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Different Sensitivity of Blocking Effects of a-Adrenoceptor Blocking Agents on Vascular Responses to Intraluminal Norepinephrine and Periarterial Stimulation in Isolated Dog Arteries
Different
Sensitivity
Agents
on
of
Vascular
and
Blocking
Effects
Responses
to
Periarterial
Stimulation
Shigetoshi Department
of
CHIBA
Pharmacology,
and
stainless
in
of
the
for
isolated an
stimulation
perfused
increase
were
3-5
phentolamine
readily
or
inhibited
Yohimbine
of
100 ƒÊg
than
the at
a
in
relatively
periarterial
stimulation
tetrodotoxin.
It method with
was is is due
useful regard
prazosin the
doses in
a
significantly that for
studying to
the
10-20
of
large
optimum periarterial
Hz.
After
to
electrical the
was
Periarterial
stimu
by 30
a to
relatively 100
times
constriction.
stimulation-induced The
by
treatment
norepinephrine
0.3-3 ƒÊg.
the dose.
the
conditions
to
norepinephrine-induced
electrical
autonomic
examine
norepinephrine
concentrations
suppressed periarterial
to
suppressed in
potentiated it
concluded
and
used
The
mmHg
significantly
or
suppressed
dog. 50
dose
also
Medicine,
intraluminal
response
small
blocking
small
rather
the
vasoconstrictor
was
for
of over volts
relative
phentolamine
required
but
vasculature
prazosin,
of
that
constriction,
inserting
40-50
vasoconstriction
dose
larger
duration,
completely
lation-induced large
msec
pressure
of
was
and
artery
perfusion
School
method
stimulation
mesenteric in
TSUKADA
7, 1985
inserting
nerve
Arteries
Japan
February
cannula
electrical
and
inducing
with
steel
periarterial
Dog
University
390,
Blocking Norepinephrine
Isolated
Miyoko
Shinshu
Accepted
effects
Intraluminal in
Matsumoto
Abstract-The
of ƒ¿-Adrenoceptor
1
vaso
constrictor and
response
10 ƒÊg
of
stimulation
pharmacology
in and
to
intraluminal the physiology
cannula in
characteristics.
There have been numerous reports of in vitro studies on the actions of sympathetic nerve stimulation on arterial smooth muscle. In 1962, Bevan (1) reported a preparation which consisted of an isolated pulmonary artery with sympathetic nerves attached for studying the physiology and pharmacology of adrenergic nerve transmission to smooth muscle. In 1965, Paterson (2) applied the transmural stimulation to arterial strips. McGregor (1965) (3) reported the effect of sympathetic nerve stimulation on vascular responses in perfused mesenteric blood vessels of the rat. He performed periarterial nerve stimulation and observed a vaso constriction. Since he separated the intestine from the mesentery by cutting close to the intestinal border of the mesentery, the preparation might contain many small vessels. In 1965, De la Lande and Rand (4) also reported that stimulation applied
periarterially on an isolated, perfused segment of rabbit artery caused a vasoconstriction. In 1970, Su and Bevan (5) used a technique of combined superfusion and transmural elec trical stimulation that permitted the simul taneous measurement of two major pre and postsynaptic events accompanying nerve excitation, the release of H3-norepinephrine and muscle contraction, in a small specimen of the artery. Recently, we introduced a new model arterial preparation for observing vascular responses of relatively large muscular vessels to vasoactive substances (6, 7). This model is suitable for measuring the con tractile response of a relatively large artery as reported previously (8-10). By using this cannula inserting method, we made an attempt to investigate characteristics of vascular responses of a relatively large mesen teric artery to periarterial electrical stimulation.
Materials and Methods Twenty-eight mongrel dogs of either sex weighing 8-17 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). After treatment with sodium heparin (200 units/ kg, i.v.), animals were sacrificed by rapid exsanguination from the right common carotid artery. Arteries (which supplied the middle portion of the small intestine) which are median branches of the cranial mesenteric artery were carefully isolated. Isolated arteries selected for study were 10-15 mm in length and 0.5-1.0 mm in outer diameter. A stainless steel cannula with small holes at a 2-5 mm distance from the distal sealed end (25-27 gauge, 0.4-0.68 mm in outer diameter and 3 cm length) was carefully inserted into each segment to avoid injury of the internal surface of the vessel as described previously (6, 7). The proximal part of each segment was tied to the stainless steel cannula which passes through the intraluminal surface of the isolated artery. The schematic representation of the cannula inserting method is shown in Fig. 1. The isolated and cannulated artery was placed in a bath which was maintained at a constant temperature of 37°C (Haake FE2) and was perfused with Krebs solution (millimolar composition: NaCl, 118; KCI, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 25 and glucose, 5.6) by means of a peristaltic pump. The perfusion solution was bubbled with 95% 02 and 5% CO2, main
1.
