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Microvascular effects of endothelin in the rabbit tenuissimus muscle and hamster cheek pouch
MICROVASCULAR
RESEARCH
37, 115-l
18 (1989)
BRIEF COMMUNICATIONS Microvascular A. *Department
Effects of Endothelin Muscle and Hamster
O)HL~~N,*“~
of
Physiology
J.
in the Rabbit Tenuissimus Cheek Pouch
P. HEuovtsr,*‘t
RAUD,“?
AND
N. P.
and flnstitute of Environmental Medicine, S-104 01 Stockholm, Sweden Received
June
WIKLUND*
Karolinska
Instituter,
22, 1988
INTRODUCTION In the last decade, the vascular endothelium has gained increased recognition as an important factor for the regulation of vascular tone. Thus, an intact endothelium is a prerequisite for vasodilatation induced by a number of stimuli, including acetylcholine and substance P (see Furchgott, 1984). One endotheliumderived relaxing factor (EDRF) has been tentatively identified as nitric oxide (Palmer et al., 1987). The release of a vasoconstrictive agent from endothelial cells in response to anoxia, stretch, increased transmural pressure (Vanhoutte, 1987; Harder, 1987), and several other stimuli has also been suggested. Recently, Yanagisawa et aE. (1988) described a novel endothelium-derived 21 amino acid peptide, endothelin, and showed that this peptide is extremely potent in contracting isolated arteries and causes a long-lasting pressor response when injected intravenously to chemically denervated rats. The aim of this study was to characterize the microvascular effects of endothelin using intravital microscopy of the rabbit tenuissimus muscle and the hamster cheek pouch. MATERIALS
AND METHODS
New Zealand white rabbits (n = 6) weighing 0.8-1.2 kg were anesthetized with urethane, 1.5 g x kg-’ via an ear vein. The tenuissimus muscle in the left hind leg was prepared for intravital microscopy as previously described (Lindbom et al., 1982). The diameter of transverse arterioles (control diameter 14-28 pm), terminal arterioles (5-12 pm), and venules (5-50 pm) was studied by intravital microscopy using a Leitz Intravital microscope and water immersion lens (Leitz SW x 25, NA 0.60). Blood flow velocity in transverse arterioles was measured by the dual slit method (Wayland and Johnson, 1967). Blood flow was calculated from the diameter and velocity values. 115 C026-2862/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
116
BRIEF
COMMUNICATION
- Log cont. (M) FIG. 1. Effect of endothelin (IO-“-3 x 10m9 A4) on blood flow in transverse arterioles of the rabbit tenuissimus muscle. Mean values 2 SEM from five experiments.
As previously described (Bjork et al., 1984), arteriolar diameters were studied in the cheek pouch of anesthetized hamsters, prepared for intravital microscopy. Plasma extravasation in the cheek pouch was quantitated using FITC-dextran (mol wt 150,000, 25 mg/lOO g body wt iv) as a tracer for plasma proteins and expressed as the peak number of leakage spots per square centimeter of cheek pouch area (Bjiirk et al., 1984). Synthetic endothelin (lo-“-3 x 1O-9 M; Peninsula Lab., Inc., Belmont, CA) was dissolved in saline and added with the buffer solutions superfusing the muscle and the cheek pouch, respectively. RESULTS Endothelin (topically for 8 min) decreased blood flow in the rabbit tenuissimus muscle in a dose-dependent manner (Fig. 1). At 3 x 10m9 M, endothelin caused flow to cease completely in transverse arterioles. The terminal arterioles were as sensitive as transverse arterioles and started to contract at a dose of 3 x lo-” M. Venules did not change in diameter (102 +- 2% of control; n = 6) even at the highest dose used (3 x 10m9 M). The effect of endothelin could be seen after l-2 min of application, reached a peak after 7-8 min, and lasted for up to 50 min. After control flow was attained, a repeated dose of endothelin (3 x 10-l’ M; n = 3) caused the same vascular response regarding both peak contraction (17 ? 5% of control diameter) and duration (40-50 min). Endothelin (topically for ‘5 min) also potently constricted arterioles (inner diameter lo-50 pm) in the hamster cheek pouch. A threshold response was observed at 10-l’ M and intense constriction with ceased blood flow occurred at l-3 x 10d9 M (n = 4) Fig. 2). The arteriolar spasm was fully developed within 1 min, and lasted up to 60 min after challenge. Thereafter, the vessels responded equally well to repeated challenge with endothelin. The vasoconstriction caused by endothelin (3 x 1O-9 M, for 10 min) was abolished and replaced by vasodilatation after addition of prostaglandin E2 (PGE*, 3 x lo-’ M topically) for 10 min, starting 5 min after the start of endothelin. However, within minutes after the PGE2 addition was stopped, the arterioles regained the constricted
BRIEF COMMUNICATION
117
FIG. 2. Micrographs of arterioles in the hamster cheek pouch before (a) and after (b) the application of endothelin (1O-9 M). Bar = 20 pm.
