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RELAXANT EFFECT OF VINCAMINE HYDROCHLORIDE AND
PYROZALIN IODIDE ALKALOIDS ON AORTIC SMOOTH MUSCLE
CELLS VIA CA2+L-CHANNELS
Z.D. Allaniyazova, N.M. Yuldashev
Tashkent Pediatric Medical Institute, Tashkent, Republic of Uzbekistan
T.T. Adilbekov, P.B. Usmanov
Institute of Biophysics and Biochemistry at National University of Uzbekistan,
Tashkent, Republic of Uzbekistan
Abstract:
In this research, the results of the effects of vincamine
hydrochloride and pyrozalin iodide alkaloids on the relaxation of aortic smooth
muscle cells in the presence of Ca
2+
L
-channels have been reported. The isometric
contraction activity of the aortic blood vessel smooth muscle derived from rabbits
was studied using mechanography. The results, under in vitro conditions,
demonstrated that the contraction activity of the rabbit aortic blood vessel smooth
muscle preparation induced by 50 mM KCl was dependent on the potential of the
plasma membrane, associated with the activity of the Ca
2+L
-channels. It was found
that vincamine and pyrozalin iodide alkaloids affected the blockage of the Ca
2+L
-
channels and a reduction in [Ca
2+
] concentration. The obtained results suggest that
the relaxing effect of vincamine hydrochloride and pyrozalin iodide alkaloids is
based on the blockage of the L-type Ca
2+
channels, and they also affect the receptor-
operated Ca
2+
channels. In conclusion, the results indicate that in providing the
relaxing effect of vincamine and pyrozalin iodide alkaloids, the blockage of
potential-dependent L-type Ca
2+
channels, along with the blockage of receptor-
operated Ca
2+
channels plays an important role.
Keywords:
Smooth muscle cell, aorta, ion channels, vasorelaxant.
Introduction
The leading causes of death worldwide are heart and vascular diseases, and
despite extensive efforts to prevent and manage them, they remain a significant
global concern [1]. Arterial hypertension is one of the most prevalent and high-
risk types of heart and vascular diseases and is considered a leading factor in both
mortality and morbidity [2]. Smooth muscle cells (SMC) of blood vessels are
essential components of the vascular wall and play a crucial role in regulating
blood pressure. The pathogenesis of hypertension is closely related to functional
and structural changes in SMC. In many cardiovascular diseases, dysfunction of
ion transport systems in vascular SMC is associated with a decrease in the
contractile activity of the muscle, which is crucial in regulating blood pressure [3].
Ion channels, including Ca
2+
, K
+
, Na
+
, and Cl
-
channels, are present in almost all
vascular cell types, including endothelial cells, smooth muscle cells, and
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fibroblasts [4]. These ion channels play a significant role in regulating various
physiological processes, such as cell membrane potential, signal transmission,
hemodynamics, and vasomotor functions [5]. Changes in ion channels can
participate in the pathologic state of the vascular system. The contractile and
relaxation functions of vascular smooth muscle cells in the vascular wall are
significantly influenced by the function of Ca
2+
transport systems located on the
plasma membrane and the sarcoplasmic reticulum membrane [6].
The purpose of the research
Investigation of the dose-dependent relaxant effect of vincamine hydrochloride
and pyrozalin iodide alkaloids on aortic smooth muscle cells
’
Ca
2+L
-channels.
Research material and methods
The isometric contraction activity of the rabbit aortic smooth muscle was
recorded using standard methodology (mechanography) [7]. Experimental
animals were anesthetized by the dislocation method, the chest cavity was
opened through a surgical incision, and the aortic blood vessel was isolated and
removed. The aorta preparation was cut into ring segments of approximately
3-4 mm in length and mounted on a specialized hook in a tissue bath, and from
the other side, it was connected to an isometric force measurement apparatus,
Grass FT-03 (Grass Instrument, USA).