Schematic
solution.
Fig.
Arrows
representation indicate
the
of the direction
perfusion of
the
taining the pH levels at 7.2-7.4. The flow rate was initially adjusted so that the perfusion pressure was between 50-100 mmHg, and then it was kept constant throughout the experiment (0.5-1.5 ml/min). The constrictor response was, therefore, observed as an increase in perfusion pressure. Electrical stimulation was applied to perivascular nerves with two platinum wires, 0.5 mm in diameter. For platinum wires that were coated with enamel, except for the cut point which was gently put on the extraluminal side of the vessel wall, served as electrodes. Stimulation was accomplished by delivering square-wave pulses of 0.5-10 msec duration, various voltages (10-80 v) and various frequencies (1-30 Hz) through platinum electrodes from an electric stimulator (Nihon Kohden, SEM 7103). Trains of pulses were given for 10 sec at 1-5 min intervals. Drugs used in this study were (+) norepinephrine hydrochloride (Sankyo), phentolamine mesylate (Ciba), prazosin hydrochloride (Pfizer-Taiyo Co.), yohimbine hydrochloride (Sigma), tetrodotoxin (TTX, Sankyo) and potassium chloride. The drug solution was administered into the perfusion line close to the cannula in a volume of 0.01 0.05 ml over 4 sec by use of a microiniector (Terumo Co.). Results When periarterial applied by different
circuit fluid.
of
an
isolated
and
electrical stimulation was pulse durations of 0.5 to
perfused
artery
with
Krebs
10 msec, at a constant frequency of 10 Hz with suprathreshold voltage, the vaso constrictor response was induced in a duration-dependent manner. At 5-8 msec duration, it reached almost the maximum. These vasoconstrictions were not sig nificantly influenced by pretreatment with a relatively small dose of 1-10 /cg of phen tolamine, a potent alpha-adrenoceptor blocking agent. With a large amount of 30 /-,g of phentolamine, these were slightly sup pressed, but not significantly. With 100 tug, phentolamine caused a significant inhibition of vasoconstriction induced by periarterial stimulation, but not completely, as shown in Fig. 2. Summarized data are shown in Fig. 3. Effects of intensity were examined from
10 to 80 v at a constant duration of 3 msec and 10 Hz. At 10 v, the perfusion pressure was not influenced in all experiments. With 40-60 v, the response became almost the maximum. The constrictor response was also suppressed by 100 ,ug of phentolamine, but still not completely, as shown in Fig. 4. With an intensity of 40-60 v and a pulse duration of 3 msec, stimulation frequency was varied between 1 to 30 Hz. At 10 Hz, almost the maximal vasoconstriction was obtained. Even in these examples, 100 ,ug of intra luminal phentolamine did not completely block the constrictions, although they were significantly suppressed as shown in Fig. 5. Summarized data are shown in Fig. 6. The responses to periarterial stimulation with
Fig. 2. Effects of 30 and 100 ug of phentolamine on vasoconstrictor responses to periarterial stimulation (10 Hz, 40 v) at different pulse durations in an isolated and perfused mesenteric artery of the dog. PP: perfusion pressure.
Fig. 3. Effects of 100 fig of phentolamine on vasoconstrictor responses to periarterial stimulation (10 Hz, 40-60 v) at different pulse durations in 4 isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors.
