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Am J Physiol Heart Circ Physiol 275: H844-H851, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 3, H844-H851, September 1998

Ablation of lung endothelial injury after pacing-induced heart failure is related to alterations in Ca2+ signaling

Claire L. Ivey, Beverly J. Roy, and Mary I. Townsley

Department of Physiology, University of South Alabama, Mobile, Alabama 36688

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that ANG II increases microvascular permeability in normal dog lungs but not after pacing-induced heart failure. This study investigated how ANG II induces permeability in isolated blood-perfused canine lung lobes and what alterations occur during heart failure. In normal lobes, the protein kinase C (PKC) inhibitors staurosporine (500 nM) or chelerythrine (10 µM) did not modify ANG II-induced increases in the capillary filtration coefficient (Kf,c, ml · min-1 · cmH2O-1 · 100 g-1; an index of microvascular permeability), suggesting that PKC is not involved. Thapsigargin (150 nM) was used to stimulate capacitative Ca2+ entry in lobes from control dogs and dogs paced at 245 beats/min for 4 wk to induce heart failure. In control lobes, Kf,c rose after thapsigargin, from 0.06 ± 0.01 to 0.17 ± 0.03 ml · min-1 · cmH2O-1 · 100 g-1 (mean ± SE, P < 0.05) but did not change in the paced group. A Ca2+ ionophore, A-23187, increased Kf,c in both control (10 µM; 0.05 ± 0.01 to 0.17 ± 0.05 ml · min-1 · cmH2O-1 · 100 g-1, P < 0.05) and pace (5 µM; 0.06 ± 0.01 to 0.21 ± 0.07 ml · min-1 · cmH2O-1 · 100 g-1, P < 0.05) lobes, indicating that increasing intracellular Ca2+ is sufficient to induce pulmonary microvascular permeability after pacing. We conclude that during heart failure, Ca2+ signaling within the pulmonary microvascular endothelium is altered.

venous hypertension; microvascular permeability; capacitative calcium entry; pulmonary endothelium; capillary filtration coefficient

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CONGESTIVE HEART FAILURE (CHF) is associated with chronic pulmonary venous hypertension. Despite this, pulmonary edema and alveolar flooding do not necessarily occur in patients with this condition, promoting the idea that the lungs from these individuals undergo adaptations that protect them from injury. One key target for such adaptation could be the pulmonary microvascular endothelium, because this barrier provides the initial and limiting resistance to transvascular fluid movement. In support of this theory, Townsley et al. (46) demonstrated an increase in the threshold for high-vascular-pressure lung injury after pacing-induced heart failure in dogs. Subsequently, in a separate study ANG II was shown to increase endothelial permeability in normal dog lung lobes, but this effect was absent in lobes from paced dogs (36).

The actions of ANG II are mediated via membrane receptors, of which there are two major isoforms, AT1 and AT2. The physiological function of the AT2 receptor is not well defined, although it is most highly expressed in the fetus, suggesting a role in cell growth and development (49). More information is available concerning the AT1 receptor, which is widely distributed and can mediate most of the known functions of ANG II. Expression of AT1 receptors has been demonstrated in endothelial cells from a variety of vascular beds (8, 29, 40). These receptors are G protein linked and can activate several signaling pathways (12). The major pathway by which ANG II exerts its actions is via stimulation of a phosphatidylinositol-specific phospholipase C promoting the generation of diacylglycerol (DAG) and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (3, 13, 23, 50). DAG activates protein kinase C (PKC), which can induce multiple cellular responses including cell contraction (43, 44). Ins(1,4,5)P3 goes on to bind to an Ins(1,4,5)P3 receptor on an intracellular Ca2+ store, most likely the endoplasmic reticulum (ER), initiating the release of Ca2+ into the cytosol. This Ca2+ release is transient but is followed by a further, more sustained rise in intracellular Ca2+ via a plasma membrane Ca2+ entry pathway (32). The depletion of the intracellular Ca2+ stores appears to be the trigger for entry of Ca2+ into the cell. This process has been termed "capacitative Ca2+ entry" (30). Several studies have demonstrated such capacitative Ca2+ entry in vascular endothelium (7, 37, 51). Finally, it is well established that increases in endothelial cell free Ca2+ play an important role in regulating endothelial barrier permeability (9, 11, 14, 25, 38). Thus it is possible that changes in Ca2+ signaling pathways occur in pulmonary endothelium during pacing-induced CHF, which may explain the lack of a permeability response to ANG II in these lungs.