state, despite the fact that endothelin was no longer present (n = 2). Likewise, nitroprusside (3 x lop6 M, Roche AG, Base& Switzerland) caused a temporary abolishment of endothelin-induced contractions (n = 3). No leakage of plasma (FITC-dextran extavasation) occurred in the cheek pouch after challenge with endothelin (3 x 10e9 M; 12 = 2). Prostaglandin E2 (3 x 10m8 M) caused a minor increase in plasma leakage (16 ? 6 leakage sites/cm’) and there was no difference when PGE2 was coadministered with endothelin (12 k 6; n = 2). In both the tenuissimus muscle and cheek pouch, a desensitization occurred when endothelin was given continuously in increasing doses (successive 8 min periods). Indeed, doses that caused maximal vasoconstriction in single doses sometimes elicited vasodilatation when applied in this way. Thus, in the tenuissimus muscle the diameter of transverse arterioles was 97 + 8% of control at 3 X lop9 M after continuous application (n = 4) as compared to 14 f 8% of control when given as single doses (n = 5). However, when the preparations were allowed to rest for 30-50 min, a normal vasoconstrictor response to endothelin was noted. DISCUSSION The novel polypeptide endothelin was extremely potent in causing vasoconstriction in the rabbit tenuissimus muscle and hamster cheek pouch. Other vasoconstrictor agents such as neuropeptide Y (NPY) and nor-adrenaline are lOO1000 times less potent than endothelin in the tenuissimus muscle (Per-now et al., 1987). Although very strong, the contraction caused by endothelin could be
overcome by two different vasodilator agents. PGE, and nitroprusside. It has been shown that preproendothelin mRNA increases in response to several stimuli known to cause endothelium-dependent contractions, and decreases in response to a chronic mechanical-fluid shear stress (Yanagisawa et rrl., 1988). These findings and the extreme potency and long-lasting effects of endothelin in V~VOmake this peptide a very interesting candidate as a mediator of endothelium-dependent vascular contractions. ACKNOWLEDGMENTS This
work
Protection Minnc.
was
\upportcd
by
the
Board (5324067-7). the and Karolinska lnstitutct.
Swedish
MRC
Swedish
Society
(14X-4342. for
Medical
14X-7YIY).
The
Rcscarch.
the
National Foundation
Environment Lars
Hiertas
cheek
pouch
REFERENCES RJ~KK,
J.. SMEDF.GARD.
G..
SVLSSJ~~.
E..
AND
AKFOKS.
K. (19X4).
The
for intravital microscopy studies of microvascular events. Prog. FURCHGOTT, R. F. (1984). The role of endothelium in the rcsponscs drugs. Amru. Rev. Phurmocd. li~xicd. 24, 1715-197. HARDFR. D. R. (1987). on intact endothelium. I.INDBOM, Influence
Pressure-induced C&c,. HP.s.
60.
myogenic 102-107.
activation
I>.. ‘~cM*. R. I-‘.. AND ARFORS. K.-E. (1982). Blood of prcparativc procedures for intravital microscopic
of llow
use Appl. of
cat
of the
hamster
Microcirc. vascular
cerebral
6, 41-53. smooth muscle
arteries
in the rabbit observations.
to
is dcpendcnt
tenuissimus Ac,/cr Plr~sid.
muscle.
Smnd.
114, 121-127. PALMER. R. M. J., FEWRIGE. A. G.. AND MONC.\DA. S. (1987). Nitric oxide release accounts for biological activity of endothelium-derived relaxing factor. Ntrrrtrcj fL~&o~~) 327, 524-526. PERNOW. J.. ~JuI.i-~. A.. HijKt.tL?, ‘f., NII SON. 0.. AND I.I:NDBFM;. J. M. (1987). Neuropep[i& Presence in perivascular noradrenergic neurons and effects on skeletal muscle blood vessels cxpcrimental
animals
VANnotIT.rE, 24, 141-144. WAYLAND. two-slit
H.. AND photometric
YANAGISAWA. 6010, K., endothelial
I’. M.
and
(1987).
man.
JOHNSON. method.
M., AND
KLKIHAKA. MASAKI.
cells.
Nnrltrc
Regal.
Pc~pri&s
Endothelium-dependent
P. C. (lY67). Erythrocytc velocity J. Appl. Physiol. 22, 333-337.
(London)
332,
41 l-415.
Y: in
19, 313-324. contractions
H.. KIMI.RA. S.. ‘l‘o~ost. T. (1988). A novel potent
the
in arteries measurement
Y.. Koa,\vnsrll. vasoconstrictor
M.. peptide
and
veins.