Fig. 4. Vasoconstrictor responses to increasing intensity of periarterial stimulation at 5 and 10 Hz (3 msec duration, 40-60 v) and blocking effects of 100 1-cgof phentolamine on vasoconstrictor responses at 10 Hz. Points represent mean values, and vertical bars represent standard errors.
various frequencies still remained as clear vasoconstriction which was more than 30% of the control responses even in preparations treated with 100 icg of phentolamine. After treatment with increasing doses of prazosin, a selective adrenergic at-blocking agent, electric stimulation induced con striction was also suppressed dose dependently, but not completely. Summarized data are shown in Fig. 7. After treatment with yohimbine, a selective a2-blocker, the striction by periarterial stimulation slightly potentiated by a relatively small
con was dose
Fig. 5. Blocking effects of 100 ttg of phentolamine on responses to 3 different frequencies of periarterial stimulation (3 msec duration and 40 v) in an isolated and perfused canine mesenteric artery.
Fig. 6. Effects of 100 ug of phentolamine on responses to different frequencies of periarterial stimulation (3 msec duration and 40-60 v) in isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors. The number for each point indicates the number of preparations perfused.
Fig. 7. Effects of increasing doses of prazosin on electrical periarterial stimulation-induced vasocon strictions at different frequencies. Electrical stimu lation was applied with a pulse duration of 3 msec, 40 v, and 10 sec periods (n=5-8). Points represent mean values, and vertical bars represent standard errors.
Fig. 8. Effects of increasing doses of yohimbine on electrical periarterial stimulation-induced vasocon strictions at different frequencies. Electrical stimu lation was applied with a pulse duration of 3 msec, 40 v, and 10 sec periods (n=7). Points represent mean values, and vertical bars represent standard errors.
of
yohimbine.
A
relatively
large
dose
of
yohimbine inhibited the stimulation-induced constriction, but not completely, although any of the used doses of yohimbine potentiated the constriction induced at a low frequency of 2 Hz. Summarized data are shown in Fig. 8.
An injection
of 1 or 10 itg of tetrodotoxin
Fig. 9. Blocking effects of 1 and 10 ug of tetrodo toxin (TTX) on vasoconstrictor responses to peri arterial stimulation (3 msec duration and 40-60 v) for a period of 10 sec in isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors.
Fig.
10.
Effects
of
3 tg
of
phentolamine
on
vasoconstriction
perfused values,
arteries. Points represent mean bars represent standard errors.
mesenteric and vertical
in isolated
nore
pinephrine-induced
and
caused no changes in perfusion pressure. The vasoconstrictor response to periarterial stimulation (3 msec, 40-60 v) was sig nificantly inhibited by tetrodotoxin treatment as shown in Fig. 9. An intraluminal injection of norepinephrine induced an increase in perfusion pressure in a dose-related manner as previously reported (8). The constriction was readily blocked by a relatively small dose of phentolamine or prazosin. The constrictor responses to 0.03 and 0.1 icg of norepinephrine were com pletely blocked by 3 ug of phentolamine or 1 gig of prazosin. Summarized data on the effects of phentolamine on norepinephrine induced vasoconstriction are shown in Fig. 10.
Discussion Recently, Hongo and Chiba (6) developed a new method for measuring the vascular responsiveness of relatively larger arteries of dogs. By use of this cannula inserting method, periarterial electrical stimulation was performed with various changes in pulse duration, intensity and frequency. Since the vasoconstrictor response to periarterial electrical stimulation was significantly sup pressed by a relatively small dose of tetrodotoxin, the constriction might be due to the neurogenic release of catecholamine, but not due to the direct electrical stimulation of vascular smooth muscle. It was confirmed that the optimum conditions for inducing vasoconstriction to periarterial stimulation were 3 to 5 msec duration, 40 to 60 volts and frequencies of 10-20 Hz. However, if this method is used for the purpose of in vestigating pharmacological and physio logical problems on adrenergic nerve terminals, it is necessary to use a low frequency stimulation because low fre quencies such as 1-5 Hz have been con sidered much closer to physiological excitation of nerve fibers (11, 12). McGregor (3) used an alpha-adrenergic blocking agent, dibenzyline, in doses which blocked norepinephrine induced vasocon striction. The periarterial stimulation-induced responses were completely blocked by dibenzyline in 8 out of 12 preparations. As he used the preparation including the small branches of mesenteric artery, the con