The aim of this study was to investigate possible mechanisms by which ANG II induces permeability changes in normal isolated, blood-perfused lung lobes and to examine what alterations may be occurring after the development of pacing-induced heart failure. Interaction of ANG II with AT1 receptors, present on endothelial cells, can promote activation of PKC. Therefore, a possible role for PKC in ANG II-induced permeability in normal lung was examined using two PKC inhibitors, staurosporine and chelerythrine. ANG II may also stimulate capacitative Ca2+ entry. To examine how this may play a role in pulmonary microvascular permeability, we chose to evaluate the effects of thapsigargin in lung lobes taken from both control and paced animals. Thapsigargin activates capacitative Ca2+ entry via an Ins(1,4,5)P3-independent pathway, by blocking the ER Ca2+-ATPase that is responsible for the uptake of free Ca2+ from the cytoplasm and thus promoting store depletion (45). In addition, the effects of a Ca2+ ionophore, A-23187, were monitored in both groups to determine whether an increase in intracellular Ca2+ alone was sufficient to increase permeability. Our data suggest that there is an alteration in the mechanism of pulmonary endothelial capacitative Ca2+ entry after pacing-induced heart failure.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pacing Model of Heart Failure

Conditioned, microfilaria-negative mongrel dogs (n = 13) were anesthetized with pentobarbital sodium (30 mg/kg iv) to allow insertion of a transvenous pacing lead (unipolar; model 4011, Medtronic) into the right ventricle via the right jugular vein. Correct placement of the pacing lead was confirmed by the ability to capture ventricular rate. A programmable generator (model 8329, Medtronic) was inserted into a subcutaneous pocket anterior to the first rib and attached to the pacing lead. All surgery was done under sterile conditions. Antibiotics (cephalexin, 500 mg, twice daily) were administered orally for 5 days postoperatively. Wound healing was checked daily. The animals were allowed to eat and drink ad libitum after the surgery and during the pacing period.

A baseline echocardiogram was performed 1-2 days after surgery, after which pacing was initiated at a ventricular rate of 245 beats/min (1, 36, 46, 48). Maintenance of pacing was checked daily by auscultation. Echocardiograms were done at regular intervals to assess cardiac function. Pacing was continued (29.6 ± 1.2 days) until the left ventricular shortening fraction (LVSF), measured in sinus rhythm, was reduced by ~50% from baseline.

Terminal Experiment

Microfilaria-negative mongrel dogs were used as controls (n = 36). Both paced and control dogs were fasted overnight before the terminal experiments. An intravenous line was inserted, and anesthesia was induced with pentobarbital sodium (<15 mg/kg iv in paced dogs; 30 mg/kg iv in controls). Previous studies showed that this regimen produces a surgical plane of anesthesia in paced dogs without accompanying cardiovascular collapse (46, 48). The dogs were orally intubated and placed on a Harvard animal respirator at a rate of 15 breaths/min and a tidal volume of 15 ml/kg. alpha -Chloralose was administered as necessary to maintain a surgical plane of anesthesia. A carotid arterial line was inserted to measure systemic arterial blood pressure (paced dogs only) and to withdraw blood. In paced dogs, a catheter was placed in the jugular vein and advanced to the pulmonary artery for measurement of pulmonary artery pressure. Placement of lines was confirmed by waveforms.