Blootl
in microvcsscls MIISUI. Y.. produced
V~.x~c~/.s by
Y,~L~KI. Y.. hy vascular
a
RESEARCH
37, 115-l
18 (1989)
BRIEF COMMUNICATIONS Microvascular A. *Department
Effects of Endothelin Muscle and Hamster
O)HL~~N,*“~
of
Physiology
J.
in the Rabbit Tenuissimus Cheek Pouch
P. HEuovtsr,*‘t
RAUD,“?
AND
N. P.
and flnstitute of Environmental Medicine, S-104 01 Stockholm, Sweden Received
June
WIKLUND*
Karolinska
Instituter,
22, 1988
INTRODUCTION In the last decade, the vascular endothelium has gained increased recognition as an important factor for the regulation of vascular tone. Thus, an intact endothelium is a prerequisite for vasodilatation induced by a number of stimuli, including acetylcholine and substance P (see Furchgott, 1984). One endotheliumderived relaxing factor (EDRF) has been tentatively identified as nitric oxide (Palmer et al., 1987). The release of a vasoconstrictive agent from endothelial cells in response to anoxia, stretch, increased transmural pressure (Vanhoutte, 1987; Harder, 1987), and several other stimuli has also been suggested. Recently, Yanagisawa et aE. (1988) described a novel endothelium-derived 21 amino acid peptide, endothelin, and showed that this peptide is extremely potent in contracting isolated arteries and causes a long-lasting pressor response when injected intravenously to chemically denervated rats. The aim of this study was to characterize the microvascular effects of endothelin using intravital microscopy of the rabbit tenuissimus muscle and the hamster cheek pouch. MATERIALS
AND METHODS
New Zealand white rabbits (n = 6) weighing 0.8-1.2 kg were anesthetized with urethane, 1.5 g x kg-’ via an ear vein. The tenuissimus muscle in the left hind leg was prepared for intravital microscopy as previously described (Lindbom et al., 1982). The diameter of transverse arterioles (control diameter 14-28 pm), terminal arterioles (5-12 pm), and venules (5-50 pm) was studied by intravital microscopy using a Leitz Intravital microscope and water immersion lens (Leitz SW x 25, NA 0.60). Blood flow velocity in transverse arterioles was measured by the dual slit method (Wayland and Johnson, 1967). Blood flow was calculated from the diameter and velocity values. 115 C026-2862/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
116
BRIEF
COMMUNICATION
- Log cont. (M) FIG. 1. Effect of endothelin (IO-“-3 x 10m9 A4) on blood flow in transverse arterioles of the rabbit tenuissimus muscle. Mean values 2 SEM from five experiments.
As previously described (Bjork et al., 1984), arteriolar diameters were studied in the cheek pouch of anesthetized hamsters, prepared for intravital microscopy. Plasma extravasation in the cheek pouch was quantitated using FITC-dextran (mol wt 150,000, 25 mg/lOO g body wt iv) as a tracer for plasma proteins and expressed as the peak number of leakage spots per square centimeter of cheek pouch area (Bjiirk et al., 1984). Synthetic endothelin (lo-“-3 x 1O-9 M; Peninsula Lab., Inc., Belmont, CA) was dissolved in saline and added with the buffer solutions superfusing the muscle and the cheek pouch, respectively. RESULTS Endothelin (topically for 8 min) decreased blood flow in the rabbit tenuissimus muscle in a dose-dependent manner (Fig. 1). At 3 x 10m9 M, endothelin caused flow to cease completely in transverse arterioles. The terminal arterioles were as sensitive as transverse arterioles and started to contract at a dose of 3 x lo-” M. Venules did not change in diameter (102 +- 2% of control; n = 6) even at the highest dose used (3 x 10m9 M). The effect of endothelin could be seen after l-2 min of application, reached a peak after 7-8 min, and lasted for up to 50 min. After control flow was attained, a repeated dose of endothelin (3 x 10-l’ M; n = 3) caused the same vascular response regarding both peak contraction (17 ? 5% of control diameter) and duration (40-50 min). Endothelin (topically for ‘5 min) also potently constricted arterioles (inner diameter lo-50 pm) in the hamster cheek pouch. A threshold response was observed at 10-l’ M and intense constriction with ceased blood flow occurred at l-3 x 10d9 M (n = 4) Fig. 2). The arteriolar spasm was fully developed within 1 min, and lasted up to 60 min after challenge. Thereafter, the vessels responded equally well to repeated challenge with endothelin. The vasoconstriction caused by endothelin (3 x 1O-9 M, for 10 min) was abolished and replaced by vasodilatation after addition of prostaglandin E2 (PGE*, 3 x lo-’ M topically) for 10 min, starting 5 min after the start of endothelin. However, within minutes after the PGE2 addition was stopped, the arterioles regained the constricted
BRIEF COMMUNICATION
117
FIG. 2. Micrographs of arterioles in the hamster cheek pouch before (a) and after (b) the application of endothelin (1O-9 M). Bar = 20 pm.