strictor response to periarterial stimulation might be mainly due to responses of small arteries and then readily, they were blocked by dibenzyline. On the other hand, we demonstrated in this study that the blocking activities of phentolamine as well as prazosin on responses to periarterial stimulation were much different from those on intraluminal administration of norepinephrine. Since a relatively large artery was used in these experiments, it may cause different results from those of McGregor (3) because the adrenergic nerve terminals are located at the advential-medial border (13, 14), and are thus more accessible to the drug in extraluminal fluid, and an injected blocking agent into the perfusion line may not readily diffuse from the intima side to the advential medial area in a relatively larger vessel. In this study, the constriction induced by periarterial stimulation was diminished by phentolamine or prazosin in concentrations 30 to 100 times that required for blocking the norepinephrine-induced vasoconstriction. The difference is understandable since the sympathetic nerves on which endogenous norepinephrine acts are mainly located in the adventitial-medial boundary of the artery, much farther to the endothelial than to the extraluminal surface. Recently, Muramatsu et al. (15) reported that electrical transmural stimulation of the isolated dog mesenteric artery produced a contractile response which was abolished by guanethidine and 6-hydroxydopamine but not by prazosin, suggesting that the non adrenergic component may play an important role. Even in this study, we could demonstrate prazosin-resistant vasoconstriction in the isolated dog mesenteric artery, although it is not ruled out that 1) norepinephrine may exert its action on adrenergic receptors that are different from the alpha-type (16), and 2) the electrical stimulation may produce a release of substances other than norepine phrine (17, 18). It seems that the lack of blocking by alpha-adrenoceptor antagonists has not been well explained at the present time.
dose, but it caused an inhibition of the constriction in larger doses. In 1984, Toda et al. (19) reported that in mesenteric and renal arteries, contractile responses to transmural electrical stimulation were poten tiated by yohimbine; the potentiation was greater in mesenteric arteries. It has been well recognized that effects of yohimbine are due mainly to an increased release of norepine phrine by the blockade of presynaptic a2 receptors (20). In 1982, Winquist et al. (21 ) reported that the vasodilator response to norepinephrine, but not that to transmural nerve stimulation, was abolished by beta-adrenoceptor antago nists in helical strips of porcine cerebral vessels. They considered that there was involvement of either adrenergic and non adrenergic nerve terminals or multiple trans mitters associated with the adrenergic nerve terminal in the relaxation to transmural nerve stimulation in porcine cerebral vessels. However, they also reported that high con centrations of propranolol began to attenuate the neurogenic dilation. Thus, it is suggested that the relative less responsiveness to a beta-blocker may in part be due to difficulty in diffusing to sympathetic nerve terminal regions. The advantages of this method are as follows: 1) the access of injected substances to vascular smooth muscle is only from the intraluminal side, differing from preparations of helical strips and rings; 2) the effect of torsion of the specimen around its axis can be eliminated, so it is closer to in vivo con ditions than a helical cut method; 3) the endothelium may not be readily injuried, differing from the helical strips; 4) the pre paration is stable for a long period (7-8 hr); and 5) the responses to vasoactive substances and electrical periarterial stimulation was repetitively obtained in the same preparations.
References 1 Bevan, J.A.: Some characteristics of the isolated sympathetic nerve-pulmonary preparation of the rabbit. J. Pharmacol. Exp. Ther. 137, 213-218 (1962) 2 Paterson, G.: The response to transmural stimu lation of isolated arterial strips and its modi fication by drugs. J. Pharm. Pharmacol. 17, 341 349 (1965) 3 McGregor, D.D.: The effect of sympathetic nerve stimulation on vasoconstrictor responses
13
14
15
16
17
18
19
20
21