Isolated Lung Preparation

The left chest was opened at the fifth intercostal space. The left upper and left middle lobes were excised for measurement of blood-free extravascular lung water (EVLW). Loose ties were placed around the left main pulmonary artery and left bronchus. All dogs were heparinized (10,000 U heparin iv). Ten minutes postheparinization, the lower left lung lobe was removed. The lobar artery, vein, and bronchus were cannulated with plastic cannulas. The lobe was then suspended from a counterbalanced force transducer (Grass FT-10), and the system was calibrated with a standard weight. Lobes were perfused with 200 ml of autologous blood mixed with 100 ml of balanced Earle's buffer and ventilated with a Harvard animal respirator using 30% O2-5% CO2-65% N2 at a rate of 6-8 breaths/min, a peak inspiratory pressure of 8-10 cmH2O, and an end-expiratory pressure of 2-3 cmH2O. Blood temperature was maintained at 37°C. Blood gases were measured, and the pH was corrected to 7.35-7.40 as necessary with the addition of sodium bicarbonate.

Lobar Hemodynamics

Thin catheters were placed in the lobar arterial and venous lines to measure arterial (Pa) and venous (Pv) pressures, respectively. The zero point was set at the lung hilum. Pressures and lobe weight were continuously recorded with a Grass model 7 polygraph. Pv was set to 4-5 cmH2O by adjusting the height of the venous reservoir. Blood flow (Q) was increased to the maximal value that would keep the lobe in an isogravimetric state (neither losing nor gaining weight) and remained at that level throughout the remainder of the experiment. Capillary pressure (Pc) was measured by the double vascular occlusion technique (35, 47, 48). At the end of the experiment, Q was measured by timed collection of blood into a graduated cylinder. Total pulmonary resistance was calculated as Rt = (Pa - Pv)/Q. Precapillary (Ra) and postcapillary (Rv) resistances were determined as Ra = (Pa - Pc)/Q and Rv = (Pc - Pv)/Q. Vascular compliance (Ct) was determined via the rapid venous occlusion technique (20) and calculated as Ct = Q/(linear rate of Delta Pv/Delta t after rapid venous occlusion), where t is time.

Evaluation of Permeability and Transvascular Fluid Exchange

The capillary filtration coefficient (Kf,c) was determined as a measure of microvascular permeability at the beginning and end of each experiment. Pv was increased by 8-10 cmH2O (i.e., from 5 to 13-15 cmH2O) for a period of 15 min, causing the lobe to gain weight. The rate of weight gain (Delta W/Delta t) became constant after ~10 min. The Delta W/Delta t calculated for minutes 13-15 was used in the following equation to calculate Kf,c: Kf,c = (Delta W/Delta t)/Delta Pc. Delta Pc was determined by the difference in Pc before and at the end of the 15-min increased-Pv period. Kf,c was expressed as milliliters per minute per centimeter of H2O per 100 g of wet weight. At the end of the Kf,c measurement, Pv was returned to the baseline level.

EVLW was measured as follows (28, 52). Lobes were homogenized with 100 ml of distilled water, and then samples of homogenate were centrifuged for 1 h at 15,000 rpm to obtain lung supernatant. Samples of blood, homogenate, and supernatant were weighed and then dried to a constant weight. Lung water was corrected for blood water using these weights and measures of total hemoglobin (2) in the blood and supernatant. EVLW was reported as milliliters per gram blood-free dry weight.

Isolated Lung Protocols

All lobes had initial pressures measured and recorded and initial and final Ct and Kf,c determined. The experimental protocols were begun after the lobes had stabilized after the initial Kf,c measurement. After completion of each protocol, the final EVLW was measured for each perfused lobe as described in Evaluation of Permeability and Transvascular Fluid Exchange.

PKC inhibition and ANG II. ANG II was administered to seven control lobes as three separate intra-arterial boluses of 1, 2.5, and 10 µg (cumulative dose 34.7 nM) as previously described (36). To investigate the effects of PKC inhibition on ANG II-induced responses, staurosporine (n = 5 lobes) or chelerythrine (n = 5 lobes) were added to the perfusate of additional control lobes to give final concentrations of 500 nM and 10 µM, respectively. After a 40-min equilibration time, the three doses of ANG II were given as above. Vascular resistances were determined before each drug administration and at the peak response after ANG II injection. Pa was allowed to return to baseline before administration of the next dose. In lobes that were pretreated with staurosporine, 2-4 mg of papaverine hydrochloride were added to the perfusate after the final ANG II dose, to aid in relaxation of the vasculature. This is a dose that does not alter pulmonary microvascular permeability (22). Once the Pa had returned to baseline value, the final measurements of resistances, Ct, and Kf,c were completed.

