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HIGHLIGHTED TOPICS
Pulmonary Circulation and Hypoxia
Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
Submitted 2 August 2004 ; accepted in final form 28 September 2004
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ABSTRACT |
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630 Torr) and CH (4 wk, barometric pressure = 380 Torr) rats were loaded with fura 2-AM and perfused with warm (37°C), aerated (21% O2-6% CO2-balance N2) physiological saline solution. Vascular smooth muscle (VSM) intracellular Ca2+ concentration ([Ca2+]i) and diameter responses to increasing intraluminal pressure were determined. Diameter and VSM cell [Ca2+]i responses to KCl were also determined. In a separate set of experiments, VSM cell membrane potential responses to increasing luminal pressure were determined in arteries from control and CH rats. VSM cell membrane potential in arteries from CH animals was depolarized relative to control at each pressure step. VSM cells from both groups exhibited a further depolarization in response to step increases in intraluminal pressure. However, arteries from both control and CH rats distended passively to increasing intraluminal pressure, and VSM cell [Ca2+]i was not affected. KCl elicited a dose-dependent vasoconstriction that was nearly identical between control and CH groups. Whereas KCl administration resulted in a dose-dependent increase in VSM cell [Ca2+]i in arteries taken from control animals, this stimulus elicited only a slight increase in VSM cell [Ca2+]i in arteries from CH animals. We conclude that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction. calcium; membrane potential; isolated vessels; chronic hypoxia
HPV has been established both as an important regulatory mechanism for reducing arterial hypoxemia resulting from a ventilation-perfusion mismatch and as a cause of pulmonary hypertension. In addition, a component of the elevated PVR following CH may be a secondary consequence of alterations in the pulmonary arterial endothelium, such as cellular hypertrophy and hyperplasia (26) as well as alterations in the synthesis and release of endothelium-derived mitogenic and vasoactive factors (13, 17, 27, 28). It has been suggested that HPV involves inhibition of K+ channels and subsequent vascular smooth muscle (VSM) cell membrane potential (Em) depolarization (25, 32). This shift in resting Em may enhance reactivity to vasoconstrictor stimuli. It is possible that other stimuli that depolarize the VSM cell resting Em may have a similar effect on vascular reactivity.
In many organs, blood flow is held constant in the face of large changes in blood pressure. This local control of blood flow is termed autoregulation. It has been proposed that autoregulation is achieved primarily through metabolic control (i.e., metabolites act on vessel to regulate flow) and myogenic control [i.e., stretch-induced vasoconstriction, also known as the "myogenic response" or "myogenic tone" first described by Bayliss in 1902 (1)], associated with stretch-induced depolarization of VSM and influx of calcium. However, the relative importance of these mechanisms in autoregulatory responses varies in different vascular beds. Indeed, an additional potential contributor to the elevated PVR and enhanced vasoconstrictor reactivity following CH is stretch-induced constriction.
Although the pulmonary circulation is normally thought of as a low-resistance vascular bed, high vascular resistance and low blood flow characterize the pulmonary vasculature of the fetus. A portion of this elevated vascular resistance may be due to myogenic tone. Indeed, pressure-induced vasoconstriction has been demonstrated in late-gestation fetal lambs (3, 38) and newborn guinea pigs (2). Interestingly, myogenic reactivity has been shown to be reduced (2, 3) or nonexistent (7) in the adult. Furthermore, the degree of stretch is limited in the adult, normoxic vasculature since pressures are low. In contrast, upon the development of pulmonary hypertension secondary to CH exposure, it is possible that pressures may reach a threshold for myogenic reactivity not achieved in the nonhypertensive circulation (14). In addition, there is evidence that isolated pulmonary vascular myocytes are depolarized following CH exposure due to altered ion channel expression (31, 34–37, 39). However, the existence of stretch-induced VSM depolarization has not been examined in intact pulmonary arteries from either control or hypertensive circulations. Because of the similarities between the pulmonary vasculature of the fetus (i.e., thickened medial layer and low arterial PO2) and the pulmonary circulation of the adult exposed to CH, we hypothesized that a portion of the elevated PVR following CH is due to the development of myogenic reactivity.