carinic agonist McN-A-343 on responses to sympathetic nerve stimulation in the rabbit ear artery. Br. J. Pharmacol. 43, 536-542 (1971) Flacke, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol. Scand. 56, Supp. 197, 1-25 (1962) Bevan, J.A., Bevan, R.D. and Duckles, S.P.: Adrenergic regulation of vascular smooth muscle. In Handbook of Physiology. Section 2: The Cardiovascular System, Vol. 2, Vascular Smooth Muscle, Edited by Bohr, D., Somlyo, A.P. and Sparks, H.V., Jr., p. 515-566, Waverly Press, Bethesda (1980) Muramatsu, I., Fujiwara, M., Miura, A. an Sakakibara, Y.: Possible involvement of adenin d nucleotides in sympathetic neuroeffector emecha nisms of dog basilar artery. J. Pharmacol. Exp. Ther. 216, 401-409 (1981) Kuriyama, H. and Makita, Y.: Modulation of noradrenergic transmission in the guinea-pig mesenteric artery: An electrophysiological study. J. Physiol. (Lond.) 335, 609-627 (1983) Langer, S.Z. and Pinto, J.E.B.: Possible involve ment of a transmitter different from norepine phrine in the residual responses to nerve stimu lation of cat nictitating membrane after pre treatment with reserpine. J. Pharmacol. Exp. Ther. 196, 697-713 (1976) Muramatsu, I., Kigoshi, S. and Oshima, M.: Nonadrenergic nature of prazosin-resistant , sym pathetic contraction in the dog mesenteric artery. J. Pharmacol. Exp. Ther. 229, 532-538 (1984) Toda, N., Okamura, T., Nakajima, M. and Miyazaki, M.: Modification by yohimbine and prazosin of the mechanical response of isolated dog mesenteric, renal and coronary arteries to transmural stimulation and norepinephrine. Eur. J. Pharmacol. 98, 69-78 (1984) Langer, S.Z.: Presynaptic regulation of the release of catecholamines. Pharmacol . Rev. 32 337-367 (1981) , Winquist, R.J., Webb, R.C. and Bohr, D.F.: Relaxation to transmural nerve stimulation and exogenously added norepinephrine in porcine cerebral vessels. A study utilizing cerebrovascular intrinsic tone. Circ. Res. 51, 767-776 (1982)
Sensitivity
Agents
on
of
Vascular
and
Blocking
Effects
Responses
to
Periarterial
Stimulation
Shigetoshi Department
of
CHIBA
Pharmacology,
and
stainless
in
of
the
for
isolated an
stimulation
perfused
increase
were
3-5
phentolamine
readily
or
inhibited
Yohimbine
of
100 ƒÊg
than
the at
a
in
relatively
periarterial
stimulation
tetrodotoxin.
It method with
was is is due
useful regard
prazosin the
doses in
a
significantly that for
studying to
the
10-20
of
large
optimum periarterial
Hz.
After
to
electrical the
was
Periarterial
stimu
by 30
a to
relatively 100
times
constriction.
stimulation-induced The
by
treatment
norepinephrine
0.3-3 ƒÊg.
the dose.
the
conditions
to
norepinephrine-induced
electrical
autonomic
examine
norepinephrine
concentrations
suppressed periarterial
to
suppressed in
potentiated it
concluded
and
used
The
mmHg
significantly
or
suppressed
dog. 50
dose
also
Medicine,
intraluminal
response
small
blocking
small
rather
the
vasoconstrictor
was
for
of over volts
relative
phentolamine
required
but
vasculature
prazosin,
of
that
constriction,
inserting
40-50
vasoconstriction
dose
larger
duration,
completely
lation-induced large
msec
pressure
of
was
and
artery
perfusion
School
method
stimulation
mesenteric in
TSUKADA
7, 1985
inserting
nerve
Arteries
Japan
February
cannula
electrical
and
inducing
with
steel
periarterial
Dog
University
390,
Blocking Norepinephrine
Isolated
Miyoko
Shinshu
Accepted
effects
Intraluminal in
Matsumoto
Abstract-The
of ƒ¿-Adrenoceptor
1
vaso
constrictor and
response
10 ƒÊg
of
stimulation
pharmacology
in and
to
intraluminal the physiology
cannula in
characteristics.
There have been numerous reports of in vitro studies on the actions of sympathetic nerve stimulation on arterial smooth muscle. In 1962, Bevan (1) reported a preparation which consisted of an isolated pulmonary artery with sympathetic nerves attached for studying the physiology and pharmacology of adrenergic nerve transmission to smooth muscle. In 1965, Paterson (2) applied the transmural stimulation to arterial strips. McGregor (1965) (3) reported the effect of sympathetic nerve stimulation on vascular responses in perfused mesenteric blood vessels of the rat. He performed periarterial nerve stimulation and observed a vaso constriction. Since he separated the intestine from the mesentery by cutting close to the intestinal border of the mesentery, the preparation might contain many small vessels. In 1965, De la Lande and Rand (4) also reported that stimulation applied
periarterially on an isolated, perfused segment of rabbit artery caused a vasoconstriction. In 1970, Su and Bevan (5) used a technique of combined superfusion and transmural elec trical stimulation that permitted the simul taneous measurement of two major pre and postsynaptic events accompanying nerve excitation, the release of H3-norepinephrine and muscle contraction, in a small specimen of the artery. Recently, we introduced a new model arterial preparation for observing vascular responses of relatively large muscular vessels to vasoactive substances (6, 7). This model is suitable for measuring the con tractile response of a relatively large artery as reported previously (8-10). By using this cannula inserting method, we made an attempt to investigate characteristics of vascular responses of a relatively large mesen teric artery to periarterial electrical stimulation.