Thapsigargin. The effects of thapsigargin on microvascular injury were assessed in eight control lobes and five lobes taken from paced animals. In these experiments ibuprofen (10 µg/ml perfusate) was added to the venous reservoir to attenuate thapsigargin-induced vasoconstriction. After 30 min, thapsigargin was added to the perfusate to give a final concentration of 150 nM. Final pressure, Ct, and Kf,c measurements were made after 1 h. In lobes that had a prolonged pressor response to thapsigargin (i.e., >1 h), final measurements were made when Pa stabilized. Vascular resistances were measured before each drug addition. All experiments involving thapsigargin were performed in the dark because of its light sensitivity.

Ca2+ ionophore. To investigate the possibility that lobes from paced animals fail to respond to increases in intracellular Ca2+ in the same fashion as control lobes, a series of experiments were performed using the Ca2+ ionophore A-23187. The effects of A-23187 were assessed in 11 control lobes and 8 lobes from paced animals. Ibuprofen (10 µg/ml) was administered to the venous reservoir, followed 30 min later by increasing doses of Ca2+ ionophore A-23187. The initial ionophore dose was 5 and 2.5 µM in the control and paced groups, respectively. This was followed by 2.5 µM increments to give cumulative doses of 5, 7.5, 10, and 12.5 µM. Kf,c was measured 1 h after each dose, and then the lobe was allowed to stabilize before the next dose was administered. The maximum dose added to any control lobe was 12.5 µM (n = 4). Lobes that demonstrated a large increase in Kf,c with earlier doses were not given this final dose. A dose of 7.5 µM A-23187 was the maximum given to any pace lobe (n = 4), because of the substantial pressor response observed. In all experiments, final measurements of resistance, Ct, and Kf,c were made 1 h after the final ionophore dose.

Drugs

ANG II, Ca2+ ionophore A-23187, ibuprofen, and staurosporine were obtained from Sigma Chemical. A stock solution of ANG II made in H2O at a concentration of 1 mg/ml was stored at -20°C. Two working dilutions were made daily (0.1 and 0.25 mg/ml). Appropriate volumes of these dilutions were then used (maximum injected volume was 40 µl) (36). A-23187 and staurosporine were both dissolved in DMSO and were kept at room temperature and 4°C, respectively. Ibuprofen was dissolved in 95% ethanol and kept at room temperature. Thapsigargin was purchased from RBI and chelerythrine from Biomol. Both were dissolved in DMSO and kept at -20°C.

Statistics

Data are presented as means ± SE. Statistical differences were evaluated by ANOVA or Student's t-test to identify specific differences within and between groups. For the A-23187-induced changes in Kf,c, the Wilcoxon signed-rank test was used because of the heterogeneity in n numbers between doses of drugs.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals in the heart failure group were paced for an average of 29.6 ± 1.2 days, resulting in a decrease in LVSF from 36.6 ± 0.4 to 21.5 ± 0.6% (P < 0.05). Body weight in this group before pacing (22.4 ± 0.8 kg) was not different from that at the time of the terminal experiment (22.8 ± 0.7 kg). Measurements of cardiovascular function in the paced group after anesthesia showed that in vivo systemic arterial pressure was 110 ± 10.7 mmHg (n = 10), central venous pressure was 11.3 ± 2.0 mmHg (n = 10), pulmonary artery pressure was 31.9 ± 3.6 mmHg (n = 10), and pulmonary wedge pressure was 21.5 ± 3.0 mmHg (n = 10). These results are comparable to those previously reported in paced dogs (36, 48). Although in vivo hemodynamic measurements were not made in the current control dogs, the initial EVLW (Table 1) is no different from that in previous controls with normal pulmonary vascular pressures (48). There was no difference between body weight of control (22.2 ± 0.4 kg) and paced dogs (22.8 ± 0.7 kg) at the time of the terminal experiment. Table 1 shows the baseline data for the isolated lung lobes. The paced animal lungs had a higher initial EVLW than the control group (Table 1). We have previously shown that baseline resistances and EVLW in the paced group are significantly higher than those in control lobes (36, 48), and the data in this study confirm that.

