The Concept of Cellular Tone: Reflections on the Endothelium, Fibroblasts, and Smooth Muscle Cells Carl A. Boswell, Isabelle Joris, Guido Majno Perspectives in Biology and Medicine, Volume 36, Number 1, Autumn 1992, pp. 79-86 (Article) Published by Johns Hopkins University Press DOI: https://doi.org/10.1353/pbm.1993.0008
For additional information about this article https://muse.jhu.edu/article/400942/summary
Access provided at 6 Jan 2020 03:57 GMT from San Francisco State University
THE CONCEPT OF CELLULAR TONE: REFLECTIONS
ON THE ENDOTHELIUM, FIBROBLASTS, AND SMOOTH MUSCLE CELLS CARL A. BOSWELL, ISABELLE JORIS, and GUIDO MAJNO*
What Is Tone?
Tone is currently understood as a property of muscular tissue, smooth
as well as striated. The purpose of this essay is to explore the meaning of tone at the level of cells, both muscular and non-muscular.
The two basic meanings of the word tone—musical as well as me-
chanical—have not changed since the tónos of the ancient Greeks, which stood for pitch as well as for tension [I]. In Guyton's authoritative Textbook
of Medical Physiology, tone is discussed in relation to smooth muscle [2]:
Smooth muscle can maintain a state of long-term, steady contraction that has
been called either tonus contraction of smooth muscle or simply smooth muscle tone. This is an important feature of smooth muscle contraction because it allows
prolonged or even indefinite continuance of the smooth muscle function. For
instance, the arterioles are maintained in a state of tonic contraction almost
throughout the entire life of the person.
Guyton then proceeds to explain that tonic contractions of smooth muscle can be caused in one of two ways: (1) sometimes by summation of individual contractile pulses, each one initiated by a separate action potential, or (2) more often by prolonged direct smooth muscle excitation without action potentials, caused by local or by circulating hormones.
All this means that the concept of tone as a prolonged or even indefinite state of contraction is well established for smooth muscle as a tissue.
But then, if the tissue of an arteriole can be in a state of semi-permanent This work was supported by NIH grant HL-25973.
The authors thank Ms. Anne H. Cutler for technical assistance and Ms. Jane M. Manzi for manuscript preparation.
""Department of Pathology, University of Massachusetts Medical Center, 55 Lake Ave-
nue North, Worcester, Massachusetts 01655.
© 1992 by The University of Chicago. All rights reserved.
003 1-5982/93/360 1-0789$0 1 .00
Perspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992 | 79
contraction, where does that contraction come from? We will argue that it can only come from the individual smooth muscle cells. In other
words, tone must exist also at the level of cells. This concept appears self-evident, but we have not seen it expressed; it seems to have been
lost in the gap between whole-organ physiologists and single-cell biologists. Since endothelial cells and fibroblasts are also known to be contractile, we will further argue that they should also be able to maintain a long-lasting tone. Tone at the Level of Cells We came across cellular tone in a series of experiments on cultured cells. Our original purpose was to challenge three types of contractile
cells (endothelium, smooth muscle cells, and fibroblasts) with con-
tracting and relaxing agents, and to compare their responses [3]. To do so we adopted a clever system devised a decade ago [4]. The principle is to seed cells on a membrane thin enough to be wrinkled if the cell
contracts. The membrane itself is prepared by spreading a drop of silicone on a glass surface, then quickly passing it through a flame, so that the surface polymerizes whereas the silicone beneath it remains fluid.
The cells, in our experience, have some difficulty in attaching to the silicone membrane, but they can be coaxed to do so if the surface is treated appropriately, i.e., with Cell-Tak (Collaborative Research, Inc., Bedford, MA) and gelatin, plus laminin and fibronectin for endothelial cells. We used aortic endothelium and smooth muscle cells from bovines
and from rats, as well as fibroblasts from rat renal capsule and from calf dermis.
