abolished by treating the arteries with the ET-1 synthesis inhibitor phosphoramidon, the ET_A-receptor blocker BQ-123, or the PKC inhibitor staurosporine and also by depleting intracellular Ca²⁺ stores.

The finding that the isolated pulmonary arteries constricted as flow increased was unexpected, since increasing pulmonary blood flow in situ often results in decreased pulmonary vascular resistance (1, 9, 23, 24). It is conceivable that the flow-induced constriction observed in the constant-transmural-pressure preparation is normally masked in the intact pulmonary vasculature by the increase in pressure that accompanies increased flow. However, pulmonary arteries may respond differently to mechanical stimuli when removed from their natural surroundings. This hypothesis is supported by a recent study by Madden et al. (20) showing that small-diameter isolated pulmonary arteries constricted in response to increased pressure, but similarly sized in situ arteries did not. Factors that might contribute to differences in response between in vitro and in situ pulmonary arteries are not known. However, if the responses revealed in the in vitro studies were realized in situ, activation of normally quiescent mechanisms might contribute to the pathogenesis of pulmonary hypertension.

Flow-induced constriction has been reported in a number of other vessel types, including cerebral (11, 25), femoral (27) and ear (3) arteries, and facial vein (12), but the mechanisms by which this response occurs are not fully understood. It has been suggested that deformation of the vascular endothelial cell layer catalyzes the production of endothelial factors (5, 7, 10, 16), and these factors contribute to the various types of flow-induced responses reported (14, 18, 22, 25). In particular, we hypothesized that the flow-induced constriction we observed might be mediated by the endothelium-derived constricting factor ET-1. The results of the present study showing that the inhibition of the ET-1 synthesis blocked flow-induced constriction suggest that, in isolated cat pulmonary arteries, increased shear stresses due to increased flow stimulate an ET-1-mediated signal-transduction pathway. This is consistent with the finding by Kuchan and Frangos (16) that cultured endothelial cells release ET-1 when exposed to shear stresses similar to those encountered in our study. Furthermore, in the present study, the absence of flow-induced constriction in arteries treated with GLA to decrease endothelial cell deformability is consistent with the hypothesis that pulmonary arteries respond to mechanical stimuli and, more specifically, contract in response to increased shear stress. The lack of a contractile response to flow after GLA perfusion does not appear attributable to smooth muscle cell damage or to changes in endothelial cell chemical reactivity, since responses to KCl, NE, and ACh were not diminished in the GLA-treated arteries. Thus the results are consistent with endothelial cell deformation as a necessary step in transducing the mechanical force into a biochemical signal.

In the intact cat lung, infusion of ET-1 increases pulmonary vascular resistance (19), suggesting that ET-1 constricts cat pulmonary arteries. Thus the isolated vessels in the present study responded like in situ vessels. Similar to results obtained in pulmonary arteries from other species (4, 8, 30), BQ-123 completely inhibited the constriction to exogenous ET-1 indicating that, in the cat pulmonary vasculature, ET_A receptors are the primary subtype involved in the ET-1-induced vasoconstriction. In addition, the lack of a flow-induced constriction in BQ-123-treated arteries suggests that activation of ET_A receptors mediates the response.

Both ET-1 synthesis and its activity depend on activation of PKC (7). Staurosporine, which at 1 nM inhibits PKC but has no significant effect on protein kinase A or G, prevented shear stress-induced ET-1 synthesis in cultured endothelial cells (16) and abolished ET-1-induced contraction in intact lungs (2). Our finding that staurosporine inhibited flow-induced contraction in isolated pulmonary arteries is a further indication that endogenous ET-1 participates in this response, although from the present study it cannot be ascertained which part of the ET-1 pathway was inhibited by the staurosporine treatment.

Elevated intracellular Ca²⁺ activates PKC, and the ET-1-induced contraction also requires Ca²⁺ mobilization (7, 13, 16, 21). Reports differ as to the relative roles of intracellular Ca²⁺ release and/or Ca²⁺ influx in ET-1 synthesis and release, and it appears that in some cases both may be involved in the contractile response to ET-1. In pulmonary arteries, Horgan et al. (13) found that ET-1 caused a biphasic contraction. The peak contraction occurred within 1-5 min and depended primarily on Ca²⁺ release from intracellular stores. A smaller contraction at 10 min was sustained primarily through extracellular Ca²⁺ influx. In the present study, we did not observe a second contraction to ET-1 and, although the removal of extracellular Ca²⁺ slightly reduced the flow-induced constriction, it did not abolish it. That flow-induced constriction typically occurred within 3 min after a change in flow, persisted in Ca²⁺-free solution, but was abolished in RYN-treated arteries supports the hypothesis that intracellular Ca²⁺ release is the initiating event in the flow-induced constriction by cat pulmonary arteries.

In summary, isolated cannulated cat pulmonary arteries exhibited flow-induced constriction that may be ET-1 mediated. How this constrictor mechanism fits into normal pulmonary arterial physiology is not clear and it may be overridden by other mechanisms in the intact lung. Further investigation to elucidate differences between in vitro and in situ responses to mechanical stimuli, in particular the mechanism by which flow-induced constriction is unmasked in the isolated vessels, may provide valuable insight into the pathogenesis of pulmonary vascular disease.

ACKNOWLEDGEMENTS

The authors thank P. A. Keller for excellent technical assistance and Christopher A. Dawson for helpful comments.

FOOTNOTES

Address for reprint requests: J. A. Madden, Neurology Research 151, VAMC, Milwaukee, WI 53295 (E-mail: madden.jane{at}milwaukee.va.gov).

Received 22 November 1996; accepted in final form 16 July 1997.

REFERENCES