Directory UMM :Data Elmu:jurnal:A:Advances in Physiology Education:Vol24.Issue1:
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LABORATORY DEMONSTRATION OF
VASCULAR SMOOTH MUSCLE
FUNCTION USING RAT
AORTIC RING SEGMENTS
Rayna J. Gonzales, Rebecca W. Carter, and Nancy L. Kanagy
Vascular Physiology Group, Department of Cell Biology and Physiology,
University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131– 0218
T
his laboratory exercise uses a simple preparation and a straightforward
protocol to illustrate many of the basic principles of vascular biology covered
in an introductory physiology course. The design of this laboratory allows
students to actively participate in an exercise demonstrating the regulation of arterial
tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows
students to gather data using well-known basic biomedical research techniques.
Specifically, students are introduced to an isolated organ-chamber technique that is
widely used to study cellular mechanisms of many tissues including vascular smooth
muscle contraction and dilation. On the basis of student evaluations, participation in
the experiments and interpreting data reinforce lecture materials on smooth muscle
and endothelial cell function and illustrate mechanisms regulating vascular tone.
Students come away with a greater understanding of vascular biology, a deeper
appreciation of integrative physiology, and an understanding of the process of
conducting tissue-chamber experiments.
ADV PHYSIOL EDUC 24: 13–21, 2000.
Key words: endothelial cell; teaching; physiology
vascular tone by various extrinsic and intrinsic factors.
Smooth muscle cell physiology is a topic represented
in major medical physiology textbooks as a small
section of muscle physiology. However, this is a rapidly changing, complex area of research that has seen
recent significant advances impacting many diverse
scientific fields. Therefore, it is advantageous for graduate students majoring in the field of biomedical sciences to understand the function and regulation of
vascular smooth muscle (VSM). In our graduate program, we address this by preparing a bibliography of
key publications, presenting lecture material covering
cellular mechanisms of VSM contraction and dilation,
and by requiring students to actively participate in a
VSM laboratory that demonstrates the regulation of
This laboratory exercise is part of a medical systems
physiology course offered for students enrolled in our
Biomedical Sciences Graduate Program. On the basis
of student reviews and exam scores, this exercise has
greatly enhanced students’ understanding of basic
physiological concepts in vascular biology. Before
participating in this laboratory exercise, students are
encouraged to review and discuss Poiseuille’s law of
fluid dynamics. This law states that resistance to flow
through a cylindrical tube is equal to (8 hL)/(pr4),
where h is the viscosity, L is the length of the tube,
1043 - 4046 / 00 – $5.00 – COPYRIGHT © 2000 THE AMERICAN PHYSIOLOGICAL SOCIETY
VOLUME 24 : NUMBER 1 – ADVANCES IN PHYSIOLOGY EDUCATION – DECEMBER 2000
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and r is the radius of the tube. Therefore, small
changes in vessel diameter have very large opposite
effects on resistance.
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lar, tissue-level, and whole animal hemodynamic responses.
METHODS AND EXPERIMENTAL PROTOCOLS
In the vasculature, the radius of a blood vessel is
altered by contraction of VSM cells (11). Thus it is
possible to predict the effect of stimuli on vascular
resistance and blood pressure by observing contraction of VSM in isolated preparations. One way to
demonstrate VSM contraction is to attach strips or
ring segments of arteries to force transducers and
record changes in tension development after exposure to different agents. These responses can subsequently be used to predict the effect of these agents
on blood pressure. Additionally, this approach can be
used to illustrate the role of intracellular calcium concentration in regulating vascular tone (3).
All protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of
Medicine. On the day of the laboratory exercise, two
male Sprague-Dawley rats (300 g; Harlan Industries)
are anesthetized with pentabarbital sodium (50
mg/kg) and exsanguinated. Thoracic aortic segments
are isolated, cleaned of adventia, and cut into rings
(6). We use aortic segments for our demonstration
because they are relatively hardy and easily prepared.
Other laboratories could easily adapt these protocols
to different VSM preparations, e.g., mesenteric or tail
blood vessels. The aortic segments are kept in cold
physiological saline solution (PSS) containing (in
mM): 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 z
7 H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2
EDTA, 1.6 CaCl2, pH 7.3. For some protocols, calcium-free PSS is prepared by omitting the CaCl2 and
adding the calcium chelator EGTA (1 mM). Once
isolated, aortas are cut into six rings (3 mm). Three
segments from each rat are denuded of endothelium
by gently rubbing the lumen with the closed tips of
microforceps. Two aortic segments from the same rat,
one endothelium intact and one endothelium denuded, are mounted on stainless steel hooks and suspended in a water-jacketed 25-ml tissue bath filled
with PSS maintained at 37°C and aerated with 95% O2
and 5% CO2. Six tissue baths are required for the two
protocols. Isometric force generation is recorded
with FT03 Grass transducers (Quincy, MA) connected
to chart recorders (Gould Instruments, Cambridge,
MA). Aortic rings are stretched to the equivalent of
2,000 mg of passive tension to allow the maximal
detection of active tension.
In this laboratory exercise, students perform two protocols. Protocol 1 demonstrates the principles of calcium regulation of vascular contraction. Agents that
elevate intracellular calcium contract VSM, and those
that lower intracellular calcium relax VSM. The
source of calcium can be determined by manipulating
extracellular calcium concentrations and by depleting
intracellular stores. The purpose of this protocol is to
determine the sources of activator calcium for three
different contractile agents: norepinephrine (NE), a
receptor-mediated vasoconstrictor that activates
phospholipase C, high potassium chloride solution
(KCl), a solution that depolarizes VSM cell membrane
potential to activate voltage-operated calcium channels, and caffeine, a methyl xanthine that penetrates
the plasma membrane and acts directly on the sarcoplasmic reticulum to release intracellular calcium
stores (1). Protocol 2 examines the ability of endothelial cell products to modulate vascular tone. In this
protocol, students examine endothelium-dependent
relaxation to ACh and endothelium-independent relaxation to a nitric oxide donor in the presence or
absence of nitric oxide synthase (NOS) inhibition,
muscarinic receptor blockade, and guanylyl cyclase
inhibition.
