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International Journal of Pharmaceutical Invention
September 2012, Volume 2(8) IJPI 20
EFFECT OF VERAPAMIL HYDROCHLORIDE ON
ACETYLCHOLINE INDUCED CONTRACTION IN FROG RECTUS
ABDOMINIS MUSCLE
K. KARTHIGADEVI*, O. MADHAVI USHARANI, I. SESHA MADHAVI,
CH.V.R.TANUJA, S. KAVIMANI
Department of Pharmacology, College of Pharmacy,
Mother Theresa Post Graduate and Research Institute of Health Sciences (Govt. of
Puducherry Institution), Indria Nagar, Gorimedu, Puducherry-605006, India.
E-mail: [email protected]
Keywords: Verapamil Hydrochloride, Calcium channel, Frog Rectus Abdominis Muscle,
Right Hand Side Shift.
INTRODUCTION
Verapamil Hydrochloride is an oral and
parenteral calcium-channel blocking
(CCB) agent belonging to the
phenylalkylamine class. Verapamil
Hydrochloride is useful for the treatment
of angina, hypertension, and for
supraventricular tachyarrhythmias. It is
considered a class IV antiarrhythmic agent
and Verapamil Hydrochloride is more
effective than digoxin for controlloing
Verapamil Hydrochloride is an oral and parenteral calcium-channel blocking (CCB)
agent belonging to the phenylalkylamine class. Verapamil Hydrochloride is useful for
the treatment of angina, hypertension, and for supraventricular tachyarrhythmias. It is
considered a class IV antiarrhythmic agent and Verapamil Hydrochloride is more
effective than digoxin for controlling ventricular rate in patients with atrial
fibrillation. Skeletal muscle contraction is explained by the excitation-contraction
coupling and is dependent on the calcium induced release of calcium from the
sarcoplasmic reticulum by Ca2+ release channel due to the previous influx of
Ca2+
through Dihydropyridine receptor present on the T-tubule which is due to the
conduction of action potential on T-tubule. So, an attempt is made to find out the
effect of Verapamil Hydrochloride on acetylcholine induced contraction in Frog
Rectus Abdominis Muscle. Maximum response produced by acetylcholine in the
absence of the blockers was taken as 100% and against that the % response was
calculated. Verapamil Hydrochloride produced significant dose dependant inhibition
of acetylcholine induced contraction in CRC.
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than digoxin for controlling ventricular
rate in patients with atrial fibrillation.
Verapamil Hydrochloride was synthesized
in 1962 and in 1981, became the first
calcium channel blocker to be approved
by the FDA. Verapamil Hydrochloride
inhibits the influx of extracellular calcium
across the myocardial and vascular
smooth muscle cell membranes. It exerts
its activity at the membrane surface of
arterial smooth muscle cells and withinconductile and contractile tissue in the
myocardium, but the serum calcium levels
remain unchanged.[ 1 ]
The calcium channel antagonists are a
mature group of drugs directed at
cardiovascular diseases including
hypertension, angina, peripheral vascular
disorders and some arrhythmic conditions.
Their sites and mechanism of actions have
been well explored over the past two
decades and their interactions at the
1subunit of L-type channels (Cav1.1-1.4)
have made them valuable molecular tools
for channel classification and localization.
With the realization that other members of
the voltage-gated calcium channel family
exist Cav 2.1-2.3 and Cav 3.1-3.3
considerable effort has been directed to
drug discovery at these channel types
where therapeutic prospects exist for a
variety of disorders including pain,
epilepsy, affective disorders,
neurodegenerative disorders, etc. In
contrast to the situation with the L-type
channel antagonists success in developing
small molecule antagonists of therapeutic
utility for these other channel types has
thus far been lacking. The reasons for this
are explored and potential new directions
are indicated including male fertility, bone
growth, immune disorders, cancer and
schistosomiasis.[ 2 ]
To explore and to investigate for new
calcium channel blockers first it wasthought to have an extensive knowledge
over calcium regulation by different
calcium channels and their structures to
have a look on binding sites of drugs.
The concentration of cytosolic free
calcium ([Ca2+
]i)is critically important for
the control of many essential cellular
responses. Changes in [Ca2+
] can control
specialized functions like excitability,
contraction, or exocytosis, due to which
neurotransmitter release takes place, while
also regulating universal cellular activities
such as metabolism and gene expression.
In most cells, elevations([Ca2+
]i)in arise
from [Ca2+
] entry via Ca2+
channels in the
surface membrane, or Ca2+
discharge from
internal stores, or both. Mechanisms that
tend to return ([Ca2+
]i) toward its low
resting value include extrusion of [Ca2+
]
across the surface membrane or
resequestration by internal [Ca2+
] stores
and by Ca2+ pump or Na+-Ca2+
exchangers.[ 3 ]
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MATERIALS AND METHOD
A pithed frog was laid on its back on the
frog dissecting board. The skin on the
abdomen was removed and the rectus
abdominis muscle was exposed. The one
side of the rectus muscle was dissected
from the pelvic girdle to the pectoral
girdle. The muscle was then pinned to the
cork so as to keep it stretched to its
normal length and soaked in frog ringer
solution. A thread was sewn through eachend and the muscle was mounted in organ
bath containing frog ringer solution at
room temperature. The concentration
response curve was obtained as described
by Kulkarni (2009). The experiment was
repeated in each set of preparation in
presence of Verapamil Hydrochloride (0.4
and 0.8 g/ml).The tissue was irrigated
for 20 minutes. Maximal response
produced by acetyl choline in the absence
of the blocker was taken as 100% and
against that the percentage response was
calculated.[ 4 ]
RESULTS
Verapamil Hydrochloride at various test
doses (0.4 and 0.8g/ml) produced
significant dose dependent inhibition of
acetylcholine induced contractions inconcentration response curves. 640g/ml
of acetylcholine was required to produce
100% of response where as in the
presence of Verapamil Hydrochloride
(0.4 and 0.8g/ml), the same
concentration of acetylcholine produced
92% and 66% ofresponses respectively.