Materials and Methods Twenty-eight mongrel dogs of either sex weighing 8-17 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). After treatment with sodium heparin (200 units/ kg, i.v.), animals were sacrificed by rapid exsanguination from the right common carotid artery. Arteries (which supplied the middle portion of the small intestine) which are median branches of the cranial mesenteric artery were carefully isolated. Isolated arteries selected for study were 10-15 mm in length and 0.5-1.0 mm in outer diameter. A stainless steel cannula with small holes at a 2-5 mm distance from the distal sealed end (25-27 gauge, 0.4-0.68 mm in outer diameter and 3 cm length) was carefully inserted into each segment to avoid injury of the internal surface of the vessel as described previously (6, 7). The proximal part of each segment was tied to the stainless steel cannula which passes through the intraluminal surface of the isolated artery. The schematic representation of the cannula inserting method is shown in Fig. 1. The isolated and cannulated artery was placed in a bath which was maintained at a constant temperature of 37°C (Haake FE2) and was perfused with Krebs solution (millimolar composition: NaCl, 118; KCI, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 25 and glucose, 5.6) by means of a peristaltic pump. The perfusion solution was bubbled with 95% 02 and 5% CO2, main
1.
Schematic
solution.
Fig.
Arrows
representation indicate
the
of the direction
perfusion of
the
taining the pH levels at 7.2-7.4. The flow rate was initially adjusted so that the perfusion pressure was between 50-100 mmHg, and then it was kept constant throughout the experiment (0.5-1.5 ml/min). The constrictor response was, therefore, observed as an increase in perfusion pressure. Electrical stimulation was applied to perivascular nerves with two platinum wires, 0.5 mm in diameter. For platinum wires that were coated with enamel, except for the cut point which was gently put on the extraluminal side of the vessel wall, served as electrodes. Stimulation was accomplished by delivering square-wave pulses of 0.5-10 msec duration, various voltages (10-80 v) and various frequencies (1-30 Hz) through platinum electrodes from an electric stimulator (Nihon Kohden, SEM 7103). Trains of pulses were given for 10 sec at 1-5 min intervals. Drugs used in this study were (+) norepinephrine hydrochloride (Sankyo), phentolamine mesylate (Ciba), prazosin hydrochloride (Pfizer-Taiyo Co.), yohimbine hydrochloride (Sigma), tetrodotoxin (TTX, Sankyo) and potassium chloride. The drug solution was administered into the perfusion line close to the cannula in a volume of 0.01 0.05 ml over 4 sec by use of a microiniector (Terumo Co.). Results When periarterial applied by different
circuit fluid.
of
an
isolated
and
electrical stimulation was pulse durations of 0.5 to
perfused
artery
with
Krebs
10 msec, at a constant frequency of 10 Hz with suprathreshold voltage, the vaso constrictor response was induced in a duration-dependent manner. At 5-8 msec duration, it reached almost the maximum. These vasoconstrictions were not sig nificantly influenced by pretreatment with a relatively small dose of 1-10 /cg of phen tolamine, a potent alpha-adrenoceptor blocking agent. With a large amount of 30 /-,g of phentolamine, these were slightly sup pressed, but not significantly. With 100 tug, phentolamine caused a significant inhibition of vasoconstriction induced by periarterial stimulation, but not completely, as shown in Fig. 2. Summarized data are shown in Fig. 3. Effects of intensity were examined from
10 to 80 v at a constant duration of 3 msec and 10 Hz. At 10 v, the perfusion pressure was not influenced in all experiments. With 40-60 v, the response became almost the maximum. The constrictor response was also suppressed by 100 ,ug of phentolamine, but still not completely, as shown in Fig. 4. With an intensity of 40-60 v and a pulse duration of 3 msec, stimulation frequency was varied between 1 to 30 Hz. At 10 Hz, almost the maximal vasoconstriction was obtained. Even in these examples, 100 ,ug of intra luminal phentolamine did not completely block the constrictions, although they were significantly suppressed as shown in Fig. 5. Summarized data are shown in Fig. 6. The responses to periarterial stimulation with
Fig. 2. Effects of 30 and 100 ug of phentolamine on vasoconstrictor responses to periarterial stimulation (10 Hz, 40 v) at different pulse durations in an isolated and perfused mesenteric artery of the dog. PP: perfusion pressure.