After the stretch, aortic rings are equilibrated 1 h,
during which time they are washed every 15 min and
treated for the final 30 min with indomethacin
(10 mmol/l, cyclooxygenase inhibitor) to eliminate
the role of cyclooxygense products in this preparation. Next, arterial segments are contracted with NE
(1 mM), and the presence or absence of endothelium
is confirmed by adding ACh (0.1 mM). A contraction
of 1,000 mg or greater to NE is considered viable,
Together, these protocols illustrate the basic mechanisms of VSM contraction and relaxation. A discussion
after the laboratory exercise puts this into the context
of blood pressure regulation providing a way to discuss vascular physiology integrating molecular, cellu-
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(1 mM), KCl (65 mM), or caffeine (30 mM). Sample
tracings of contractile responses are shown in Figs.
2–4. Next, rings are washed for 30 min with calciumcontaining PSS to remove the contractile agents. After
the wash period, artery rings are stimulated again
with the same agents. The first response demonstrates
contraction mediated by release of intracellular calcium stores because there is no calcium available for
influx, whereas the second response is dependent on
calcium release and calcium influx.
Description of Responses
FIG. 1.
Initial norepinephrine (NE) contractile response used
to determine tissue viability in 2 aortic rings. NE contraction is greater in the segment without endothelium (2; bottom trace). Only the segment with endothelium-intact (1) relaxed in response to ACh (top
trace).
VSM response to NE. In the absence of extracellular
calcium, NE stimulates a transient contraction that
returns nearly to baseline. This contraction is caused
by production of the second messenger inositol
trisphosphate, which binds to receptors on the sarcoplasmic reticulum to release calcium from this intracellular store (8). Washing with calcium-containing
buffer stimulates contraction dependent on calcium
entry through open calcium channels such as the
voltage-gated calcium channel (VGCC) (2, 13). This
contraction diminishes as the ligand is washed off the
receptor. In the presence of extracellular calcium, NE
causes a rapid, sustained contraction that is significantly larger than that obtained in the absence of
extracellular calcium. The differences in time course,
duration, and magnitude between the two contractions illustrate the contribution both of intracellular
and extracellular calcium stores in mediating receptor-activated contraction.
relaxation of 60% or more to ACh is considered endothelium-intact (Fig. 1, top trace), whereas relaxation of ,5% is considered successfully denuded (Fig.
1, bottom trace).
Preparation of tissues and the endothelium check are
completed before student arrival to shorten the time
of the experiment. When the students arrive, they are
divided into groups of four to six to allow active
participation. The tracings from the curves generated
by the addition of NE and ACh are left on the chart
recorder, and students are instructed to observe and
record the magnitude of the responses to these agents
on a worksheet (Table 1). Students are now ready to
participate in protocols 1 and 2.
The endothelium-denuded artery segments develop a
larger contraction compared with the endotheliumintact segment. The diminished response in the intact
segment is caused by NE binding to a2-adrenergic
receptors on endothelial cells to stimulate the release
of nitric oxide and other vasodilators (5). The release
of these vasodilators opposes and attenuates the VSM
contraction.
Protocol 1
Calcium sources for VSM contraction in response to NE, KCl, and caffeine. Three sets of
two-ring segments, one endothelium-intact and one
denuded, are mounted in separate tissue baths as
described above. The purpose of this protocol is to
evaluate the contribution of intracellular and extracellular calcium on VSM contraction for three different
agents, as described previously (4). Briefly, after the
equilibration period, PSS in tissue baths is replaced
with calcium-free PSS containing EGTA. Aortic rings
are then stimulated with one of three agonists: NE
VSM response to KCl. In the absence of extracellular calcium, KCl did not stimulate a contractile response. However, in the presence of extracellular
calcium, KCl produced a sustained contraction. Increasing extracellular KCl from 7 to 72 mM reduces
the driving force for K1 across the sarcolemma causing membrane depolarization. This more positive
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activate calcium-sensitive Cl2 channels to further depolarize the cell (7).
membrane potential increases the open probability
for VGCC. Increasing the extracellular concentration
of KCl should not cause a contraction in the calciumfree PSS in either the endothelium-intact or -denuded
aortic rings, because no calcium is available to enter
through the open VGCC. In the presence of extracellular calcium, however, there is calcium influx. This
influx increases intracellular free calcium and increases calcium-calmodulin binding to activate myosin light chain kinase and cause contraction. In addition, the increased calcium may stimulate calciumsensitive calcium release from intracellular stores and
VSM response to caffeine. Millimolar concentrations of caffeine penetrate the cell membrane to activate calcium release from the sarcoplasmic reticulum.
Caffeine contraction of VSM is entirely dependent on
calcium release from these intracellular stores (1). In
the absence of extracellular calcium, there is a transient contraction that diminishes as calcium is removed from the cytosol by the cytosolic calcium
transporters. In the presence of extracellular calcium,
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FIG. 2.
Contractile response to NE in 2 aortic segments in the
absence of extracellular calcium (initial challenge)
and in the presence of extracellular calcium (second
challenge). The segments contracted to NE under both
conditions; however, the response in the absence of
extracellular calcium was not maintained. In the presence of extracellular calcium, the contraction to NE
was greater in the segment without endothelium. PSS,
physiological saline solution.
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FIG. 4.
Contractile responses to caffeine in 2 aortic segments
in the absence and presence of extracellular calcium.
The segments contracted to caffeine under both conditions, but the response in the presence of extracellular Ca21 was greater compared with the response in
the absence of extracellular Ca21.
Protocol 2
Examination of endothelium-dependent dilation. Six aortic segments are mounted in three tissue
baths on stainless steel hooks with two segments
(endothelium intact and denuded) in each tissue bath.