                              
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  		Table 1.   Baseline hemodynamic measurements in isolated lung lobes

As previous studies showed (36), ANG II induced a dose-dependent increase in Ra (Fig. 1) but had little effect on Rv (change after 1 µg: -0.03 ± 0.35; 2.5 µg: 0.31 ± 0.14; 10 µg: 0.14 ± 0.36 cmH2O · l-1 · min · 100 g). Addition of staurosporine (500 nM) did not affect baseline Ra (prestaurosporine: 8.41 ± 1.43; poststaurosporine: 7.25 ± 0.97 cmH2O · l-1 · min · 100 g) or Rv (pre: 7.32 ± 0.52; post: 7.23 ± 0.6 cmH2O · l-1 · min · 100 g). However, staurosporine did block the vasoconstrictor effect of 2.5- and 10-µg ANG II (P < 0.05 and P < 0.01, respectively). To confirm that this was caused by inhibition of PKC, the effects of a more specific inhibitor, chelerythrine (10 µM), were studied. Chelerythrine did not change baseline Ra (pre: 7.07 ± 1.22; post: 7.16 ± 1.36 cmH2O · l-1 · min · 100 g) or Rv (pre: 6.65 ± 0.57; post: 6.64 ± 0.52 cmH2O · l-1 · min · 100 g) but did reduce the 10 µg ANG II-induced increase in Ra. Neither staurosporine nor chelerythrine influenced Rv after ANG II administration (data not shown).


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  		Fig. 1.   Effect of increasing concentrations of ANG II on change (Delta ) in arterial resistance (Ra) in lung lobes from control dogs. ANG II was administered alone (n = 7 lobes) or 40 min after pretreatment with either staurosporine (n = 5) or chelerythrine (n = 5). At 10 µg ANG II, * and + illustrate a significant increase in Ra vs. 1 (P < 0.01) and 2.5 (P < 0.05) µg ANG II in same group, respectively. # P < 0.05, ANG II alone vs. ANG II after staurosporine or chelerythrine at same ANG II concentration.

ANG II administration promoted a significant increase in Kf,c in control lobes (P < 0.05; Fig. 2), confirming previous findings (36). There was no difference in baseline Kf,c between lobes that received staurosporine or chelerythrine and ones that did not. Staurosporine tended to increase Kf,c, although neither staurosporine nor chelerythrine significantly influenced the permeability response to ANG II (i.e., the final Kf,c) compared with lobes that received ANG II alone.


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  		Fig. 2.   Baseline vs. final capillary filtration coefficients (Kf,c) measured in lung lobes taken from control dogs. ANG II was administered alone (n = 7 lobes) or 40 min after pretreatment with either staurosporine (n = 5) or chelerythrine (n = 5). * P < 0.05, final vs. baseline within same group.

The above results suggest that ANG II is not increasing permeability in control lobes via PKC. To examine the role of capacitative Ca2+ entry in pulmonary microvascular permeability, we studied the effects of thapsigargin. In control lobes thapsigargin did not affect Ra (Fig. 3A) but did increase Rv (Fig. 3B; P < 0.05). Baseline Ra and Rv were both accentuated in the pace lobes compared with the controls as previously shown (36, 48). After thapsigargin in the paced group, there was a significant rise in both Ra (P < 0.01) and Rv (P < 0.05). In these lobes there was often a severe increase in Pa that took a long time to return to baseline, so that the final Kf,c measurement was delayed. However, there was no difference in the mean time between thapsigargin administration and Kf,c for pace (80.2 ± 7.0 min) vs. control (72.6 ± 4.7 min) lobes. Baseline Kf,c was similar between the control and pace lobes, despite an increase in EVLW in the paced group. In contrast, although thapsigargin induced a prominent increase in Kf,c in control lobes (P < 0.01), Kf,c in the pace lobes remained unchanged (Fig. 4).