Fig 1: Effect of Verapamil Hydrochloride on Ach Induced Contractions in Frog Rectus
Abdominis Muscle.
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Fig 2: The graph represents the log dose- response curves of Acetylcholine (100 g/ml)
in presence of various concentrations of Verapamil Hydrochloride (0.4 and 0.8g/ml).
The progressive shift to the right hand side was noted indicating the antagonistic effect
of Verapamil Hydrochloride.
DISCUSSION
Any muscle contraction is possible only
through the cellular calcium signaling and
cellular calcium regulation is the
substantial preoccupation of the cell.
Heilbrunn described the calcium is the
most ubiquitous intracellular signaling
molecule, which serves both physiological
and pathologically. It plays an important
role in signal transduction pathways,
where it acts as a second messenger, in
neurotransmitter release from neurons by
exocytosis, contraction of all muscle cell
types. Many enzymes require calcium
ions as a cofactor; those of the blood-
clotting cascade being notable examples.
Extracellular calcium is also important for
maintaining the potential difference across
excitable cell membranes, as well as
proper bone formation.[ 3,5 ]
Regulation of Ca2+
is under very tight and
dynamic control in all major cell
compartments (cytoplasm, nucleus,
endoplasmic reticulum, mitochondria). In
each compartment, this control is
achieved through the interplay of
transmembrane entry, extrusion systems
(channels, exchanger, transporters) and of
buffering systems (Ca2+
binding protein).
It is this role of calcium as an
information-bearing second messenger
together with the control processes that
make possible the existence of calcium
controlling drugs. A triumvirate of
hormones-vitamin D, parathyroid
hormone and calcitonin- that serve to
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regulate calcium absorption, calcium
excretion and bone deposition and
resorption (remodelling), achieves this
control.[6 ]
THE KEY ROLE OF Ca2+
and Ca2+
CHANNEL IN CELLULAR
SIGNALING
Ca2+
channels allow passage of Ca2+
ions
into the cytoplasm through a selective
pore which is opened in response to
depolarization of the cell membrane. TheCa
2+flux creates a net inward,
depolarizing current and the resulting
accumulation of Ca2+
in the cytoplasm can
act as a chemical trigger for secretion of
hormones and neurotransmitters,
contraction of muscle and a variety of
other Ca2+
sensitive events. Thus, upon
sensing membrane potential changes,
Ca2+
channels simultaneously generate an
electrical signal while directly creating an
intracellular chemical messenger. This
dual ability is unique among the family of
ion channels and allows the Ca2+
channel
to play a variety of roles in excitation-
secretion and excitation-contraction
coupling.
Excitation-Contraction Coupling
Skeletal muscle contraction is explained
by the excitation-contraction coupling and
is dependent on the calcium induced
release of calcium from the Sarcoplasmic
reticulum by Ca2+ release channel (RyR)
due to the previous influx of Ca2+
through
Dihydropyridine Receptor (DHPR)
present on the T-tubule which is due to
the conduction of action potential on T-
tubule. The inhibitory effect of Verapamil
Hydrochloride and diltiazem may be due
to the blockade of receptor operated
calcium channel and voltage dependent
calcium channel.Sarcoplasmic reticulum
and single channel studies have provided
evidence that phenylalkylamine calcium
antagonists inhibit calcium releasethrough the sarcoplasmic reticulum
calcium channel/ryanodine receptor. This
action has not been observed with
dihydropyridine calcium antagonists.[ 7 ]
CONCLUSION
The acetylcholine induced contractions in
isolated skeletal muscle preparation was
effectively antagonised by Verapamil
Hydrochloride. It also showed dose
dependent antagonistic effect. The log
dose response curves of acetylcholine in
presence of Verapamil Hydrochloride was
shifted to the right hand side indicating
the antagonistic effect. The antagonistic
effect of Verapamil Hydrochloride
(phenylalkylamine) may be due to the
blocking of both L-type Ca2+
channel
(DHPR) and Ryanodine Ca2+
sensitive
channel (RyR) which are involved in the
skeletal muscle contractions.
REFERENCES1. http://www.druginfosys.com
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2. TriggleDJ, Calcium ChannelAntagonists: Clinical uses past,
present and future, Biochem
Pharmacology, 74(1), 2007 June 30,
1-9.
3. Richard W. Tsien, Rogery Y. Tsien,Calcium Channels, Stores and
Osallation, Annual Review, Cell
Biology, 6, 1990: 715-60.
4. S.K.Kulkarini, Handbook ofExperimental Pharmacology, 3
rd
edition, 2009, VallabhPrakashan
Publications
5. http://en.wikipedia.org/wiki/Calcium_channel_blockers
6. Stefan I McDonough, CalciumChannel Pharmacology, Springer,
2004.
7. Ricardo Zucchi, Effect of gallopamilon excitation-contraction coupling,
General Pharmacology: The vascular
system, 27(5), July 1996, 749-7