The experimental bath (5 ml) was continuously circulated with Krebs-
Henseleit physiological solution, and the temperature of the physiological
solution was maintained constantly by a thermostat. Additionally, the solution
was aerated with a gas mixture consisting of 95 % O
2
and 5 % CO
2
. The isometric
contraction activity of the aortic blood vessel preparation was recorded using a
Grass FT-03 mechanotransducer (Grass Instrument, USA) and a signal amplifier,
Endim 621.02 (Czech Republic).
The physiological Krebs-Henseleit solution used for the continuous
circulation in the experimental bath had the following composition (in mM): NaCl
–
118; KCl
–
4.8; MgSO
4
–
1.2; KH
2
PO
4
–
1.2; CaCl
2
–
2.5; NaHCO
3
–
25; glucose
–
11 (pH=7.4). Furthermore, the physiological solution was aerated with carbogen
(95 % O
2
and 5 % CO
2
), and the temperature was maintained at a constant level
(t = 37±0.5 °C) using a thermostat. Initially, the rabbit aortic preparation was
incubated for about 45-60 minutes under a tension of 1 g = 9.8 mN before
recording the myogenic electromechanical activity.
The isometric contraction activity of the vascular smooth muscle
preparation was recorded automatically using the Endim 621.02 (Czech
Republic) mechanical writing apparatus. To induce the contraction of the aortic
smooth muscle preparation under isometric conditions, an α
1
-adrenoreceptor
(α
1
-
AR) agonist, phenylephrine (1 µM), was used.
In the experiments, vincamine hydrochloride, pyrozalin iodide, L
–
NAME
(Sigma-Aldrich, Germany), NaHCO
3
, CaCl
2
, MgSO
4
, glucose, NaCl, KCl, NaH
2
PO
4
(Russia) were used. The obtained results were statistically analyzed using
Student
’
s t-test.
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Research results and their analysis
The membrane potential of smooth muscle cells (SMC) is one of the main
factors in controlling their contraction and functional activity. In a resting state,
the membrane potential of SMC is primarily determined by the permeability of
their cytoplasmic membrane to K
+
ions. Consequently, changes in the
concentration of extracellular K
+
ions lead to alterations in membrane potential,
which in turn affects the functional activity of SMC. Specifically, an increase in
extracellular K
+
ions in the incubation environment leads to depolarization of the
SMC plasma membrane, which is associated with the development of their
sustained contraction. This is why high-potassium solutions are widely used in
experiments to study SMC contraction.
For instance, the contraction activity of an aortic preparation incubated in
a KCl (50 mM) environment is associated with the activation of Ca
2+
channels in
the SMC plasma membrane, which is dependent on the membrane potential. In
this case, an increase in the extracellular K
+
ion concentration in the environment
leads to changes in the membrane potential. As a result, membrane depolarization
occurs, and this, in turn, activates potential-dependent Ca
2+
channels and
increases the intracellular Ca
2+
ion concentration in the cytoplasm of the cells,
contributing to muscle contraction.
In this context, the impact of the indole alkaloids vincamine and pyrozalin
iodide on the contraction of the aortic preparation stimulated by KCl (50 mM) was
investigated in these experiments. The alkaloids were found to have a potent
concentration-dependent vasorelaxant effect, with concentrations ranging from 5 to
200 µM. Specifically, at a concentration of 5 µM, vincamine was observed to reduce
the amplitude of aortic preparation contraction by approximately 15.7±4.2
%.
Furthermore, at a concentr
ation of 50 µM, it was noted that this value
constituted 93.6±3.6
% (Figure 1A). Similarly, pyrozalin iodide demonstrated a
maximum vasorelaxant effect of 85.3±4.1
% at a concentration of 200 µM (Figure 1B).