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  		Fig. 3.   Baseline vs. final (1 h after thapsigargin) measurements of Ra (A) and venous resistance (Rv; B) as a function of wet weight in lung lobes taken from control (n = 8) and paced (n = 5) dogs. ** P < 0.01, final vs. baseline within same group; # P < 0.05, paced vs. control at same time point.


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  		Fig. 4.   Baseline vs. final (1 h after thapsigargin) Kf,c measured in lung lobes taken from control (n = 8) and paced (n = 5) dogs. Baseline Kf,c was not different between control and pace lobes. ** P < 0.01, final vs. baseline within same group.

The effects of a Ca2+ ionophore, A-23187, on vascular resistance in both control lobes and lobes from paced animals is shown in Fig. 5. In control lobes, there was no predominant rise in either Ra (baseline: 5.2 ± 0.5; final: 6.9 ± 1.0 cmH2O · l-1 · min · 100 g) or Rv (baseline: 5.8 ± 0.4; final: 9.0 ± 1.2 cmH2O · l-1 · min · 100 g), although A-23187 did induce a dose-related increase in total resistance, which was significantly above baseline at all concentrations. A similar pattern is shown in the pace lobes, in which 5 µM A-23187 significantly increased Rt. Furthermore, the change in Rt tended to be higher in the paced group, although this was not statistically significant (P = 0.052) because of the large variability in the pressor response in the paced group.


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  		Fig. 5.   Effect of increasing concentrations of Ca2+ ionophore A-23187 on change in total resistance (Delta Rt) in lung lobes taken from control (n = 11) and paced (n = 8) dogs. * P < 0.05, Delta Rt at individual doses.

In the control group, Kf,c rose in a dose-dependent fashion in response to A-23187. Kf,c was significantly higher than baseline at 10 and 12.5 µM A-23187 (Fig. 6). Because of the accentuated pressor response in lobes taken from paced animals, a lower initial dose of the ionophore (2.5 µM) was administered. A-23187 also significantly increased Kf,c in these lobes at a dose of 5 µM (Fig. 6).


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  		Fig. 6.   Effect of increasing concentrations of Ca2+ ionophore A-23187 on Kf,c measured in lung lobes taken from control (n = 11) and paced (n = 8) dogs. Baseline Kf,c was not different between control and pace lobes. * P < 0.05, individual doses vs. baseline within same group.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The endothelium is instrumental in governing microvascular permeability. Endothelial disruption by cell damage and/or contraction leads to intercellular gap formation, promoting increased fluid accumulation and pulmonary edema. During heart failure venous pressures are markedly increased, promoting a rise in fluid flux into the interstitium and increased EVLW. From an increase in EVLW alone, however, one cannot draw conclusions as to the permeability properties of the endothelial barrier. In the lung, the ease with which endothelial permeability can be measured has proved instrumental in allowing investigators to distinguish between edema caused by increased vascular resistance and capillary pressure and that caused by endothelial injury. Kf,c, the measure used in this study, evaluates the hydraulic conductance of the entire pulmonary microvascular exchange area and thus is indicative of the state of the endothelial barrier alone. We have found that in the isolated, perfused lung neither passively increasing vascular pressure (venous pressures <= 35 cmH2O) nor marked active vasoconstriction induced by a variety of agonists (e.g., norepinephrine, histamine, serotonin, or arachidonic acid) alter Kf,c (34, 46-48), substantiating the fact that we can differentiate between smooth muscle- and endothelium-mediated events in this model. In the present study, all agonists used increased pulmonary vascular resistance in the control group in conjunction with a rise in Kf,c. In contrast, lung lobes from paced animals displayed an enhanced vasoactive response to the same drugs. Furthermore, we have seen similar enhancement in the pulmonary vascular responsiveness to norepinephrine in paced dogs (48). These changes could be caused by upregulation of receptor populations, increased Ca2+ stores, and/or enhanced coupling of store depletion to Ca2+ entry in smooth muscle cells. However, the marked tendency toward an increased pressor response to A-23187 (Fig. 5) suggests that a more fundamental enhancement in the responsiveness of smooth muscle contractile proteins to increases in Ca2+ may underlie these observations. It is clear that an understanding of the adaptations in smooth muscle function in pacing-induced heart failure will require further work, although we do know that the enhanced pressor responses in the canine lung after pacing are not caused by decrements in endothelium-derived nitric oxide (48) or prostacyclin (unpublished observations). In contrast to the enhanced pressor responses in the paced group, both thapsigargin and ANG II failed to induce increases in permeability, suggesting a markedly different adaptation in endothelial function.