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METHODS |
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70 Torr). Control animals were housed in ambient air conditions (barometric pressure = 630 Torr, inspired PO2 122 Torr). Our laboratory has previously demonstrated that this hypoxic exposure protocol results in the development of pulmonary hypertension in these animals (33).
Isolated pulmonary artery preparation.
Rats were anesthetized with pentobarbital sodium (50 mg ip). A midsternal incision was made to expose the heart, and 100 units of heparin were injected directly into the left ventricle. The left lobe of the lung was excised and placed in ice-cold physiological saline solution [(PSS) composed of (in mM) 129.8 NaCl, 5.4 KCl, 0.5 NaH2PO4, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose], and it was aerated with a 21% O2-6% CO2-73% N2 gas mixture. To separate the effects of stretch on Em and vasoconstriction from those of acute hypoxia, all experiments were performed under normoxic conditions (5, 39). The left lung lobe was secured in a Silastic-coated petri dish that contained cold aerated PSS. Small pulmonary arteries (200–350 µm) free of side branches were dissected away from the adjacent airway and transferred to a vessel chamber (Living Systems). Arteries were cannulated and pressurized to 12 Torr with PSS using a servo-controlled peristaltic pump (Living Systems) and were superfused (5 ml/min) with warmed (37°C), aerated PSS for 30 min. To avoid flow-dependent changes in vessel diameter, experiments were performed with the distal cannula closed. To verify that there was no luminal flow, the servo-controlled peristaltic pump was switched to manual mode. Arterial segments that did not maintain pressure were discarded. To demonstrate the presence of an intact endothelium, arteries were preconstricted to 30% of their baseline diameter with UTP, and the vasodilation in response to acetylcholine (1 µM) was assessed, followed by a 30-min washout period.
Pulmonary VSM cell Em response to increasing intraluminal pressure.
VSM cell Em values were recorded using intracellular sharp electrodes from pressurized small pulmonary arteries from both control and CH rats, prepared as described above. VSM cells were impaled with microelectrodes (tip resistance, 100 M
) filled with 3 M KCl. A neuroprobe model 1600 amplifier (A-M Systems) was used to record Em. Analog output from the amplifier was low-pass filtered at 1 kHz and routed to a Tektronix RM502A oscilloscope and a Gould chart recorder. Em recordings were made in isolated pulmonary arteries pressurized at each of three intraluminal pressures (5, 12, and 45 Torr). The highest pressure tested represents pulmonary artery pressure measured in vivo in hypertensive rats following 4 wk of hypoxia (33). Criteria for acceptance of Em recordings were 1) an abrupt negative deflection in potential as the microelectrode was advanced into the cell, 2) stable Em for at least 1 min, and 3) an abrupt change in potential to 0 mV after the electrode was retracted from the cell.