These segments are equilibrated similarly to those in
protocol 1, and the viability of segments and endothelium were determined as described above. After a
viability and endothelium check, aortic rings in the
three tissue baths are treated with inhibitors or vehicle for 30 min to demonstrate the mechanism of
endothelium-derived nitric oxide-mediated relaxation
of VSM.
caffeine produces a more pronounced contraction
that is augmented by recycling of calcium through the
sarcoplasmic reticulum. The presence of endothelial
cells may slightly diminish the contractile response
due to basal release of endothelial relaxing factors.
One bath is treated with Nv-nitro-L-arginine (LNNA;
100 mM), an NOS inhibitor, one with atropine (30
mM; muscarinic antagonist), and one with vehicle
(ddH2O). The tissue segments are all contracted with
NE (0.1 mM), and contractile response is allowed to
plateau. Then, without washing out the NE, increasing concentrations of the cholinergic agonist ACh are
added to the bath (0.01 to 10 mM). After finishing the
concentration response curve, the nitric oxide donor
S-nitrosylacetylpenicillimine (SNAP, 1 mM) is added to
the baths. Next, after the dilation reaches a plateau,
the guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 1 mM) is added to the
FIG. 3.
Tracings of force generated in aortic segments depolarized with high concentrations of extracellular potassium chloride. In the absence of extracellular calcium
(initial challenge), there was a negligible contraction. In
the presence of extracellular calcium (second challenge),
there was a sustained contraction in both segments.
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FIG. 5.
Contractile responses to NE, ACh, S-nitrosylacetylpenicillimine (SNAP), and 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ) in aortic segments
treated with the nitric oxide synthase (NOS) inhibitor
Nv-nitro-L-arginine (LNNA). Segments were treated
with LNNA for 30 min before contraction with NE.
After NE contraction plateaued, a concentration response curve (CRC) to ACh was generated. Neither
segment exhibited relaxation to ACh, demonstrating
complete blockade of endothelial NOS activity by
LNNA. However, both tissues responded to the nitric
oxide donor SNAP, with relaxation nearly reaching
baseline. The addition of the guanylyl cyclase inhibitor ODQ restored contraction back to the level observed before treatment with SNAP.
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FIG. 6.
Responses to NE, ACh, SNAP, and ODQ in aortic segments treated with the muscarinic receptor antagonist, atropine. Segments were treated with atropine
for 30 min before contraction with NE. After NE contraction plateaued, ACh was added to the bath in increasing concentrations. Neither segment exhibited
relaxation to ACh, demonstrating complete blockade
of muscarinic receptors. However, contraction was
not maintained in the endothelium-intact segment
(top trace). Both tissues relaxed to the nitric oxide
donor SNAP. Addition of the guanylyl cyclase inhibitor ODQ restored contraction to pre-SNAP tension in
the endothelium-denuded segment and above preSNAP tension in the endothelium-intact segment,
demonstrating basal nitric oxide stimulation of gaunylyl cyclase.
bath (10). Again, the final tension at the plateau is
recorded.
Relaxation in atropine-treated aortic segments.
Atropine treatment will not affect NE contraction, and
there should be a greater contraction in the endothelium-denuded segment compared with the endothelium-intact segment. Relaxation to ACh will be blocked
by the muscarinic receptor antagonist. However,
both denuded and intact segments will relax after
addition of the nitric oxide donor (Fig. 6). The nitric
oxide relaxation is reversed by the guanylyl cyclase
inhibitor ODQ. This ODQ-mediated contraction
should be augmented in the endothelium-intact segment compared with the contraction elicited by NE in
the absence of ODQ. The contraction is augmented
due to the loss of guanylyl cyclase stimulation by
endothelium-derived nitric oxide.
Description of Responses
Relaxation in LNNA-treated aortic segments. In
the bath treated with the NOS inhibitor, the response
to NE is augmented compared with the response of
segments treated with either atropine or vehicle. The
augmented response is due to inhibition of basal nitric
oxide production. The response should not be different between the endothelium-intact and -denuded
segments in the presence of the NOS inhibitor. Furthermore, addition of ACh will not elicit relaxation in
either segment in contrast to the initial check of
viability when relaxation was observed in the endothelium-intact segment (Fig. 1). The addition of SNAP
causes maximal relaxation in both segments that is
reversed by the addition of the guanylyl cyclase inhibitor ODQ (Fig. 5). Because the contribution of nitric
oxide should be eliminated by LNNA, the final contraction should be equivalent to the initial contraction
to NE in both segments.
Relaxation in vehicle-treated aortic segments.
NE contraction should be greater in the endotheliumdenuded segment. ACh will elicit relaxation only in
the endothelium-intact segment, whereas SNAP will
relax both segments to baseline (Fig. 7). The addition
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“Participation of the whole group was an important
aspect of the lab. Not only could I relate to the
material visually, but I could also use the dialogue
between myself and the other students as a supplemental way of grasping the material. I felt it was easy
to consult with my peers as well as with a mentoring
professor to clarify the material.”
“Being able to see the instantaneous response of the
vasculature via the chart recording to various pharmacological agents was important in understanding how
quickly and dramatically the contraction happens!”
FIG. 7.
Responses to NE, ACh, SNAP, and ODQ in vehicletreated aortic segments. Segments were equilibrated
in PSS for 30 min before contraction with NE. After NE
contraction plateaued, an ACh CRC was generated.
Only the endothelium-intact segment (top trace) relaxed to ACh, demonstrating complete removal of endothelium in the denuded segment. Both tissues relaxed to baseline after addition of the nitric oxide
donor SNAP. Addition of the guanylyl cyclase inhibitor ODQ restored contraction to pre-SNAP tension in
the endothelium-denuded segment (bottom trace) and
above pre-SNAP tension in the endothelium-intact segment, demonstrating basal nitric oxide stimulation of
gaunylyl cyclase.
STUDY GUIDE QUESTIONS
These questions are designed to be used during the
laboratory session to stimulate discussion among the
students. Written responses are turned in after the
laboratory session.
Question 1: Why must segments be stretched to detect “active tension generation?” Describe “passive
tension” and the elements of the cell that contribute
to it.
of ODQ will restore the NE contraction with an augmented response in the endothelium-intact segment.