Previously we showed that although the basal permeability of the canine pulmonary microvasculature is unchanged after pacing-induced heart failure (36, 46, 48), the ANG II-induced elevation in permeability seen in control canine lungs is abolished after 1 mo of pacing (36). ANG II binds to AT1 receptors on vascular endothelium (5, 41) and results in the release of Ins(1,4,5)P3 and DAG. Ins(1,4,5)P3 induces depletion of intracellular Ca2+ stores, leading to capacitative Ca2+ entry. DAG activates the PKC enzymatic cascade (49), which in the endothelium can mediate numerous cellular functions, including stimulation of adenylyl cyclases, regulation of intracellular Ca2+ levels, and cell contraction via filament rearrangement (19, 43-44). Any of these events could promote changes in microvascular permeability.

Because some extracellular mediators have been shown to increase endothelial permeability via PKC activation (21), our first experiments were designed to assess whether PKC was involved in ANG II-mediated responses in control dog lungs. Thus lobes were pretreated with one of two PKC inhibitors, staurosporine or chelerythrine, before ANG II administration. The results in untreated control lobes are in agreement with our earlier investigations in that ANG II administration increases the pulmonary arterial resistance but has little effect on venous resistance (36). Similar findings have also been reported by Hyman (15). The addition of staurosporine abolished the ANG II-induced vasoconstriction. However, at the dose used (500 nM) staurosporine is not a selective PKC inhibitor and can also affect protein kinases A and G and myosin light chain kinase (24, 42), any or all of which may influence the pressor response to ANG II. Further experiments were therefore performed using chelerythrine, which is selective for PKC at the dose used (10 µM). At the highest concentration of ANG II (10 µg), chelerythrine also inhibited arterial vasoconstriction. This suggests that the ANG II-induced rise in arterial resistance is largely mediated by PKC. Attenuation of ANG II-induced arteriolar constriction in rat cremaster muscle by PKC inhibition, using staurosporine and bisindolylmaleimide I, has recently been reported by Kulenovic et al. (18). They used enzyme activity assays to demonstrate that the drugs were reducing PKC activity, although the activity of other kinases was not tested. In the present study, ANG II also induced an increase in Kf,c in the control group, corresponding to our previous data (36). However, unlike their effects on the pressor response, neither staurosporine nor chelerythrine attenuated the permeability response after ANG II, indicating that PKC activation is not involved in ANG II-mediated pulmonary permeability responses.

Another mechanism by which ANG II may change lung permeability is by stimulating Ins(1,4,5)P3 production (30). The effects of increasing endothelial cell Ca2+ levels on permeability and barrier function are well documented. A rise in intracellular Ca2+ can promote endothelial cell contraction (9, 11, 25, 39, 53) and uncouple focal adhesions (11, 26), producing intercellular gap formation and increased microvascular permeability. We hypothesized that after pacing-induced heart failure there may be an alteration in pulmonary endothelial cell Ca2+ handling and/or specific target proteins that respond to changes in intracellular Ca2+. Thapsigargin is a plant alkaloid that inhibits the Ca2+-ATPase of the intracellular Ca2+ stores and limits refilling, thereby mimicking Ins(1,4,5)P3-activating agonists and stimulating capacitative Ca2+ entry (31). In the control lobes, thapsigargin induced a significant augmentation in Kf,c after 1 h. This rise in permeability is unlikely to be caused by any corresponding venoconstriction, because the change in Pc was far below that previously reported to be required to induce changes in Kf,c (46). Interestingly, despite a significant rise in both venous and arterial resistances after thapsigargin administration to lobes from paced animals, there was no increase in Kf,c. This provides further evidence that mechanisms responsible for changing permeability in normal dog lungs are altered after heart failure. Our control data are in agreement with a recent study that showed an increase in pulmonary microvascular permeability mediated by thapsigargin in isolated, buffer-perfused rat lungs, which required the presence of extracellular Ca2+ (4).