Accordingly, we seeded the three types of cells on silicone membranes and waited for them to settle, that is, to become firmly attached. As soon as that happened, the plan was to stimulate the cells with suitable contracting agents, such as bradykinin or serotonin. However, an un-
foreseen event frustrated our plans: the cells did settle, but in doing so they also contracted. Beautiful wrinkles appeared in the silicone sub-
strate (Figure 1). Similar findings were reported by Kelley, et al. [5], in
their study of endothelial cells, pericytes, and smooth muscle cells. At first we reacted to this turn of events with dismay: if the cells were already in a state of contraction at the start, how could we ever study the effect of contracting agents? Then it dawned on us that this observation was a finding in itself: perhaps these three types of cells have an
inborn tendency to maintain a basal level of contraction—a tone. How could tone arise in cultured cells? In the passage quoted above, Guyton suggests that tissues can be kept in a state of tone by local or general hormones. Cultured cells could be stimulated in similar fashion by agents in the medium; a perfect candidate would be calf serum, 80 Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
Fig. 1.—An example of rat endothelial cells on a polymerized silicone membrane. The
cells have maintained this level of wrinkling for seven days without a change of medium.
which we routinely added in the proportion of 10 percent. Although serum is often thought of as a physiologic medium, it is in fact highly abnormal. It contains the factors released by platelets, including serotonin, the products of the coagulation cascade, such as the fibrinopeptides which are known to induce vascular leakage [6-8], and thrombin [9], which is a powerful stimulant of endothelial contraction [3, 10]. We
therefore decreased the concentration of serum to 0.5 percent, but all three cell types still contracted. Most powerful, as judged by the extent of wrinkling, were the smooth muscle cells and the fibroblasts; even single cells produced wrinkling, whereas endothelial cells wrinkled more effectively when they formed confluent patches. Assuming that the active ingredients in serum would be of low molecular weight, we tested the effect of 2 percent dialyzed (12-14 kilodalton cut-off) calf serum on cells for three days; the degree of wrinkling did not change. To extend this reasoning, cells were washed thoroughly with phosphate-buffered saline, then incubated in serum-free medium
for three days with only minor loss of wrinkling. Finally, cells in complete medium were placed in anoxic conditions to assess their requirements for oxygen in the contractile process. The cells maintained wrinPerspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992
81
kling for as long as four days. Currently we are testing the hypothesis that the maintenance of tone requires expenditure of energy; experiments with anoxic cultures indicate that this is indeed the case.
At this stage of our study we do not know which factors in the culture medium maintain the state of contraction of the three cell types; perhaps they differ for each type. The experiments with serum-free medium suggest three possible conclusions: (1) the stimulating agent is an unknown autocrine factor; (2) the cells tested can develop an intrinsic tone without a specific stimulus from the medium; (3) persistent contraction in serum-free medium does not rule out the need for a stimulus—
the cells could still be under the effect of some agent(s) present in the serum before that time. Whichever may be the correct answer, we can say for sure that all three types of cells—endothelium, smooth muscle,
and fibroblasts—are able to maintain a state of contraction for days on end.
An analogy comes to mind, although it may not apply at the metabolic
level: the catch muscles of bivalve molluscs can sustain isometric contrac-
tion for over an hour, while apparently maintaining their intracellular calcium at basal levels [11] and oxygen consumption at a fraction above resting levels [12]. Preliminary studies of contraction in serum-free anoxic conditions indicate that calf aortic endothelial cells can maintain
tension without oxygen for at least 33 hours, as long as glucose is present.
Implications of Cellular Tone In Vivo In discussing the implications of cellular tone, we will consider only the three types of contractile cells that we have been studying: 1)For smooth muscle cells, the implications are well established: arteriolar tone helps maintain blood pressure; tone in the wall of hollow viscera such as the gut or the urinary bladder is an integral part of function in those organs [2].
2)For fibroblasts, the principal implication of the tone concept regards the healing of open wounds, as well as the slow contraction of many fibrotic lesions [13] and possibly embryonic morphogenesis [14]. Indeed rather than fibroblasts we should say myofibroblasts, the accepted name of fibroblasts when they modulate toward a contractile mode [15]. This modulation is observed also in cultured fibroblasts [16, 17], presumably because the conditions of cell culture are basically those of an open wound. During the closure of a wound, the myofibroblasts develop traction by means of cell-to-cell contacts, as well as by forming "microtendons" which pull on the stroma [18]. Two types of contraction have been proposed and appear to coexist: (1) an immediate and reversible
response similar to that of smooth muscle, easily recorded in vitro [13], 82
Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
and (2) a "hand-over-hand" type of traction, which should be much
slower and irreversible [14]. Both mechanisms should be able to sustain
a constant pull, i.e., a tone that is maintained for days and possibly weeks. Conceivably, the bundles known as "stress fibers" might come
into play in maintaining the tone; it has been said that they are able to sustain an isometric contraction [19].