Myogenic and KCl-induced vasoconstrictor and Ca2+ responses. Myogenic vasoconstrictor responses were determined in small pulmonary arteries from both control and CH rats. Following the test for endothelial viability, pressurized arteries were loaded with the cell-permeant ratiometric Ca2+-sensitive fluorescent indicator fura 2-AM (Molecular Probes). Fura 2-AM was dissolved in anhydrous DMSO at a concentration of 1 mM. Immediately before loading, fura 2-AM was mixed with 0.5 volume of a 20% solution of pluronic acid in DMSO, and this mixture was diluted with loading buffer [3 mM MOPS (pH 7.4), 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2, 1 mM NaH2PO4, 0.02 mM EDTA, 2 mM pyruvate, 5 mM glucose, and 1% bovine serum albumin] to yield a final concentration of 2 µM fura 2-AM and 0.05% pluronic acid. Vessels were incubated in this solution for 45 min at room temperature in the dark. Administration of fura 2 to the abluminal surface in this manner has been shown to preferentially load VSM cells (20). Before experimentation, vessels were reequilibrated for 15 min with warmed, aerated PSS following the loading period to wash out excess dye and to allow hydrolysis of AM groups by intracellular esterases. Pressure-induced vasoconstrictor responses were determined by exposing fura-loaded vessels to a series of 10-Torr pressure steps beginning at 5 Torr and ending at 45 Torr. Each pressure step was held for 5 min. To determine the passive diameter at each pressure step, vessels were superfused for 1 h with Ca2+-free PSS that contained (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 5.5 glucose, and 3 EGTA. Another pressure-response curve was then performed under Ca2+-free conditions. Separation of the diameters measured in Ca2+-replete and Ca2+-free PSS is indicative of the degree of myogenic tone at each pressure step. In a separate set of vessels, vasoconstrictor responses to increasing concentrations of KCl (15–85 mM) were determined in fura-loaded pulmonary arteries from both control and CH rats. The internal diameter (ID) for all studies was continuously monitored using video microscopy and edge-detection software (IonOptix). Fura-loaded vessels were alternatively excited at 340 and 380 nm, and the respective 510-nm emissions were quantified using a photomultiplier tube and recorded using IonWizard software (IonOptix). Vessel wall intracellular Ca2+ concentration ([Ca2+]i) was calculated at each pressure step, and concentration of KCl as the mean F340/F380 from the background-subtracted 510-nm signal was collected over the last 1 min.
Data analysis.
Variances about the means will be quantified by using standard deviations (SD). Data were analyzed by using two-way repeated-measures ANOVA. Where significant, main effects occurred, individual groups were compared by using Student-Newman-Keuls post hoc test. A probability of P 
0.05 was accepted as statistically significant for all comparisons.
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RESULTS |
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DISCUSSION |
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The present study is the first to investigate myogenic vasoconstriction in the pulmonary circulation using pressurized, intact small intrapulmonary arteries (removing possible neural or humoral influences), thus facilitating a direct assessment of the ability of pulmonary VSM cells to respond to stretch. We have shown that, although pulmonary VSM cells depolarize in response to stretch, vessel diameters under Ca2+-replete and Ca2+-free PSS were identical, suggesting no active vasoconstriction (Fig. 2). Because pressure-induced vasoconstriction is dependent on Ca2+ influx, our finding that vessel wall Ca2+ concentration ([Ca2+]) did not increase in response to increasing intraluminal pressure (Fig. 3) is consistent with the absence of myogenic tone. Although there are similarities between the pulmonary circulation of the fetus and that of the CH adult, the results of the present study suggest that the pulmonary arterial circulation does not demonstrate stretch-induced constriction within a physiological pressure range. Previous investigators have shown a myogenic response in the pulmonary circulation of both the fetus and newborn. For example, Belik has demonstrated myogenic tone in the pulmonary circulation of newborn guinea pigs (2) as well as fetal and newborn sheep (3) using arterial rings. In addition, Storme et al. (38) were able to unmask an autoregulatory response to the partial occlusion of the ductus arteriosus in fetal sheep in vivo by inhibiting nitric oxide synthase. However, in these studies, stretch-induced contraction was not existent in the adult (2, 3). In addition, Davis et al. (7, 9) transplanted neonatal hamster pulmonary tissue into adult female hamster cheek pouch. These arteries have been shown to constrict to hypoxia and dilate to sodium nitroprusside (9); however, they respond passively to either increases or decreases in transmural pressure (7). Interestingly, renal arteries transplanted in this manner constricted when transmural pressure was increased and dilated upon a reduction in transmural pressure [i.e., respond actively (15)]. Moreover, PVR has been shown to decrease in response to elevated left atrial pressure, suggesting that they respond passively to stretch (4, 22). To date, only one study has demonstrated a myogenic response in the pulmonary circulation of the adult. Kulik et al. (21) demonstrated active vasoconstriction in response to stretch in small pulmonary arteries of adult cats using an arterial ring preparation. Discrepancies between the findings of Kulik et al. and those of the present study may be due to either species differences or possibly the degree and direction of stretch employed in these different experimental preparations. Because VSM cells wrap circumferentially around arteries, in a pressurized artery preparation, all VSM cells will be exposed to a circumferential force. However, in a ring preparation, the force applied is different depending on the location relative to the point of attachment of the ring to the force transducer. The applied force will be circumferential near the point of attachment, but it will change to a tangential force as one moves away from this point.