Fig. 3. Effects of 100 fig of phentolamine on vasoconstrictor responses to periarterial stimulation (10 Hz, 40-60 v) at different pulse durations in 4 isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors.
Fig. 4. Vasoconstrictor responses to increasing intensity of periarterial stimulation at 5 and 10 Hz (3 msec duration, 40-60 v) and blocking effects of 100 1-cgof phentolamine on vasoconstrictor responses at 10 Hz. Points represent mean values, and vertical bars represent standard errors.
various frequencies still remained as clear vasoconstriction which was more than 30% of the control responses even in preparations treated with 100 icg of phentolamine. After treatment with increasing doses of prazosin, a selective adrenergic at-blocking agent, electric stimulation induced con striction was also suppressed dose dependently, but not completely. Summarized data are shown in Fig. 7. After treatment with yohimbine, a selective a2-blocker, the striction by periarterial stimulation slightly potentiated by a relatively small
con was dose
Fig. 5. Blocking effects of 100 ttg of phentolamine on responses to 3 different frequencies of periarterial stimulation (3 msec duration and 40 v) in an isolated and perfused canine mesenteric artery.
Fig. 6. Effects of 100 ug of phentolamine on responses to different frequencies of periarterial stimulation (3 msec duration and 40-60 v) in isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors. The number for each point indicates the number of preparations perfused.
Fig. 7. Effects of increasing doses of prazosin on electrical periarterial stimulation-induced vasocon strictions at different frequencies. Electrical stimu lation was applied with a pulse duration of 3 msec, 40 v, and 10 sec periods (n=5-8). Points represent mean values, and vertical bars represent standard errors.
Fig. 8. Effects of increasing doses of yohimbine on electrical periarterial stimulation-induced vasocon strictions at different frequencies. Electrical stimu lation was applied with a pulse duration of 3 msec, 40 v, and 10 sec periods (n=7). Points represent mean values, and vertical bars represent standard errors.
of
yohimbine.
A
relatively
large
dose
of
yohimbine inhibited the stimulation-induced constriction, but not completely, although any of the used doses of yohimbine potentiated the constriction induced at a low frequency of 2 Hz. Summarized data are shown in Fig. 8.
An injection
of 1 or 10 itg of tetrodotoxin
Fig. 9. Blocking effects of 1 and 10 ug of tetrodo toxin (TTX) on vasoconstrictor responses to peri arterial stimulation (3 msec duration and 40-60 v) for a period of 10 sec in isolated and perfused canine mesenteric arteries. Points represent mean values, and vertical bars represent standard errors.
Fig.
10.
Effects
of
3 tg
of
phentolamine
on
vasoconstriction
perfused values,
arteries. Points represent mean bars represent standard errors.
mesenteric and vertical
in isolated
nore
pinephrine-induced
and
caused no changes in perfusion pressure. The vasoconstrictor response to periarterial stimulation (3 msec, 40-60 v) was sig nificantly inhibited by tetrodotoxin treatment as shown in Fig. 9. An intraluminal injection of norepinephrine induced an increase in perfusion pressure in a dose-related manner as previously reported (8). The constriction was readily blocked by a relatively small dose of phentolamine or prazosin. The constrictor responses to 0.03 and 0.1 icg of norepinephrine were com pletely blocked by 3 ug of phentolamine or 1 gig of prazosin. Summarized data on the effects of phentolamine on norepinephrine induced vasoconstriction are shown in Fig. 10.