Experiments were subsequently performed with the Ca2+ ionophore, A-23187, to determine whether simply raising intracellular Ca2+ concentrations would in itself result in increased permeability in lungs from paced animals. Our results show that in control lobes A-23187 produced a dose-dependent increase in Kf,c. Khimenko et al. (17) reported similar findings in buffer-perfused rat lungs. In contrast to thapsigargin, A-23187 also produced a significant increase in Kf,c in lobes from paced animals. The mechanism of ionophore-induced permeability is not clear but appears to be independent of myosin light chain phosphorylation by a Ca2+/calmodulin-dependent myosin light chain kinase (10, 17, 38).

Regardless of mechanism, our data indicate that increasing endothelial Ca2+ is sufficient to induce a change in lung permeability after heart failure. This suggests that adaptations are likely to be occurring either at the Ca2+ entry step or in the signaling pathway linking store depletion and channel opening to allow Ca2+ entry into the cell. Similarly, the fact that both ANG II and thapsigargin fail to induce a permeability response in pace lobes suggests that a downregulation of the ANG II AT1 receptor in endothelium per se is unlikely to be the sole cause of this phenomenon. Because thapsigargin induces Ca2+ entry independently of Ins(1,4,5)P3, stunted Ins(1,4,5)P3 production after heart failure is also unlikely. The precise mechanism responsible for capacitative Ca2+ entry is still unclear, but two major theories have been proposed. One suggests that depletion of the intracellular Ca2+ pool triggers the release of a factor that diffuses to the plasma membrane to open the Ca2+-release-activated Ca2+ (CRAC) channel (6, 27, 33). If this was indeed the case, it is plausible that the production of this mediator is inhibited in lung lobes from paced animals. The second hypothesis concerning capacitative Ca2+ entry is that the emptying of intracellular Ca2+ store produces a conformational change in the organelle and/or its surface proteins that is relayed to the CRAC channel either by direct coupling (16) or via the cytoskeleton (31). This would suggest that mechanisms coupling the Ca2+ store and the CRAC channel could be disrupted during pacing, thus preventing the rise in intracellular Ca2+ and subsequent increase in permeability, or that heart failure results in a downregulation of the CRAC channel itself. Finally, one could argue that an increased rate of Ca2+ extrusion in the paced group might explain the resistance of the lung endothelium to injury. However, if this were true, one could expect attenuation of the A-23187-induced permeability change as well in this group. Obviously, more studies are required to clarify the exact mechanism of capacitative Ca2+ entry and to identify the component of the pathway that may change after pacing-induced heart failure.

In summary, this report shows that ANG II-induced increase in pulmonary microvascular permeability is not PKC-mediated, suggesting the involvement of an Ins(1,4,5)P3-dependent mechanism. Administration of thapsigargin, a stimulator of endothelial capacitative Ca2+ entry, to isolated blood-perfused lung lobes resulted in increased permeability in controls but not after pacing-induced heart failure. It is hypothesized that ANG II and thapsigargin induce permeability changes in control lobes via a common mechanism, raising intracellular Ca2+ through capacitative Ca2+ entry. Because increasing intracellular Ca2+ concentration using a Ca2+ ionophore is sufficient to promote vascular permeability in lung lobes from paced animals, we conclude that after heart failure the pulmonary vasculature adapts in such a way that Ca2+ signaling is altered and lung injury is avoided.

    ACKNOWLEDGEMENTS

The authors thank Dr. D. Lynn Dyess and Jimmy Lakey for invaluable assistance.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-39045.

Address for reprint requests: M. I. Townsley, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688.

Received 27 October 1997; accepted in final form 19 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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