Ca²⁺ Dependence of Mechanical Injury to Lung Capillaries

	LETTER

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Ca²⁺ Dependence of Mechanical Injury to Lung Capillaries

To the Editor: We appreciate the invited editorial by Fitz-Roy E. Curry (1) on our recent article "Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs" (6). Curry concurred with our hypothesis that endothelium, rather than the basement membrane, regulates fluid filtration at pressures below those required for capillary rupture (5, 9) and agreed that the active participation of endothelium in stress injury might be attributed to the well-accepted involvement of increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activation of myosin light chain kinase (MLCK) (3). However, he then proposed an alternate mechanism for mechanical stress-induced permeability increases based on a [Ca²⁺]_i-independent decrease in intracellular cAMP, possibly related to a dearth of plasmalemmal membrane, as the endothelium thins to follow the basement membrane strain (9).

Although we did not directly measure [Ca²⁺]_i in our study, we believe that the involvement of such a [Ca²⁺]_i-independent mechanism underlying these vascular permeability increases is highly unlikely for two reasons. First, Gd³⁺ completely blocked the permeability increase at high airway pressures at a dose that is known to inhibit cation entry into cells without significantly affecting other cellular processes (8). Second, an increase in [Ca²⁺]_i is known to decrease intracellular cAMP in endothelial cells by inhibiting a Ca²⁺-sensitive adenylyl cyclase (3). On the other hand, cellular cAMP could theoretically be decreased to increase vascular permeability without an increase in [Ca²⁺]_i by 1) a stretch-induced activation of G_i proteins; 2) an increase extrusion of cAMP; 3) an increased activity of cAMP-specific phosphodiesterases; 4) an activation of Ca²⁺-independent protein kinase C (PKC) isozymes that affect cytoskeletal integrity; or 5) an increased tyrosine phosphorylation of focal adhesion and cytoskeletal proteins involved in cell adhesion. There is evidence that shear stress activates G_i protein, but this has not been shown to be Ca²⁺ independent (4). Although stretch of endothelial cells has been shown to activate PKC and mitogen-activated protein kinase pathways independent of increases in [Ca²⁺]_i (10), tyrosine phosphorylation of cytoskeletal proteins is generally dependent on an increase in [Ca²⁺]_i (2). In fact, we have recently demonstrated (7) attenuation of high airway pressure-induced increases in permeability in the isolated rat lung model using genistein, a tyrosine kinase inhibitor, as well as augmentation of injury using the tyrosine phosphatase inhibitor phenylarsine oxide. However, both of these drugs also affect endothelial Ca²⁺ entry, so the exact contribution of each mechanism is uncertain (6).

In conclusion, it is not necessary to invoke a highly unlikely hypothesis for the active augmentation of high pressure-induced increases in vascular permeability. Rather, the proposed mechanism of Gd³⁺ in preventing Ca²⁺ entry through known stretch-activated cation channels with activation of calcium calmodulin, depression of cAMP, and inhibition of protein kinase A, with subsequent activation of MLCK and contraction of actin-myosin filaments, is the mechanism best supported by the available cell studies. Thus the proposed "stretch recoil" of endothelial cells, which contributes to an increased vascular permeability during mechanical stress, implicates many of the same pathways involved in receptor-mediated permeability responses. However, further studies are needed to detail these pathways.

	REFERENCES

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1.   Curry, F. E. Invited Editorial on "Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs." J. Appl. Physiol. 84: 1111-1112, 1998[Free Full Text].

2.   Fleming, I., B. Fisslthaler, and R. Busse. Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. J. Biol. Chem. 271: 11009-11015, 1996[Abstract/Free Full Text].

3.   Moore, T. M., P. M. Chetham, J. J. Kelly, and T. Stevens. Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and AMP (Review). Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 17): L203-L222, 1998[Abstract/Free Full Text].

4.   Ohno, M., G. H. Gibbons, V. J. Dzau, and J. P. Cooke. Shear stress elevates endothelial cGMP role of a potassium channel and G protein coupling. Circulation 88: 193-197, 1993[Abstract/Free Full Text].

5.   Parker, J. C., and C. L. Ivey. Isoproterenol attenuates high vascular pressure-induced permeability increases in isolated rat lungs. J. Appl. Physiol. 83: 1962-1967, 1998[Abstract/Free Full Text].

6.   Parker, J. C., C. L. Ivey, and A. Tucker. Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs. J. Appl. Physiol. 84: 1113-1118, 1998[Abstract/Free Full Text].