Discussion Recently, Hongo and Chiba (6) developed a new method for measuring the vascular responsiveness of relatively larger arteries of dogs. By use of this cannula inserting method, periarterial electrical stimulation was performed with various changes in pulse duration, intensity and frequency. Since the vasoconstrictor response to periarterial electrical stimulation was significantly sup pressed by a relatively small dose of tetrodotoxin, the constriction might be due to the neurogenic release of catecholamine, but not due to the direct electrical stimulation of vascular smooth muscle. It was confirmed that the optimum conditions for inducing vasoconstriction to periarterial stimulation were 3 to 5 msec duration, 40 to 60 volts and frequencies of 10-20 Hz. However, if this method is used for the purpose of in vestigating pharmacological and physio logical problems on adrenergic nerve terminals, it is necessary to use a low frequency stimulation because low fre quencies such as 1-5 Hz have been con sidered much closer to physiological excitation of nerve fibers (11, 12). McGregor (3) used an alpha-adrenergic blocking agent, dibenzyline, in doses which blocked norepinephrine induced vasocon striction. The periarterial stimulation-induced responses were completely blocked by dibenzyline in 8 out of 12 preparations. As he used the preparation including the small branches of mesenteric artery, the con
strictor response to periarterial stimulation might be mainly due to responses of small arteries and then readily, they were blocked by dibenzyline. On the other hand, we demonstrated in this study that the blocking activities of phentolamine as well as prazosin on responses to periarterial stimulation were much different from those on intraluminal administration of norepinephrine. Since a relatively large artery was used in these experiments, it may cause different results from those of McGregor (3) because the adrenergic nerve terminals are located at the advential-medial border (13, 14), and are thus more accessible to the drug in extraluminal fluid, and an injected blocking agent into the perfusion line may not readily diffuse from the intima side to the advential medial area in a relatively larger vessel. In this study, the constriction induced by periarterial stimulation was diminished by phentolamine or prazosin in concentrations 30 to 100 times that required for blocking the norepinephrine-induced vasoconstriction. The difference is understandable since the sympathetic nerves on which endogenous norepinephrine acts are mainly located in the adventitial-medial boundary of the artery, much farther to the endothelial than to the extraluminal surface. Recently, Muramatsu et al. (15) reported that electrical transmural stimulation of the isolated dog mesenteric artery produced a contractile response which was abolished by guanethidine and 6-hydroxydopamine but not by prazosin, suggesting that the non adrenergic component may play an important role. Even in this study, we could demonstrate prazosin-resistant vasoconstriction in the isolated dog mesenteric artery, although it is not ruled out that 1) norepinephrine may exert its action on adrenergic receptors that are different from the alpha-type (16), and 2) the electrical stimulation may produce a release of substances other than norepine phrine (17, 18). It seems that the lack of blocking by alpha-adrenoceptor antagonists has not been well explained at the present time.
dose, but it caused an inhibition of the constriction in larger doses. In 1984, Toda et al. (19) reported that in mesenteric and renal arteries, contractile responses to transmural electrical stimulation were poten tiated by yohimbine; the potentiation was greater in mesenteric arteries. It has been well recognized that effects of yohimbine are due mainly to an increased release of norepine phrine by the blockade of presynaptic a2 receptors (20). In 1982, Winquist et al. (21 ) reported that the vasodilator response to norepinephrine, but not that to transmural nerve stimulation, was abolished by beta-adrenoceptor antago nists in helical strips of porcine cerebral vessels. They considered that there was involvement of either adrenergic and non adrenergic nerve terminals or multiple trans mitters associated with the adrenergic nerve terminal in the relaxation to transmural nerve stimulation in porcine cerebral vessels. However, they also reported that high con centrations of propranolol began to attenuate the neurogenic dilation. Thus, it is suggested that the relative less responsiveness to a beta-blocker may in part be due to difficulty in diffusing to sympathetic nerve terminal regions. The advantages of this method are as follows: 1) the access of injected substances to vascular smooth muscle is only from the intraluminal side, differing from preparations of helical strips and rings; 2) the effect of torsion of the specimen around its axis can be eliminated, so it is closer to in vivo con ditions than a helical cut method; 3) the endothelium may not be readily injuried, differing from the helical strips; 4) the pre paration is stable for a long period (7-8 hr); and 5) the responses to vasoactive substances and electrical periarterial stimulation was repetitively obtained in the same preparations.
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