3) For endothelial cells, the concept of tone is relevant at the level of the capillaries and venules, in which the entire wall consists of endothelium, partially supported by pericytes but devoid of smooth muscle cells. In larger vessels the contractile properties of the endothelial cells, as regards vascular tone, are probably outweighed by those of the smooth muscle, but it was interesting to see—in our experiments—that the endothelium from the aorta also showed the capacity to contract. The presence of actin, myosin [20, 21], and myosin light chain kinase [22] all point to the similarities between smooth muscle and endothelial cells. Oddly enough, all discussions of vascular tone in the contemporary
physiologic literature exclude or at least omit any discussion of the capillaries [23], presumably because they are widely thought to have "no intrinsic constrictor mechanism of their own" [24]. The only exception
known to us is the role assigned to endothelial sphincters in regulating flow through the liver, spleen, bone marrow, and other organs [25].
The tone of the arterioles is thought to play the key role in determining blood pressure; the only critique of this concept has been that larger arteries may also be involved [26]. The endothelium is thought to be involved in vascular tone only by influencing the smooth muscle of the media [27, 28]. Yet the capillary wall must be able to maintain its own tone, because it offers considerable resistance to blood flow. This is
reflected in the pressure drop along the capillary proper, which according to Guyton is of the order of 20 mm Hg [2]. A study of the pressure profile in the vessels of the hamster cheek pouch has reported a drop of 10 mm in capillaries [29]; another study reported a percentage pressure drop varying from 22 to 44 percent of the total drop [30]. Whatever the exact figures may be in any given animal model, it seems clear that capillaries contribute significantly to the peripheral resistance, perhaps amounting to 15—20 percent. The inescapable conclusion is that capillaries must have a tone. This idea has surfaced before. Capillary tone was taken for granted in the
classic 1918 studies of Dale and Richards, on the vasodilator responses
to histamine [31]. Subsequently, the notion prevailed that capillary walls were inert, and the concept of capillary tone disappeared. In the 1960s endothelial contraction was demonstrated in the venules, by electron
microscopy as well as in vivo (reviewed in [32]), and the topic regained credibility. Eventually, in the 1980s it became possible to demonstrate cellular contraction in vitro, using the retraction of collagen lattices as Perspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992 | 83
well as the silicone method. The elegant study of Kelley, et al. [5], based on these techniques, offered direct proof that three types of vascular cells can contract (retinal pericytes, smooth muscle cells, and pulmonary microvascular endothelial cells); it was concluded that these cells could contribute to "microvascular tonus." However, Sims [33] suggests that,
in the case of dermal and choroidal capillaries, the arrangement of pericytes along the vessel wall argues against a contractile function for these cells.
The concept of capillary tone should be applicable not only in normal physiology (in relation to blood pressure) but also in pathologic situations, both local and general. Although capillaries are usually said to change little in caliber, they do dilate in response to cold [34], and some dilatation certainly occurs in inflammation, ischemia, and reperfusion injury; pathologists acknowledge capillary tone indirectly when they apply the term "paralyzed" to the greatly dilated capillaries of ischemic tissues. Capillary tone is potentially important in shock, regarding the maldistribution of blood [35] as well as the overall pressure drop.
To sum up: if tissues have tone, cells must have tone. Once this is accepted, many questions arise: Which cells can develop tone and which can not? What factors trigger tone, and how is it sustained? How does cellular tone come into play in human disease? We hope that answers will soon be at hand. REFERENCES
1.Liddell, H. G., and Scott, R. A Greek-English Lexicon. Oxford: Clarendon Press, 1968.
2.Guyton, A. C. Textbook ofMedical Physiology, 7th ed. Philadelphia: Saunders, 1986.
3.Boswell, C. ?.; Majno, G.; Joris, I.; and Ostrom, K. A. Acute endothelial cell contraction in vitro: A comparison with fibroblasts and vascular smooth
muscle cells. Microvasc. Res. 43:178-191, 1992. 4.Harris, A. K.; Wild, P.; and Stopak, D. Silicone rubber substrata: A new
wrinkle in the study of cell locomotion. Science 208:177-179, 1980. 5.Kelley, C; D'Amore, P.; Hechtman, H. B.; and Shepro, D. Microvascular
pericyte contractility in vitro: Comparison with other cells of the vascular
wall.). Cell Biol. 104:483-490, 1987.