Answer: The intermediate filaments of the cell make
up the cytoskeleton of the smooth muscle cell and
maintain the cell in an elongated shape under resting
conditions. These filaments also anchor the contractile units (sarcomeres) to the cytoskeleton at the
dense bands and dense bodies. Initial contraction of
the thin and thick filaments are thought to stretch
these intermediate, elastic filaments without generating cell shortening. This contraction cannot be observed experimentally unless the cells are first loaded
with a passive force to fully stretch the intermediate
elastic filaments (9).
DISCUSSION AND SUMMARY
The laboratory exercise is designed to encourage
thought and discussion about the mechanisms involved in vascular contraction and relaxation. Furthermore, students are provided with the opportunity to
actively participate in generating data and interpreting results. This laboratory exercise complements any
smooth muscle physiology lecture and encourages
students to relate experimental results to lecture topics. The interaction of students during this exercise
also allows students to generate discussions and share
ideas about the mechanisms of intracellular regulation
and endothelium regulation of vascular tone. Most
students have very favorable comments on this exercise. Several comments that we have received include:
Question 2: Why must the strips be contracted first to
evaluate the presence of endothelial cells?
Answer: Relaxation of the smooth muscle cells cannot
occur in the absence of active tone. The initial stretch
provides passive tone; however, only increasing intracellular calcium above resting levels will generate
active tone. Active tone is generated in this experiment by stimulating a-adrenergic receptors with NE
(12).
“Explaining what was going to happen first and then
letting us experience it ourselves was a very effective
and straightforward way of learning.”
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strate arginine. The untreated endothelial-intact aortic
ring will not relax to ACh because there is no NOS
present to be activated. Rather, ACh will bind to
muscarinic receptors on the VSM cells and cause
contraction. In the aortic segment containing endothelial cells, there will be a concentration-dependent
relaxation to the ACh as endothelial NOS is activated
by the graded increase in calcium levels in the endothelial cells.
Question 3: What agents caused a contraction in the
absence of extracellular calcium? If the agent
caused a contraction, was it dependent or independent on the presence of endothelium?
Answer: Only caffeine and NE contracted arteries in
the absence of extracellular calcium. The presence of
endothelium attenuated the contraction to the NE
due to the release of nitric oxide from the endothelium.
SUGGESTED READINGS
Question 4: What do the results in protocol 1 indicate regarding the source of calcium for each of the
three agents examined?
Barany M. Biochemistry of Smooth Muscle Contraction. 1996.
TB Bolton and T Toomita. Smooth Muscle Excitiation. 1996.
Answer: The results suggest that both NE and caffeine
can stimulate calcium release from intracellular
stores. The augmented contraction in every case by
the addition of extracellular calcium indicates that all
three contractile stimuli stimulate calcium entry into
the VSM cells.
Morgan JP, Perreault CL, and Morgan KG. The cellular
basis of contraction and relaxation in cardiac and
vascular smooth muscle. Am Heart J 121: 961–968,
1991.
Question 5: What would have happened in each
case if an antagonist to voltage-gated Ca21 channels
had been present?
Frehn C. Mechanisms of vasoconstriction. Am Heart J
121: 958 –960, 1991.
Hiinura JT, et al. Evidence for increased myofilament
Ca21 sensitivity in norepinephrine-activated vascular
smooth muscle. Am J Physiol Heart Circ Physiol 259:
H2–H8, 1990.
Answer: A voltage-gated calcium-channel blocker
would have prevented contraction to KCl and would
have diminished contraction to NE and caffeine. The
resulting contraction should have looked much like
the one in zero calcium solution.
Kitazawa T, et al. G protein-mediated sensitization of
smooth muscle contraction through myosin light
chain phosphorylation. J Biol Chem 266: 1708 –1715,
1991.
Question 6: What effect on ACh relaxation do you
observe with each of the inhibitors, and how is it
working?
Answer: The muscarinic antagonist atropine blocks
relaxation by ACh. This is through competitive antagonism at the muscarinic receptors on endothelial
cells. In these cells, there will be neither receptor
activation nor an increase in intracellular calcium and
NOS activation. LNNA (NOS inhibitor) blocks AChinduced relaxation by competing with the endogenous NOS substrate L-arginine. In this case, ACh will
bind to its receptor, activate Gai, and increase intracellular calcium. This will increase endothelial NOS
affinity for substrate, but no nitric oxide will be produced because NOS will be bound to the inhibitor
LNNA, which is in excess of the endogenous sub-
Address for reprint requests and other correspondence: N. L. Kanagy, 237 Biomedical Research Facility, Dept. of Cell Biology and
Physiology, Univ. of New Mexico Health Sciences Center, Albuquerque, NM 87131–5218 (E-mail: [email protected]).
Received 26 May 2000; accepted in final form 19 September 2000
References
1. Alexander PB and Cheung DW. Ca21 mobilization by caffeine in single smooth muscle cells of the rat tail artery. Eur
J Pharmacol Mol Pharmacol 288: 79 – 88, 1994.
2. Blatter LA. Depletion and filling of intracellular calcium stores
in vascular smooth muscle. Am J Physiol Cell Physiol Cell
Physiol 268: C503–C512, 1995.
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3. Fukuizumi Y, Kobayashi S, Nishimura J, and Kanaide H.
Cytosolic calcium concentration-force relation during contractions in the rabbit femoral artery: time-dependency and stimulus specificity. Br J Pharmacol 114: 329 –338, 1995.
4. Gonzales RJ and Kanagy NL. Endothelium-independent relaxation of vascular smooth muscel by 17b-estradiol. Cardiovasc Pharmacol Therapeut 4: 227–234, 1999.
5. Ishibashi Y, Duncker DJ, and Bache RJ. Endogenous
nitric oxide masks alpha 2-adrenergic coronary vasoconstriction during exercise in the ischemic heart. Circ Res 80:
196 –207, 1997.
6. Kanagy NL. Increased vascular responsiveness to alpha 2-adrenergic stimulation during NOS inhibition-induced hypertension. Am J Physiol Heart Circ Physiol 273: H2756 –H2764,
1997.