7.   Parker, J. C., C. L. Ivey, and A. Tucker. Phosphotyrosine phosphatase and tyrosine inhibition modulate airway pressure-induced lung injury. J. Appl. Physiol. 85: 1753-1761, 1998[Abstract/Free Full Text].

8.   Sackin, H. Mechanosensitive channels. Annu. Rev. Physiol. 57: 333-353, 1995[Medline].

9.   Townsley, M. I., C. R. Neal, and C. C. Michel. High-pressure vascular injury. In: Connective Tissue Biology, edited by R. K. Reed, and K. Rubin. London: Portland, 1998, p. 207-219.

10.   Traub, O., B. P. Monia, N. M. Dean, and B. C. Berk. PKC-epsilon is required for mechano-sensitive activation of ERK 1/2 in endothelial cells. J. Biol. Chem. 272: 31251-31257, 1997[Abstract/Free Full Text].

James C. Parker, Mary I. Townsley, Troy Stevens, Departments of Physiology and Pharmacology University of South Alabama Mobile, Alabama 36688

	REPLY

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To the Editor: I stand by my editorial comments (1), which supported the conclusion of Parker's group (4) that mechanisms within the endothelial cells forming part of the blood-gas barrier participate actively in the formation of the high permeability state with increased pulmonary vascular or increased alveolar volume. This is an important contribution, and it opens the way for new experimental studies of these effects of high airway pressure on increased permeability. However, the nature of the endothelial cell mechanisms that may lead to increased permeability when the endothelium and associated basement membrane are stretched remains unclear. In my editorial, I emphasized this issue by describing alternative mechanisms that may contribute to an increase in permeability.

The model that Parker and colleagues (4) used to account for the observation that gadolinium blocked the increase in permeability with high airway pressure is very similar to that used to account for the increased permeability when venular endothelium is exposed to acute inflammatory mediators. The main features of the model are Ca²⁺ activation of MLCK, resulting in tension development within the endothelium and gap formation within the endothelial barrier. Agents that attenuate increased permeability are hypothesized to act by reducing the extent of contraction within endothelial cells. Thus gadolinium was assumed to decrease Ca²⁺ influx, and agents that raise intracellular cAMP and attenuate the permeability response were assumed to reduce MLCK activity. I do not disagree that these ideas provide a useful working hypothesis.

However, the novel nature of the stretch-activated response, and experimental observations that are not easily accounted for by the above paradigm, suggest that some alternative hypotheses need to be evaluated. For example, in intact microvessels, cAMP directly and rapidly modifies the number of tight junction strands between endothelial cells. Thus a process that lowers cAMP may reduce adhesion between adjacent endothelial cells. In this state, the stress developed within endothelial cells as the result of mechanical deformation (as opposed to active contraction) may be sufficient to cause cell retraction. The lowered cAMP may be due to inhibition of adenylate cyclase by Ca²⁺, as noted by Parker and colleagues in their letter, or to a number of signal-transduction pathways that result in increased cAMP phosphodiesterase activity. I also drew attention to the fascinating new observations that some of the gaps formed as a result of increased pressure and exposure to inflammatory mediators are transcellular, not between adjacent endothelial cells. These mechanisms may involve reorganization of vesicles in the region of the junction in response to applied tension, as suggested by Neal and Michel (3) and Feng et al. (2).

The important point is that we have much to learn about the mechanisms regulating microvessel permeability under a variety of physiological and pathological conditions. Endothelial failure under high pressure is a useful new experimental model to evaluate the parallels between stretch-activated endothelial mechanisms that may increase permeability and the better known acute inflammatory responses.

	REFERENCES

Top Letter References Reply R-References

1.   Curry, F. E. Invited Editorial on "Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lung." J. Appl. Physiol. 84: 1111-1112, 1998.

2.   Feng, D., J. A. Nagy, J. Hipp, K. Pyne, H. F. Dvorak, and A. M. Dvorak. Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-pig, mouse, and rat: many are transcellular pores. J. Physiol. (Lond.) 504: 747-761, 1997[Abstract/Free Full Text].

3.   Neal, C. R., and C. C. Michel. Transcellular gaps in microvascular walls of frog and rat when permeability is increased by perfection with the ionophore A-23187. J. Physiol. (Lond.) 488: 427-437, 1995[Abstract/Free Full Text].

4.   Parker, J. C., C. L. Ivey, and J. A. Tucker. Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lung. J. Appl. Physiol. 84: 1113-1118, 1998.

Fitz-Roy E. Curry, Department of Human Physiology University of California, Davis, School of Medicine Davis, California 95615