6.Rowland, F. N.; Donovan, M. J.; Picciano, P. T.; et al. Fibrin-mediated
vascular injury. Identification of fibrin peptides that mediate endothelial cell retraction. Am. J. Pathol. 117:418-428, 1984.
7.Busch, C, and Gerdin, B. Effect of low molecular weight fibrin degrada-
tion products on endothelial cells in culture. Thromb. Res. 22:33-39, 1981. 8.Kadish, J. L.; Butterfield, C. E.; and Folkman, J. The effect of fibrin on cultured vascular endothelial cells. Tissue ¿f Cell 11:99-108, 1979.
9.Laposata, M.; Dovnarsky, D. K.; and Shin, H. S. Thrombin-induced gap
formation in confluent endothelial cell monolayers in vitro. Blood 62:549556, 1983.
10. Morel, N. M. L.; Petruzzo, P. P.; Hechtman, H. B.; and Shepro, D. 84
Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
Inflammatory agonists that increase microvascular permeability in vivo stimulate cultured pulmonary microvessel endothelial cell contraction. Inflammation 14:571-583, 1990.
11.Atsumi, S., and Sugi, H. Localization of calcium-accumulating structures in the anterior byssal retractor muscle of Mytilus edulis and their role in the regulation of active and catch contractions./. Physiol. 257:549-560, 1976. 12.Baguet, F., and Gillis, J. M. Energy cost of tonic contraction in a lamellibranch catch muscle. /. Physiol. 198:127-143, 1968.
13.Majno, G.; Gabbiani, G.; Hirschel, B. J.; et al. Contraction of granulation tissue in vitro: Similarity to smooth muscle. Science 173:548-550, 1971. 14.Lewis, J. Morphogenesis by fibroblast traction. Nature 307:413-414, 1984.
15.Majno, G. The story of the myofibroblasts. Am. J. Surg. Pathol. 3:535-542, 1979.
16.Goldberg, B., and Green, H. An analysis of collagen secretion by estab-
lished mouse fibroblast lines./. Cell Biol. 22:227-258, 1964. 17.Devis, R., and James, D. W. Close associations between adult guinea-pig fibroblasts in tissue culture, studied with the electron microscope. /. Anal. 98:63-68, 1964.
18.Ryan, G. B.; Cliff W. J.; Gabbiani, G; et al. Myofibroblasts in human granulation tissue. Hum. Pathol. 5:55-67, 1974. 19.Byers, H. R.; White, G. E.; and Fujiwara, K. Organization and function of stress fibers in cells in vitro and in situ. A review. Cell Muscle Motil.
5:83-137, 1984.
20.Drenckhahn, D., and Wagner, J. Stress fibers in the splenic sinus endothelium in situ: Molecular structure, relationship to the extracellular matrix, and contractility. /. Cell Biol. 102:1738-1747, 1986. 21.Wong, M. K. K., and Gotlieb, A. I. Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments. Arteriosclerosis 6:212-219, 1986.
22.Wysolmerski, R. B., and Lagunoff, D. Involvement of myosin light-chain kinase in endothelial cell retraction. Proc. Natl. Acad. Sci. USA 87:16—20, 1990.
23.Joshua, I. G; Fleming, J. T.; and Dowe, J. P. The influence of extracellular calcium on microvascular tone in the rat cremaster muscle (42817). Proc. Soc. Exp. Biol. Med. 189:344-352, 1988. 24.Webb, W. R., and Brunswick, R. A. Microcirculation in shock—clinical
review. In Pathophysiology of Shock, Anoxia, and Ischemia, edited by R. A. Cowley and B. F. Trump. Baltimore: Williams and Wilkins, 1982.
25.McCuskey, R. S. Microcirculation—basic considerations. In Pathophysiology of Shock, Anoxia, and Ischemia, edited by R. A. Cowley and B. F. Trump. Baltimore: Williams and Wilkins, 1982.
26.Grega, G. J. Constrictor responses in large vessels. Fed. Proc. 46:264-265, 1987.
27.Busse, R.; Trogisch, G.; and Bassenge, E. The role of endothelium in the control of vascular tone. Basic Res. Cardiol. 80:475-490, 1985.
28.Rubanyi, G. M. Maintenance of "basal" vascular tone may represent a physiologic role for endothelin./. Vase. Med. Biol. 6:315-316, 1989.