7. Lamb FS and Barna TJ. Chloride ion currents contribute
functionally to norepinephrine-induced vascular contraction.
Am J Physiol Heart Circ Physiol 275: H151–H160, 1998.
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8. Nahorski SR, Wilcox RA, Mackrill JJ, and Challiss RAJ.
Phosphoinositide-derived second messengers and the regulation of Ca21 in vascular smooth muscle. J Hypertens 12, Suppl
10: S133–S143, 1994.
9. Small JV and Gimona M. The cytoskeleton of the vertebrate
smooth muscle cell. Acta Physiol Scand 164: 341–348, 1998.
10. Tseng CM, Tabrizi-Fard MA, and Fung HL. Differential sensitivity among nitric oxide donors toward ODQ-mediated inhibition of vascular relaxation. J Pharmacol Exp Ther 292: 737–
742, 2000.
11. Van Breeman C. Cellular mechanisms regulating [Ca21]i in
smooth muscle. Ann Rev Physiol 51: 315–329, 1989.
12. Walsh MP, Kargacin GJ, Kendrick-Jones J, and Lincoln
TM. Intracellular mechanisms involved in the regulation of
vascular smooth muscle tone. Can J Physiol Pharmacol 73:
565–573, 1995.
13. Xiong Z and Sperelakis N. Regulation of L-type calcium
channels of vascular smooth muscle cells. J Mol Cell Cardiol
27: 75–91, 1995.
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LABORATORY DEMONSTRATION OF
VASCULAR SMOOTH MUSCLE
FUNCTION USING RAT
AORTIC RING SEGMENTS
Rayna J. Gonzales, Rebecca W. Carter, and Nancy L. Kanagy
Vascular Physiology Group, Department of Cell Biology and Physiology,
University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131– 0218
T
his laboratory exercise uses a simple preparation and a straightforward
protocol to illustrate many of the basic principles of vascular biology covered
in an introductory physiology course. The design of this laboratory allows
students to actively participate in an exercise demonstrating the regulation of arterial
tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows
students to gather data using well-known basic biomedical research techniques.
Specifically, students are introduced to an isolated organ-chamber technique that is
widely used to study cellular mechanisms of many tissues including vascular smooth
muscle contraction and dilation. On the basis of student evaluations, participation in
the experiments and interpreting data reinforce lecture materials on smooth muscle
and endothelial cell function and illustrate mechanisms regulating vascular tone.
Students come away with a greater understanding of vascular biology, a deeper
appreciation of integrative physiology, and an understanding of the process of
conducting tissue-chamber experiments.
ADV PHYSIOL EDUC 24: 13–21, 2000.
Key words: endothelial cell; teaching; physiology
vascular tone by various extrinsic and intrinsic factors.
Smooth muscle cell physiology is a topic represented
in major medical physiology textbooks as a small
section of muscle physiology. However, this is a rapidly changing, complex area of research that has seen
recent significant advances impacting many diverse
scientific fields. Therefore, it is advantageous for graduate students majoring in the field of biomedical sciences to understand the function and regulation of
vascular smooth muscle (VSM). In our graduate program, we address this by preparing a bibliography of
key publications, presenting lecture material covering
cellular mechanisms of VSM contraction and dilation,
and by requiring students to actively participate in a
VSM laboratory that demonstrates the regulation of
This laboratory exercise is part of a medical systems
physiology course offered for students enrolled in our
Biomedical Sciences Graduate Program. On the basis
of student reviews and exam scores, this exercise has
greatly enhanced students’ understanding of basic
physiological concepts in vascular biology. Before
participating in this laboratory exercise, students are
encouraged to review and discuss Poiseuille’s law of
fluid dynamics. This law states that resistance to flow
through a cylindrical tube is equal to (8 hL)/(pr4),
where h is the viscosity, L is the length of the tube,
1043 - 4046 / 00 – $5.00 – COPYRIGHT © 2000 THE AMERICAN PHYSIOLOGICAL SOCIETY
VOLUME 24 : NUMBER 1 – ADVANCES IN PHYSIOLOGY EDUCATION – DECEMBER 2000
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and r is the radius of the tube. Therefore, small
changes in vessel diameter have very large opposite
effects on resistance.
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lar, tissue-level, and whole animal hemodynamic responses.
METHODS AND EXPERIMENTAL PROTOCOLS
In the vasculature, the radius of a blood vessel is
altered by contraction of VSM cells (11). Thus it is
possible to predict the effect of stimuli on vascular
resistance and blood pressure by observing contraction of VSM in isolated preparations. One way to
demonstrate VSM contraction is to attach strips or
ring segments of arteries to force transducers and
record changes in tension development after exposure to different agents. These responses can subsequently be used to predict the effect of these agents
on blood pressure. Additionally, this approach can be
used to illustrate the role of intracellular calcium concentration in regulating vascular tone (3).
All protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of
Medicine. On the day of the laboratory exercise, two
male Sprague-Dawley rats (300 g; Harlan Industries)
are anesthetized with pentabarbital sodium (50
mg/kg) and exsanguinated. Thoracic aortic segments
are isolated, cleaned of adventia, and cut into rings
(6). We use aortic segments for our demonstration
because they are relatively hardy and easily prepared.
Other laboratories could easily adapt these protocols
to different VSM preparations, e.g., mesenteric or tail
blood vessels. The aortic segments are kept in cold
physiological saline solution (PSS) containing (in
mM): 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 z
7 H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2
EDTA, 1.6 CaCl2, pH 7.3. For some protocols, calcium-free PSS is prepared by omitting the CaCl2 and
adding the calcium chelator EGTA (1 mM). Once
isolated, aortas are cut into six rings (3 mm). Three
segments from each rat are denuded of endothelium
by gently rubbing the lumen with the closed tips of
microforceps. Two aortic segments from the same rat,
one endothelium intact and one endothelium denuded, are mounted on stainless steel hooks and suspended in a water-jacketed 25-ml tissue bath filled
with PSS maintained at 37°C and aerated with 95% O2
and 5% CO2. Six tissue baths are required for the two
protocols. Isometric force generation is recorded
with FT03 Grass transducers (Quincy, MA) connected
to chart recorders (Gould Instruments, Cambridge,
MA). Aortic rings are stretched to the equivalent of
2,000 mg of passive tension to allow the maximal
detection of active tension.