The obtained results indicate that vincamine and pyrozalin iodide alkaloids
exhibit their vasorelaxant effects through the involvement of potential-dependent
Ca
2+
channels in SMC. To further elucidate these results, in subsequent
experiments, the effects of vincamine and pyrozalin iodide on [Ca
2+
]out
concentration were investigated. In this condition, the alkaloids were found to
reduce the contraction activity of the aortic preparation by approximately
18.1±4.2
% for vincamine and 17.9±3.9
% for pyrozalin iodide (Figure 2).
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Figure 1. Concentration-dependent relaxant effects of vincamine
hydrochloride (A) and pyrozalin iodide (B) alkaloids on the contraction
activity induced by KCl (50 mM) in the rabbit aortic smooth muscle
preparation.
The contraction force induced by KCl (50 mM) was considered as 100% control
(significant differences in all cases: *p<0.05; and **p<0.01; n=6).
Figure 2. Relaxant effects of vincamine and pyrozalin iodide alkaloids on
potential-dependent L-type Ca
2+
channels to [Ca
2+
]
out
concentration
The contraction force of the rabbit aortic preparation was induced by the
cumulative addition of CaCl
2
to Krebs solutions in the presence of alkaloids.
The contraction force induced by 50 mM KCl was considered as 100% along the
ordinate axis, and the mM concentration of CaCl
2
was plotted along the abscissa
axis (significant differences in all cases: *p<0.05, **p<0.01; n=5).
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Additional experiments were conducted using the specific blocker of
potential-dependent Ca
2+
channels, verapamil (IC
50
= 0.1 µM), and analyzed To
further clarify the above-mentioned results.
In this condition, under verapamil (0.1 µM) incubation, the contraction
force of the aortic smooth muscle preparation was observed to decrease by
50±3.2
%, and under this condition, vincamine (IC
50
= 16.3 µM) concentration
further
reduced the contraction force by an additional 15.5±4
%, while pyrozalin
iodide (IC
50
= 82.2 µM) led to an additional decrease of 11.9±4.2
% (Figure 3).
Figure 3. Relaxant effects of vincamine and pyrozalin iodide alkaloids on
the contraction induced by KCl (50 mM) in the rabbit aortic smooth muscle
preparation in the presence of the Ca2
+L
-channel specific blocker,
verapamil (0.1 µM) incubation.
The contraction force induced by KCl (50 mM) was considered as 100 % control
(significant differences in all cases: *p<0.05; and **p<0.01; n=5).
The obtained results indicate that the vasorelaxant effects of the studied
indole alkaloids are associated with the blockade of Ca
2+L
-channels. Additionally,
the fact that the alkaloids further reduced the effect of verapamil suggests their
additional mechanism of action beyond Ca
2+L
-channel blockade. It is suggested
that indole alkaloids can modulate Ca
2+
homeostasis through the function of
receptor-operated Ca
2+
channels (Ca
2+
R).
In conjunction with potential-dependent L-type Ca
2+
channels, receptor-
operated Ca
2+
channels that are active in response to α
-adrenoreceptor agonist
stimulation also play a significant role in regulating Ca
2+
homeostasis in SMC. For
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instance, phenylephrine (0.1-
100 µM) induces contraction in b
lood vessel SMC
through a cascade of reactions as a result of α
1
-AR activation. In in vitro
conditions, α
1
-AR agonist phenylephrine is typically used to induce SMC
contraction. After the development of contraction to a steady state with
phenylephrine, we added the alkaloids at various concentrations (ranging from
1 µM to 250 µM) and studied their relaxant effects. In the experiments, we
investigated whether vincamine and pyrozalin iodide indole alkaloids play a role
in relaxing the receptor-operated Ca
2+
c
hannels. Using the α
1
-AR agonist
phenylephrine (1 µM) to induce contraction, we observed that vincamine exerted
a significant relaxant effect, reducing the contraction force by 18.2±4.4
% at 3 µM
concentration. Vincamine had a pronounced effect on the contraction induced by
1 µM phenylephrine in the aortic preparation in the concentration range of
3-
35 µM, reducing the contraction force by 95.6±3.8
% at a concentration of
30 µM, and an IC
50
value of 12.9 µM was determined. On the other hand, pyrozalin
iod
ide alkaloid reduced the contraction force by approximately 81.4±4.1
% at
concentrations ranging from 10 to 250 µM, with a half
-maximal concentration of
76.5 µM.