29.Davis, M. J.; Ferrer, P. N.; and Gore, R. W. Vascular anatomy and hydrostatic pressure profile in the hamster cheek pouch. Am. J. Physiol. 250: H291-H303, 1986.
30.Joyner, W. L., and Davis, M. J. Pressure profile along the microvascular network and its control. Fed. Proc. 46:266-269, 1987.
Perspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992
85
31.Dale, H. H., and Richards, A. N. The vasodilator action of histamine and
of some other substances./. Physiol. 52:110-165, 1918. 32.Joris, I.; Majno, G.; Corey, E. J.; and Lewis, R. A. The mechanism of vascular leakage induced by leukotriene E4. Endothelial contraction. Am. J. Pathol. 126:19-24, 1987.
33.Sims, D. E. The pericyte—a review. Tissue and Cell 18:153-174, 1986. 34.Sandison, J. C. Contraction of blood vessels and observations on the circulation in the transparent chamber in the rabbit's ear. Anal. Record 54:105—127, 1932.
35.Chaudry, I. H., and Baue, A. E. Overview of hemorrhagic shock. In Pathophysiology of Shock, Anoxia, and Ischemia, edited by R. A. Cowley and B. F. Trump. Baltimore: Williams and Wilkins, 1982.
86 Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
For additional information about this article https://muse.jhu.edu/article/400942/summary
Access provided at 6 Jan 2020 03:57 GMT from San Francisco State University
THE CONCEPT OF CELLULAR TONE: REFLECTIONS
ON THE ENDOTHELIUM, FIBROBLASTS, AND SMOOTH MUSCLE CELLS CARL A. BOSWELL, ISABELLE JORIS, and GUIDO MAJNO*
What Is Tone?
Tone is currently understood as a property of muscular tissue, smooth
as well as striated. The purpose of this essay is to explore the meaning of tone at the level of cells, both muscular and non-muscular.
The two basic meanings of the word tone—musical as well as me-
chanical—have not changed since the tónos of the ancient Greeks, which stood for pitch as well as for tension [I]. In Guyton's authoritative Textbook
of Medical Physiology, tone is discussed in relation to smooth muscle [2]:
Smooth muscle can maintain a state of long-term, steady contraction that has
been called either tonus contraction of smooth muscle or simply smooth muscle tone. This is an important feature of smooth muscle contraction because it allows
prolonged or even indefinite continuance of the smooth muscle function. For
instance, the arterioles are maintained in a state of tonic contraction almost
throughout the entire life of the person.
Guyton then proceeds to explain that tonic contractions of smooth muscle can be caused in one of two ways: (1) sometimes by summation of individual contractile pulses, each one initiated by a separate action potential, or (2) more often by prolonged direct smooth muscle excitation without action potentials, caused by local or by circulating hormones.
All this means that the concept of tone as a prolonged or even indefinite state of contraction is well established for smooth muscle as a tissue.
But then, if the tissue of an arteriole can be in a state of semi-permanent This work was supported by NIH grant HL-25973.
The authors thank Ms. Anne H. Cutler for technical assistance and Ms. Jane M. Manzi for manuscript preparation.
""Department of Pathology, University of Massachusetts Medical Center, 55 Lake Ave-
nue North, Worcester, Massachusetts 01655.
© 1992 by The University of Chicago. All rights reserved.
003 1-5982/93/360 1-0789$0 1 .00
Perspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992 | 79
contraction, where does that contraction come from? We will argue that it can only come from the individual smooth muscle cells. In other
words, tone must exist also at the level of cells. This concept appears self-evident, but we have not seen it expressed; it seems to have been
lost in the gap between whole-organ physiologists and single-cell biologists. Since endothelial cells and fibroblasts are also known to be contractile, we will further argue that they should also be able to maintain a long-lasting tone. Tone at the Level of Cells We came across cellular tone in a series of experiments on cultured cells. Our original purpose was to challenge three types of contractile
cells (endothelium, smooth muscle cells, and fibroblasts) with con-
tracting and relaxing agents, and to compare their responses [3]. To do so we adopted a clever system devised a decade ago [4]. The principle is to seed cells on a membrane thin enough to be wrinkled if the cell
contracts. The membrane itself is prepared by spreading a drop of silicone on a glass surface, then quickly passing it through a flame, so that the surface polymerizes whereas the silicone beneath it remains fluid.