In this laboratory exercise, students perform two protocols. Protocol 1 demonstrates the principles of calcium regulation of vascular contraction. Agents that
elevate intracellular calcium contract VSM, and those
that lower intracellular calcium relax VSM. The
source of calcium can be determined by manipulating
extracellular calcium concentrations and by depleting
intracellular stores. The purpose of this protocol is to
determine the sources of activator calcium for three
different contractile agents: norepinephrine (NE), a
receptor-mediated vasoconstrictor that activates
phospholipase C, high potassium chloride solution
(KCl), a solution that depolarizes VSM cell membrane
potential to activate voltage-operated calcium channels, and caffeine, a methyl xanthine that penetrates
the plasma membrane and acts directly on the sarcoplasmic reticulum to release intracellular calcium
stores (1). Protocol 2 examines the ability of endothelial cell products to modulate vascular tone. In this
protocol, students examine endothelium-dependent
relaxation to ACh and endothelium-independent relaxation to a nitric oxide donor in the presence or
absence of nitric oxide synthase (NOS) inhibition,
muscarinic receptor blockade, and guanylyl cyclase
inhibition.
After the stretch, aortic rings are equilibrated 1 h,
during which time they are washed every 15 min and
treated for the final 30 min with indomethacin
(10 mmol/l, cyclooxygenase inhibitor) to eliminate
the role of cyclooxygense products in this preparation. Next, arterial segments are contracted with NE
(1 mM), and the presence or absence of endothelium
is confirmed by adding ACh (0.1 mM). A contraction
of 1,000 mg or greater to NE is considered viable,
Together, these protocols illustrate the basic mechanisms of VSM contraction and relaxation. A discussion
after the laboratory exercise puts this into the context
of blood pressure regulation providing a way to discuss vascular physiology integrating molecular, cellu-
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(1 mM), KCl (65 mM), or caffeine (30 mM). Sample
tracings of contractile responses are shown in Figs.
2–4. Next, rings are washed for 30 min with calciumcontaining PSS to remove the contractile agents. After
the wash period, artery rings are stimulated again
with the same agents. The first response demonstrates
contraction mediated by release of intracellular calcium stores because there is no calcium available for
influx, whereas the second response is dependent on
calcium release and calcium influx.
Description of Responses
FIG. 1.
Initial norepinephrine (NE) contractile response used
to determine tissue viability in 2 aortic rings. NE contraction is greater in the segment without endothelium (2; bottom trace). Only the segment with endothelium-intact (1) relaxed in response to ACh (top
trace).
VSM response to NE. In the absence of extracellular
calcium, NE stimulates a transient contraction that
returns nearly to baseline. This contraction is caused
by production of the second messenger inositol
trisphosphate, which binds to receptors on the sarcoplasmic reticulum to release calcium from this intracellular store (8). Washing with calcium-containing
buffer stimulates contraction dependent on calcium
entry through open calcium channels such as the
voltage-gated calcium channel (VGCC) (2, 13). This
contraction diminishes as the ligand is washed off the
receptor. In the presence of extracellular calcium, NE
causes a rapid, sustained contraction that is significantly larger than that obtained in the absence of
extracellular calcium. The differences in time course,
duration, and magnitude between the two contractions illustrate the contribution both of intracellular
and extracellular calcium stores in mediating receptor-activated contraction.
relaxation of 60% or more to ACh is considered endothelium-intact (Fig. 1, top trace), whereas relaxation of ,5% is considered successfully denuded (Fig.
1, bottom trace).
Preparation of tissues and the endothelium check are
completed before student arrival to shorten the time
of the experiment. When the students arrive, they are
divided into groups of four to six to allow active
participation. The tracings from the curves generated
by the addition of NE and ACh are left on the chart
recorder, and students are instructed to observe and
record the magnitude of the responses to these agents
on a worksheet (Table 1). Students are now ready to
participate in protocols 1 and 2.
The endothelium-denuded artery segments develop a
larger contraction compared with the endotheliumintact segment. The diminished response in the intact
segment is caused by NE binding to a2-adrenergic
receptors on endothelial cells to stimulate the release
of nitric oxide and other vasodilators (5). The release
of these vasodilators opposes and attenuates the VSM
contraction.
Protocol 1
Calcium sources for VSM contraction in response to NE, KCl, and caffeine. Three sets of
two-ring segments, one endothelium-intact and one
denuded, are mounted in separate tissue baths as
described above. The purpose of this protocol is to
evaluate the contribution of intracellular and extracellular calcium on VSM contraction for three different
agents, as described previously (4). Briefly, after the
equilibration period, PSS in tissue baths is replaced
with calcium-free PSS containing EGTA. Aortic rings
are then stimulated with one of three agonists: NE
VSM response to KCl. In the absence of extracellular calcium, KCl did not stimulate a contractile response. However, in the presence of extracellular
calcium, KCl produced a sustained contraction. Increasing extracellular KCl from 7 to 72 mM reduces
the driving force for K1 across the sarcolemma causing membrane depolarization. This more positive
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activate calcium-sensitive Cl2 channels to further depolarize the cell (7).
membrane potential increases the open probability
for VGCC. Increasing the extracellular concentration
of KCl should not cause a contraction in the calciumfree PSS in either the endothelium-intact or -denuded
aortic rings, because no calcium is available to enter
through the open VGCC. In the presence of extracellular calcium, however, there is calcium influx. This
influx increases intracellular free calcium and increases calcium-calmodulin binding to activate myosin light chain kinase and cause contraction. In addition, the increased calcium may stimulate calciumsensitive calcium release from intracellular stores and
VSM response to caffeine. Millimolar concentrations of caffeine penetrate the cell membrane to activate calcium release from the sarcoplasmic reticulum.