We have currently demonstrated that pulmonary VSM cell resting Em in intact pressurized arteries from CH rats are depolarized compared with VSM cells in arteries from control animals. These results are consistent with the findings of previous studies performed in isolated pulmonary artery myocytes (31, 34–37, 39). For example, Shimoda et al. (36) demonstrated that resting Em in single pulmonary myocytes isolated from male Wistar rats exposed to 17–21 days of normobaric hypoxia (10% O2) were depolarized compared with control. In the systemic circulation, Harder (16) has demonstrated pressure-dependent VSM cell depolarization in intact cat middle cerebral arteries between 10 and 150 mmHg. In this study, Em at lower intraluminal pressures were similar to those of control pulmonary arteries in the present study (16). In addition, Davis et al. (10) demonstrated that dispersed coronary artery VSM cells depolarized by 20 mV when cell length was increased by 25%, demonstrating that mechanosensitivity is an inherent property of VSM cells. Stretch-induced VSM cell depolarization opens voltage-dependent calcium channels (VDCC), resulting in Ca2+ influx and increased [Ca2+]i (30). Blockade of VDCC inhibits the myogenic response (16, 18, 19, 23), illustrating that pressure-induced vasoconstriction results from Ca2+ influx via these channels in systemic arteries. Moreover, the Em measured at 45 Torr in the present experiments is near that observed at 40 Torr in mesenteric arteries in an earlier study from our laboratory using identical techniques (11). This observation suggests that the mechanosensitivity of pulmonary VSM may be similar to that of cells from the systemic vasculature. Em recorded from unpressurized VSM cells from main pulmonary artery strips from control and CH rats were –60 mV (SD 3) and –47 mV (SD 6), respectively (unpublished observations). These values are similar to those of the present study at 5 Torr, suggesting that there is a threshold for stretch-sensitive ion channel activation in pulmonary artery VSM cells. Although we have demonstrated that pulmonary VSM cells depolarize in response to stretch, VSM cell Ca2+ did not increase. In addition, a pressure-induced decrease in luminal diameter was not observed. Taken together, these results suggest that, under the conditions of the present study, small pulmonary arteries do not exhibit myogenic vasoconstriction. To demonstrate our ability to measure changes in [Ca2+]i in response to depolarizing stimuli in our preparation, we examined KCl-mediated changes in ID and [Ca2+]i. Although vasoconstriction to KCl was similar in arteries taken from control and CH rats, increases in vessel wall [Ca2+]i were blunted in arteries from CH animals compared with control. This blunted [Ca2+]i response to KCl is consistent with the findings of Shimoda et al. (35), demonstrating altered Ca2+ handling following CH. These investigators have shown that the VDCC-mediated increase in VSM cell [Ca2+]i in response to endothelin-1 is blunted in pulmonary myocytes from animals exposed to 3 wk of normobaric hypoxia compared with control. In addition, these authors showed that, while the elevated basal VSM cell [Ca2+]i seen in CH myocytes is dependent on extracellular Ca2+, inhibition of VDCC with nifedipine had no effect on [Ca2+]i. Taken together, these findings and those of the present study support the postulate that Ca2+ influx mechanisms in VSM cells are altered following CH.