(Figure 4).
Figure 4. Effects of vincamine (A) and pyrozalin iodide (B) alkaloids on the
contraction induced by phenylephrine in the rabbit aortic smooth muscle.
The contraction force induced by 1 µM phenylephrine is considered as 100%
control (significant differences in all cases: *p<0.05; and **p<0.01; n=5).
The results obtained suggest that the observed effects of the studied
alkaloids may be attributed to receptor-operated Ca
2+
channel blockade. Such
blockade of receptor-operated Ca
2+
channels leads to reduced Ca
2+
ion entry, a
decrease in [Ca
2+
]
in
levels, and as a consequence, relaxation of smooth muscle cells
(SMC). These findings indicate that the relaxant effect of the studied alkaloids may
be directly associated with the blockade of receptor-operated Ca
2+
channels.
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Figure 5 illustrates the influence of phentolamine (10 µM) on the relaxa
nt effect
of vincamine and pyrozalin iodide (5th figure). In conditions where phentolamine was
not present, the relaxant effect of vincamine (35 µM) and pyrozalin iodide (250 µM)
alkaloids was significantly reduced compared to the control conditions with 1
0 µM
phentolamine, showing reductions of approximately 43.4±4.7 % and 56.7±4.3 %,
respectively, under the influence of phentolamine.
These results indicate that the relaxation effect of indole alkaloids may
involve both the blockade of voltage-gated L-type Ca
2+
channels and the blockade
of receptor-operated Ca
2+
channels. Such combined effects can lead to a noticeable
decrease in [Ca
2+
]
in
levels in SMCs, potentially contributing to the significant
development of the relaxant effect of the studied alkaloids.
Conclusion
The results obtained suggest that the relaxant effect of vincamine and
pyrozalin iodide alkaloids on isolated rat aortic smooth muscle preparations
under in vitro conditions can be explained by the activity of plasma membrane
voltage-gated Ca
2+L
-channels that are sensitive to changes in membrane potential.
It is possible to infer that their relaxant effect is associated with the blockade of
L-type Ca
2+
channels and a reduction in [Ca
2+
]
in
concentration.
It appears that vincamine and pyrozalin iodide alkaloids exert their relaxant
effect primarily through the blockade of L-type Ca
2+
channels. However, the
preservation of the relaxant effect of the studied alkaloids under the presence of
verapamil implies the involvement of alternative mechanisms beyond the
blockade of L-type Ca
2+
channels in the regulation of intracellular Ca
2+
ion levels
in smooth muscle cells.
Figure 5. Influence of phentolamine (10 µM) on the relaxant effect of
vincamine and pyrozalin iodide.
The effects of alkaloids in the
presence of verapamil (0.1 µM) and phentolamine
(10 µM) are shown. The contraction induced by 1 µM phenylephrine is considered
as 100% control (significant differences in all cases: *p<0.05; **p<0.01; n=3).
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Our experiments indicate that vincamine and pyrozalin iodide exhibit a
notable relaxant effect on rat aortic preparations pre-contracted with
phenylephrine, with vincamine showing a stronger relaxant effect relative to
pyrozalin iodide. Consequently, our results also suggest the involvement of
receptor-operated Ca
2+
channels in the relaxant effect of these alkaloids.
These findings indicate that the relaxant effect of the studied alkaloids
involves both voltage-gated L-type Ca
2+
channel blockade and receptor-operated
Ca
2+
channel blockade. This dual mechanism may lead to a substantial decrease in
[Ca
2+
]
in
in SMCs, contributing to the significant development of the relaxant effect.
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