Caffeine contraction of VSM is entirely dependent on
calcium release from these intracellular stores (1). In
the absence of extracellular calcium, there is a transient contraction that diminishes as calcium is removed from the cytosol by the cytosolic calcium
transporters. In the presence of extracellular calcium,
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FIG. 2.
Contractile response to NE in 2 aortic segments in the
absence of extracellular calcium (initial challenge)
and in the presence of extracellular calcium (second
challenge). The segments contracted to NE under both
conditions; however, the response in the absence of
extracellular calcium was not maintained. In the presence of extracellular calcium, the contraction to NE
was greater in the segment without endothelium. PSS,
physiological saline solution.
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I D E A S
FIG. 4.
Contractile responses to caffeine in 2 aortic segments
in the absence and presence of extracellular calcium.
The segments contracted to caffeine under both conditions, but the response in the presence of extracellular Ca21 was greater compared with the response in
the absence of extracellular Ca21.
Protocol 2
Examination of endothelium-dependent dilation. Six aortic segments are mounted in three tissue
baths on stainless steel hooks with two segments
(endothelium intact and denuded) in each tissue bath.
These segments are equilibrated similarly to those in
protocol 1, and the viability of segments and endothelium were determined as described above. After a
viability and endothelium check, aortic rings in the
three tissue baths are treated with inhibitors or vehicle for 30 min to demonstrate the mechanism of
endothelium-derived nitric oxide-mediated relaxation
of VSM.
caffeine produces a more pronounced contraction
that is augmented by recycling of calcium through the
sarcoplasmic reticulum. The presence of endothelial
cells may slightly diminish the contractile response
due to basal release of endothelial relaxing factors.
One bath is treated with Nv-nitro-L-arginine (LNNA;
100 mM), an NOS inhibitor, one with atropine (30
mM; muscarinic antagonist), and one with vehicle
(ddH2O). The tissue segments are all contracted with
NE (0.1 mM), and contractile response is allowed to
plateau. Then, without washing out the NE, increasing concentrations of the cholinergic agonist ACh are
added to the bath (0.01 to 10 mM). After finishing the
concentration response curve, the nitric oxide donor
S-nitrosylacetylpenicillimine (SNAP, 1 mM) is added to
the baths. Next, after the dilation reaches a plateau,
the guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 1 mM) is added to the
FIG. 3.
Tracings of force generated in aortic segments depolarized with high concentrations of extracellular potassium chloride. In the absence of extracellular calcium
(initial challenge), there was a negligible contraction. In
the presence of extracellular calcium (second challenge),
there was a sustained contraction in both segments.
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FIG. 5.
Contractile responses to NE, ACh, S-nitrosylacetylpenicillimine (SNAP), and 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ) in aortic segments
treated with the nitric oxide synthase (NOS) inhibitor
Nv-nitro-L-arginine (LNNA). Segments were treated
with LNNA for 30 min before contraction with NE.
After NE contraction plateaued, a concentration response curve (CRC) to ACh was generated. Neither
segment exhibited relaxation to ACh, demonstrating
complete blockade of endothelial NOS activity by
LNNA. However, both tissues responded to the nitric
oxide donor SNAP, with relaxation nearly reaching
baseline. The addition of the guanylyl cyclase inhibitor ODQ restored contraction back to the level observed before treatment with SNAP.
A N D
I D E A S
FIG. 6.
Responses to NE, ACh, SNAP, and ODQ in aortic segments treated with the muscarinic receptor antagonist, atropine. Segments were treated with atropine
for 30 min before contraction with NE. After NE contraction plateaued, ACh was added to the bath in increasing concentrations. Neither segment exhibited
relaxation to ACh, demonstrating complete blockade
of muscarinic receptors. However, contraction was
not maintained in the endothelium-intact segment
(top trace). Both tissues relaxed to the nitric oxide
donor SNAP. Addition of the guanylyl cyclase inhibitor ODQ restored contraction to pre-SNAP tension in
the endothelium-denuded segment and above preSNAP tension in the endothelium-intact segment,
demonstrating basal nitric oxide stimulation of gaunylyl cyclase.
bath (10). Again, the final tension at the plateau is
recorded.
Relaxation in atropine-treated aortic segments.
Atropine treatment will not affect NE contraction, and
there should be a greater contraction in the endothelium-denuded segment compared with the endothelium-intact segment. Relaxation to ACh will be blocked
by the muscarinic receptor antagonist. However,
both denuded and intact segments will relax after
addition of the nitric oxide donor (Fig. 6). The nitric
oxide relaxation is reversed by the guanylyl cyclase
inhibitor ODQ. This ODQ-mediated contraction
should be augmented in the endothelium-intact segment compared with the contraction elicited by NE in
the absence of ODQ. The contraction is augmented
due to the loss of guanylyl cyclase stimulation by
endothelium-derived nitric oxide.
Description of Responses
Relaxation in LNNA-treated aortic segments. In
the bath treated with the NOS inhibitor, the response
to NE is augmented compared with the response of
segments treated with either atropine or vehicle. The
augmented response is due to inhibition of basal nitric
oxide production. The response should not be different between the endothelium-intact and -denuded
segments in the presence of the NOS inhibitor. Furthermore, addition of ACh will not elicit relaxation in
either segment in contrast to the initial check of
viability when relaxation was observed in the endothelium-intact segment (Fig. 1). The addition of SNAP
causes maximal relaxation in both segments that is
reversed by the addition of the guanylyl cyclase inhibitor ODQ (Fig. 5). Because the contribution of nitric
oxide should be eliminated by LNNA, the final contraction should be equivalent to the initial contraction
to NE in both segments.
Relaxation in vehicle-treated aortic segments.
NE contraction should be greater in the endotheliumdenuded segment. ACh will elicit relaxation only in
the endothelium-intact segment, whereas SNAP will
relax both segments to baseline (Fig. 7). The addition
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“Participation of the whole group was an important
aspect of the lab. Not only could I relate to the
material visually, but I could also use the dialogue
between myself and the other students as a supplemental way of grasping the material. I felt it was easy
to consult with my peers as well as with a mentoring
professor to clarify the material.”