state, despite the fact that endothelin was no longer present (n = 2). Likewise, nitroprusside (3 x lop6 M, Roche AG, Base& Switzerland) caused a temporary abolishment of endothelin-induced contractions (n = 3). No leakage of plasma (FITC-dextran extavasation) occurred in the cheek pouch after challenge with endothelin (3 x 10e9 M; 12 = 2). Prostaglandin E2 (3 x 10m8 M) caused a minor increase in plasma leakage (16 ? 6 leakage sites/cm’) and there was no difference when PGE2 was coadministered with endothelin (12 k 6; n = 2). In both the tenuissimus muscle and cheek pouch, a desensitization occurred when endothelin was given continuously in increasing doses (successive 8 min periods). Indeed, doses that caused maximal vasoconstriction in single doses sometimes elicited vasodilatation when applied in this way. Thus, in the tenuissimus muscle the diameter of transverse arterioles was 97 + 8% of control at 3 X lop9 M after continuous application (n = 4) as compared to 14 f 8% of control when given as single doses (n = 5). However, when the preparations were allowed to rest for 30-50 min, a normal vasoconstrictor response to endothelin was noted. DISCUSSION The novel polypeptide endothelin was extremely potent in causing vasoconstriction in the rabbit tenuissimus muscle and hamster cheek pouch. Other vasoconstrictor agents such as neuropeptide Y (NPY) and nor-adrenaline are lOO1000 times less potent than endothelin in the tenuissimus muscle (Per-now et al., 1987). Although very strong, the contraction caused by endothelin could be
overcome by two different vasodilator agents. PGE, and nitroprusside. It has been shown that preproendothelin mRNA increases in response to several stimuli known to cause endothelium-dependent contractions, and decreases in response to a chronic mechanical-fluid shear stress (Yanagisawa et rrl., 1988). These findings and the extreme potency and long-lasting effects of endothelin in V~VOmake this peptide a very interesting candidate as a mediator of endothelium-dependent vascular contractions. ACKNOWLEDGMENTS This
work
Protection Minnc.
was
\upportcd
by
the
Board (5324067-7). the and Karolinska lnstitutct.
Swedish
MRC
Swedish
Society
(14X-4342. for
Medical
14X-7YIY).
The
Rcscarch.
the
National Foundation
Environment Lars
Hiertas
cheek
pouch
REFERENCES RJ~KK,
J.. SMEDF.GARD.
G..
SVLSSJ~~.
E..
AND
AKFOKS.
K. (19X4).
The
for intravital microscopy studies of microvascular events. Prog. FURCHGOTT, R. F. (1984). The role of endothelium in the rcsponscs drugs. Amru. Rev. Phurmocd. li~xicd. 24, 1715-197. HARDFR. D. R. (1987). on intact endothelium. I.INDBOM, Influence
Pressure-induced C&c,. HP.s.
60.
myogenic 102-107.
activation
I>.. ‘~cM*. R. I-‘.. AND ARFORS. K.-E. (1982). Blood of prcparativc procedures for intravital microscopic
of llow
use Appl. of
cat
of the
hamster
Microcirc. vascular
cerebral
6, 41-53. smooth muscle
arteries
in the rabbit observations.
to
is dcpendcnt
tenuissimus Ac,/cr Plr~sid.
muscle.
Smnd.
114, 121-127. PALMER. R. M. J., FEWRIGE. A. G.. AND MONC.\DA. S. (1987). Nitric oxide release accounts for biological activity of endothelium-derived relaxing factor. Ntrrrtrcj fL~&o~~) 327, 524-526. PERNOW. J.. ~JuI.i-~. A.. HijKt.tL?, ‘f., NII SON. 0.. AND I.I:NDBFM;. J. M. (1987). Neuropep[i& Presence in perivascular noradrenergic neurons and effects on skeletal muscle blood vessels cxpcrimental
animals
VANnotIT.rE, 24, 141-144. WAYLAND. two-slit
H.. AND photometric
YANAGISAWA. 6010, K., endothelial
I’. M.
and
(1987).
man.
JOHNSON. method.
M., AND
KLKIHAKA. MASAKI.
cells.
Nnrltrc
Regal.
Pc~pri&s
Endothelium-dependent
P. C. (lY67). Erythrocytc velocity J. Appl. Physiol. 22, 333-337.
(London)
332,
41 l-415.
Y: in
19, 313-324. contractions
H.. KIMI.RA. S.. ‘l‘o~ost. T. (1988). A novel potent
the
in arteries measurement
Y.. Koa,\vnsrll. vasoconstrictor
M.. peptide
and
veins.
Blootl
in microvcsscls MIISUI. Y.. produced
V~.x~c~/.s by
Y,~L~KI. Y.. hy vascular
a