The cells, in our experience, have some difficulty in attaching to the silicone membrane, but they can be coaxed to do so if the surface is treated appropriately, i.e., with Cell-Tak (Collaborative Research, Inc., Bedford, MA) and gelatin, plus laminin and fibronectin for endothelial cells. We used aortic endothelium and smooth muscle cells from bovines
and from rats, as well as fibroblasts from rat renal capsule and from calf dermis.
Accordingly, we seeded the three types of cells on silicone membranes and waited for them to settle, that is, to become firmly attached. As soon as that happened, the plan was to stimulate the cells with suitable contracting agents, such as bradykinin or serotonin. However, an un-
foreseen event frustrated our plans: the cells did settle, but in doing so they also contracted. Beautiful wrinkles appeared in the silicone sub-
strate (Figure 1). Similar findings were reported by Kelley, et al. [5], in
their study of endothelial cells, pericytes, and smooth muscle cells. At first we reacted to this turn of events with dismay: if the cells were already in a state of contraction at the start, how could we ever study the effect of contracting agents? Then it dawned on us that this observation was a finding in itself: perhaps these three types of cells have an
inborn tendency to maintain a basal level of contraction—a tone. How could tone arise in cultured cells? In the passage quoted above, Guyton suggests that tissues can be kept in a state of tone by local or general hormones. Cultured cells could be stimulated in similar fashion by agents in the medium; a perfect candidate would be calf serum, 80 Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
Fig. 1.—An example of rat endothelial cells on a polymerized silicone membrane. The
cells have maintained this level of wrinkling for seven days without a change of medium.
which we routinely added in the proportion of 10 percent. Although serum is often thought of as a physiologic medium, it is in fact highly abnormal. It contains the factors released by platelets, including serotonin, the products of the coagulation cascade, such as the fibrinopeptides which are known to induce vascular leakage [6-8], and thrombin [9], which is a powerful stimulant of endothelial contraction [3, 10]. We
therefore decreased the concentration of serum to 0.5 percent, but all three cell types still contracted. Most powerful, as judged by the extent of wrinkling, were the smooth muscle cells and the fibroblasts; even single cells produced wrinkling, whereas endothelial cells wrinkled more effectively when they formed confluent patches. Assuming that the active ingredients in serum would be of low molecular weight, we tested the effect of 2 percent dialyzed (12-14 kilodalton cut-off) calf serum on cells for three days; the degree of wrinkling did not change. To extend this reasoning, cells were washed thoroughly with phosphate-buffered saline, then incubated in serum-free medium
for three days with only minor loss of wrinkling. Finally, cells in complete medium were placed in anoxic conditions to assess their requirements for oxygen in the contractile process. The cells maintained wrinPerspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992
81
kling for as long as four days. Currently we are testing the hypothesis that the maintenance of tone requires expenditure of energy; experiments with anoxic cultures indicate that this is indeed the case.
At this stage of our study we do not know which factors in the culture medium maintain the state of contraction of the three cell types; perhaps they differ for each type. The experiments with serum-free medium suggest three possible conclusions: (1) the stimulating agent is an unknown autocrine factor; (2) the cells tested can develop an intrinsic tone without a specific stimulus from the medium; (3) persistent contraction in serum-free medium does not rule out the need for a stimulus—
the cells could still be under the effect of some agent(s) present in the serum before that time. Whichever may be the correct answer, we can say for sure that all three types of cells—endothelium, smooth muscle,
and fibroblasts—are able to maintain a state of contraction for days on end.
An analogy comes to mind, although it may not apply at the metabolic
level: the catch muscles of bivalve molluscs can sustain isometric contrac-
tion for over an hour, while apparently maintaining their intracellular calcium at basal levels [11] and oxygen consumption at a fraction above resting levels [12]. Preliminary studies of contraction in serum-free anoxic conditions indicate that calf aortic endothelial cells can maintain
tension without oxygen for at least 33 hours, as long as glucose is present.
Implications of Cellular Tone In Vivo In discussing the implications of cellular tone, we will consider only the three types of contractile cells that we have been studying: 1)For smooth muscle cells, the implications are well established: arteriolar tone helps maintain blood pressure; tone in the wall of hollow viscera such as the gut or the urinary bladder is an integral part of function in those organs [2].