“Being able to see the instantaneous response of the
vasculature via the chart recording to various pharmacological agents was important in understanding how
quickly and dramatically the contraction happens!”
FIG. 7.
Responses to NE, ACh, SNAP, and ODQ in vehicletreated aortic segments. Segments were equilibrated
in PSS for 30 min before contraction with NE. After NE
contraction plateaued, an ACh CRC was generated.
Only the endothelium-intact segment (top trace) relaxed to ACh, demonstrating complete removal of endothelium in the denuded segment. Both tissues relaxed to baseline after addition of the nitric oxide
donor SNAP. Addition of the guanylyl cyclase inhibitor ODQ restored contraction to pre-SNAP tension in
the endothelium-denuded segment (bottom trace) and
above pre-SNAP tension in the endothelium-intact segment, demonstrating basal nitric oxide stimulation of
gaunylyl cyclase.
STUDY GUIDE QUESTIONS
These questions are designed to be used during the
laboratory session to stimulate discussion among the
students. Written responses are turned in after the
laboratory session.
Question 1: Why must segments be stretched to detect “active tension generation?” Describe “passive
tension” and the elements of the cell that contribute
to it.
of ODQ will restore the NE contraction with an augmented response in the endothelium-intact segment.
Answer: The intermediate filaments of the cell make
up the cytoskeleton of the smooth muscle cell and
maintain the cell in an elongated shape under resting
conditions. These filaments also anchor the contractile units (sarcomeres) to the cytoskeleton at the
dense bands and dense bodies. Initial contraction of
the thin and thick filaments are thought to stretch
these intermediate, elastic filaments without generating cell shortening. This contraction cannot be observed experimentally unless the cells are first loaded
with a passive force to fully stretch the intermediate
elastic filaments (9).
DISCUSSION AND SUMMARY
The laboratory exercise is designed to encourage
thought and discussion about the mechanisms involved in vascular contraction and relaxation. Furthermore, students are provided with the opportunity to
actively participate in generating data and interpreting results. This laboratory exercise complements any
smooth muscle physiology lecture and encourages
students to relate experimental results to lecture topics. The interaction of students during this exercise
also allows students to generate discussions and share
ideas about the mechanisms of intracellular regulation
and endothelium regulation of vascular tone. Most
students have very favorable comments on this exercise. Several comments that we have received include:
Question 2: Why must the strips be contracted first to
evaluate the presence of endothelial cells?
Answer: Relaxation of the smooth muscle cells cannot
occur in the absence of active tone. The initial stretch
provides passive tone; however, only increasing intracellular calcium above resting levels will generate
active tone. Active tone is generated in this experiment by stimulating a-adrenergic receptors with NE
(12).
“Explaining what was going to happen first and then
letting us experience it ourselves was a very effective
and straightforward way of learning.”
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strate arginine. The untreated endothelial-intact aortic
ring will not relax to ACh because there is no NOS
present to be activated. Rather, ACh will bind to
muscarinic receptors on the VSM cells and cause
contraction. In the aortic segment containing endothelial cells, there will be a concentration-dependent
relaxation to the ACh as endothelial NOS is activated
by the graded increase in calcium levels in the endothelial cells.
Question 3: What agents caused a contraction in the
absence of extracellular calcium? If the agent
caused a contraction, was it dependent or independent on the presence of endothelium?
Answer: Only caffeine and NE contracted arteries in
the absence of extracellular calcium. The presence of
endothelium attenuated the contraction to the NE
due to the release of nitric oxide from the endothelium.
SUGGESTED READINGS
Question 4: What do the results in protocol 1 indicate regarding the source of calcium for each of the
three agents examined?
Barany M. Biochemistry of Smooth Muscle Contraction. 1996.
TB Bolton and T Toomita. Smooth Muscle Excitiation. 1996.
Answer: The results suggest that both NE and caffeine
can stimulate calcium release from intracellular
stores. The augmented contraction in every case by
the addition of extracellular calcium indicates that all
three contractile stimuli stimulate calcium entry into
the VSM cells.
Morgan JP, Perreault CL, and Morgan KG. The cellular
basis of contraction and relaxation in cardiac and
vascular smooth muscle. Am Heart J 121: 961–968,
1991.
Question 5: What would have happened in each
case if an antagonist to voltage-gated Ca21 channels
had been present?
Frehn C. Mechanisms of vasoconstriction. Am Heart J
121: 958 –960, 1991.
Hiinura JT, et al. Evidence for increased myofilament
Ca21 sensitivity in norepinephrine-activated vascular
smooth muscle. Am J Physiol Heart Circ Physiol 259:
H2–H8, 1990.
Answer: A voltage-gated calcium-channel blocker
would have prevented contraction to KCl and would
have diminished contraction to NE and caffeine. The
resulting contraction should have looked much like
the one in zero calcium solution.
Kitazawa T, et al. G protein-mediated sensitization of
smooth muscle contraction through myosin light
chain phosphorylation. J Biol Chem 266: 1708 –1715,
1991.
Question 6: What effect on ACh relaxation do you
observe with each of the inhibitors, and how is it
working?
Answer: The muscarinic antagonist atropine blocks
relaxation by ACh. This is through competitive antagonism at the muscarinic receptors on endothelial
cells. In these cells, there will be neither receptor
activation nor an increase in intracellular calcium and
NOS activation. LNNA (NOS inhibitor) blocks AChinduced relaxation by competing with the endogenous NOS substrate L-arginine. In this case, ACh will
bind to its receptor, activate Gai, and increase intracellular calcium. This will increase endothelial NOS
affinity for substrate, but no nitric oxide will be produced because NOS will be bound to the inhibitor
LNNA, which is in excess of the endogenous sub-
Address for reprint requests and other correspondence: N. L. Kanagy, 237 Biomedical Research Facility, Dept. of Cell Biology and
Physiology, Univ. of New Mexico Health Sciences Center, Albuquerque, NM 87131–5218 (E-mail: [email protected]).
Received 26 May 2000; accepted in final form 19 September 2000
References
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