Our present findings indicate that KCl-induced vasoconstriction is similar between groups, despite differences in [Ca2+]i. One possible explanation for these findings is that, following CH, there is a switch from a dependence on Ca2+ influx for vasoconstriction to a mechanism that relies on increases in Ca2+ sensitivity. The small GTPase RhoA and its downstream effector Rho kinase (ROK) produce an augmented vasoconstrictor response for a given concentration of intracellular Ca2+ by inhibiting myosin light chain phosphatase. There is recent evidence to suggest that the RhoA/ROK pathway is involved in regulating vascular reactivity in the pulmonary circulation (12, 29). Indeed, KCl-induced increases in perfusion pressure in isolated saline-perfused lungs were nearly eliminated by the ROK inhibitor Y-27632 in lungs from CH rats, with no effect in controls. In this same study, these investigators provided evidence that, although lungs from CH rats have higher basal vascular resistance compared with controls, nifedipine had no effect on perfusion pressure. In contrast, Y-27632 decreased basal perfusion pressure in a concentration-dependent manner in lungs from CH rats but was without effect in controls (29). Moreover, nifedipine has been shown to have a minimal effect on endothelin-1-induced contraction in pulmonary artery rings from CH rats (35). Interestingly, Ledvora et al. (24) have demonstrated in carotid artery strips that phosphorylation of the 20-kDa myosin light chain in response to stretch can still occur in Ca2+-free PSS. Taken together, these results suggest that, following CH, changes in Ca2+ sensitivity play a greater role in regulating vascular tone than does Ca2+ influx through voltage-gated Ca2+ channels.
Although we did not observe stretch-induced constriction in small pulmonary arteries (250–300 µm ID), it is possible that this response is restricted to more distal arterioles. Indeed, Davis and colleagues (6, 8) have shown heterogeneity along the vascular tree, with greater myogenic reactivity being observed in more distal segments. In addition, the present experiments were performed under normoxic conditions. Although pulmonary artery VSM cells depolarize in response to stretch under normoxic conditions, it is possible that hypoxia initiates other signaling cascades, which are required for vasoconstriction to occur in response to stretch.
In summary, the present study provides evidence that pulmonary VSM cell Em is depolarized following CH compared with controls and that VSM cells from both control and CH animals depolarize in response to stretch. However, arteries from either control or CH animals do not exhibit stretch-induced vasoconstriction. Thus these findings suggest that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction.
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ACKNOWLEDGMENTS |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 281: L202–L208, 2001.This article has been cited by other articles:
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  N. L. Jernigan, B. R. Walker, and T. C. Resta Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L515 - L529. [Abstract] [Full Text] [PDF]
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 J. Yin, J. Hoffmann, S. M. Kaestle, N. Neye, L. Wang, J. Baeurle, W. Liedtke, S. Wu, H. Kuppe, A. R. Pries, et al. Negative-Feedback Loop Attenuates Hydrostatic Lung Edema via a cGMP-Dependent Regulation of Transient Receptor Potential Vanilloid 4 Circ. Res., April 25, 2008; 102(8): 966 - 974. [Abstract] [Full Text] [PDF]
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B. R. S. Broughton, B. R. Walker, and T. C. Resta Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L797 - L806. [Abstract] [Full Text] [PDF]
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 K. J. Allahdadi, L. C. Duling, B. R. Walker, and N. L. Kanagy Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKC{delta} Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H920 - H927. [Abstract] [Full Text] [PDF]
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M. L. Paffett, J. S. Naik, T. C. Resta, and B. R. Walker Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1135 - L1142. [Abstract] [Full Text] [PDF]
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K. J. Allahdadi, B. R. Walker, and N. L. Kanagy ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2911 - H2918. [Abstract] [Full Text] [PDF]
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 H. A. Lee, E. B. Baek, K. S. Park, H. J. Jung, J. I. Kim, S. J. Kim, and Y. E Earm Mechanosensitive nonselective cation channel facilitation by endothelin-1 is regulated by protein kinase C in arterial myocytes Cardiovasc Res, November 1, 2007; 76(2): 224 - 235. [Abstract] [Full Text] [PDF]
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 S. de Frutos, R. Spangler, D. Alo, and L. V. G. Bosc NFATc3 Mediates Chronic Hypoxia-induced Pulmonary Arterial Remodeling with {alpha}-Actin Up-regulation J. Biol. Chem., May 18, 2007; 282(20): 15081 - 15089. [Abstract] [Full Text] [PDF]
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 G. C. Sieck Commentary J Appl Physiol, March 1, 2005; 98(3): 1091 - 1091. [Full Text] [PDF]
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Significantly different than 12 and 5 Torr (P < 0.05).
) in F340/F380. Values are means ± SD. *Significantly different than control (P < 0.05).