2)For fibroblasts, the principal implication of the tone concept regards the healing of open wounds, as well as the slow contraction of many fibrotic lesions [13] and possibly embryonic morphogenesis [14]. Indeed rather than fibroblasts we should say myofibroblasts, the accepted name of fibroblasts when they modulate toward a contractile mode [15]. This modulation is observed also in cultured fibroblasts [16, 17], presumably because the conditions of cell culture are basically those of an open wound. During the closure of a wound, the myofibroblasts develop traction by means of cell-to-cell contacts, as well as by forming "microtendons" which pull on the stroma [18]. Two types of contraction have been proposed and appear to coexist: (1) an immediate and reversible
response similar to that of smooth muscle, easily recorded in vitro [13], 82
Carl A. Boswell, Isabelle Joris, and Guido Majno ¦ Concept of Cellular Tone
and (2) a "hand-over-hand" type of traction, which should be much
slower and irreversible [14]. Both mechanisms should be able to sustain
a constant pull, i.e., a tone that is maintained for days and possibly weeks. Conceivably, the bundles known as "stress fibers" might come
into play in maintaining the tone; it has been said that they are able to sustain an isometric contraction [19].
3) For endothelial cells, the concept of tone is relevant at the level of the capillaries and venules, in which the entire wall consists of endothelium, partially supported by pericytes but devoid of smooth muscle cells. In larger vessels the contractile properties of the endothelial cells, as regards vascular tone, are probably outweighed by those of the smooth muscle, but it was interesting to see—in our experiments—that the endothelium from the aorta also showed the capacity to contract. The presence of actin, myosin [20, 21], and myosin light chain kinase [22] all point to the similarities between smooth muscle and endothelial cells. Oddly enough, all discussions of vascular tone in the contemporary
physiologic literature exclude or at least omit any discussion of the capillaries [23], presumably because they are widely thought to have "no intrinsic constrictor mechanism of their own" [24]. The only exception
known to us is the role assigned to endothelial sphincters in regulating flow through the liver, spleen, bone marrow, and other organs [25].
The tone of the arterioles is thought to play the key role in determining blood pressure; the only critique of this concept has been that larger arteries may also be involved [26]. The endothelium is thought to be involved in vascular tone only by influencing the smooth muscle of the media [27, 28]. Yet the capillary wall must be able to maintain its own tone, because it offers considerable resistance to blood flow. This is
reflected in the pressure drop along the capillary proper, which according to Guyton is of the order of 20 mm Hg [2]. A study of the pressure profile in the vessels of the hamster cheek pouch has reported a drop of 10 mm in capillaries [29]; another study reported a percentage pressure drop varying from 22 to 44 percent of the total drop [30]. Whatever the exact figures may be in any given animal model, it seems clear that capillaries contribute significantly to the peripheral resistance, perhaps amounting to 15—20 percent. The inescapable conclusion is that capillaries must have a tone. This idea has surfaced before. Capillary tone was taken for granted in the
classic 1918 studies of Dale and Richards, on the vasodilator responses
to histamine [31]. Subsequently, the notion prevailed that capillary walls were inert, and the concept of capillary tone disappeared. In the 1960s endothelial contraction was demonstrated in the venules, by electron
microscopy as well as in vivo (reviewed in [32]), and the topic regained credibility. Eventually, in the 1980s it became possible to demonstrate cellular contraction in vitro, using the retraction of collagen lattices as Perspectives in Biology and Medicine, 36, 1 ¦ Autumn 1992 | 83
well as the silicone method. The elegant study of Kelley, et al. [5], based on these techniques, offered direct proof that three types of vascular cells can contract (retinal pericytes, smooth muscle cells, and pulmonary microvascular endothelial cells); it was concluded that these cells could contribute to "microvascular tonus." However, Sims [33] suggests that,
in the case of dermal and choroidal capillaries, the arrangement of pericytes along the vessel wall argues against a contractile function for these cells.
The concept of capillary tone should be applicable not only in normal physiology (in relation to blood pressure) but also in pathologic situations, both local and general. Although capillaries are usually said to change little in caliber, they do dilate in response to cold [34], and some dilatation certainly occurs in inflammation, ischemia, and reperfusion injury; pathologists acknowledge capillary tone indirectly when they apply the term "paralyzed" to the greatly dilated capillaries of ischemic tissues. Capillary tone is potentially important in shock, regarding the maldistribution of blood [35] as well as the overall pressure drop.
To sum up: if tissues have tone, cells must have tone. Once this is accepted, many questions arise: Which cells can develop tone and which can not? What factors trigger tone, and how is it sustained? How does cellular tone come into play in human disease? We hope that answers will soon be at hand. REFERENCES
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