PARADOXICAL EFFECTS OF METHANOL EXTRACTS OF RICINUSCOMMUNISSEEDS ON SMOOTH MUSCLE PREPARATIONS

ABSTRACT

The biphasic pharmacological activity of the aqueous methanol extract of Ricinuscommunisseeds on smooth muscles was investigated in this research. The qualitative and quantitative phytochemical analysis for bioactive compounds in the methanol extract of the fermented and unfermented Ricinuscommunisseeds were carried out by the method of Trease and Evans  and Harborne. The median lethal doses of the two forms of extracts were determined by the method described by Lorke.The qualitative phytochemical screening of the extracts showed relative presence of alkaloids, flavonoids, hydrogen cyanides, steroids, soluble carbohydrates, tannins and phenol in the fermented and unfermented extracts; while glycosides, saponins and reducing sugars were not present in the fermented extract. The quantitative phytochemical screening of the extracts showed the presence of high quantities of reducing sugars, 39.60±0.00mg/100g, soluble carbohydrates, steroids and alkaloids for the unfermented methanol extracts of Ricinuscommuniswhile the fermented extract revealed the presence of high quantities of tannins,

15.16±0.04mg/100g; flavonoids, 4.94±0.03mg/100g; and  phenolics 12.62±0.04mg/100g. The median lethal dose of the unfermented methanol seed extracts of Ricinuscommunisrecorded nodeath  at concentration of 5000mg/kg body weight while the fermented extract recorded death at the same concentration.The smooth muscle effects of the extracts were determined on the rabbit jejunum and pregnant rat uterus. A membrane depolarizing drug,acetycholinewas used to initiate the normal rhythmic contraction and it contracted the rabbit jejunum at the concentration of 1µg. Adrenaline, adrenergic receptor substance relaxed the jejunum at the concentration of

1µg. The unfermented extract relaxed the jejunum at different doses of 0.1, 0.2 and 0.4 ml at the same concentration of 0.5µg/ml. The fermented extract also relaxed the jejunum at different doses of 0.1, 0.2, 0.4 and 0.8 ml. Prazosin, an α- adrenergic antagonist, blocked the relaxing effect of adrenaline at increasing doses of 0.4, 0.8 and 1.0 ml at a working concentration of 20 µg/ml. The extracts were also added in the bath with prazosin, it also blocked the relaxant effects of the extracts at the doses of 0.2 and 0.4 ml. Indomethacin, a non-steroidal anti-inflammatory drug, at a concentration of 20µg/ml, had no effect on the relaxant effect of adrenaline even at higher dose of 1.0 ml. Indomethacin had no antagonistic effect on the extracts, at concentrations of 0.5µg/ml. Oxytocin, a standard drug known for uterine contraction initiated a normal rhythmic contraction on the uterus at a concentration of 10iu/ml and a dose of 0.1 ml. The unfermented extract had no significant effect on the uterine tissue at a concentration of 0.5µg/ml dose of 0.1 ml while the fermented extract contracted the uterine tissue at the same concentration and dose. Indomethacin, a prostaglandin synthesis blocker, also had no significant effect on the uterine tissue against the standard drugs and the extracts at the concentrations of 20µg/ml and 0.5µg/ml respectively. Ergotamine blocked the effects of the extracts at the concentration of 10µg/ml and a dose of 1.0 ml. From these results, it can be concluded that the fermented methanol extracts of Ricinuscommunisseeds can serve as an oxytocic agents for pregnant women during delayed labour as claimed by the traditional birth attendants.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.0          Medicinal Plants

Plants serveas rich sources of organic compounds, many of which have been used for medicinal purposes. Medicinal plants are the plants whose parts (leaves, seeds, stems, roots, fruits, foliage etc), extracts, infusions, decoctions or powders are used in the treatment of different diseases of humans, plants and animals (Jamil et al.,2007). In the last few decades, there has been an exponential growth in the field of herbal medicine. It is getting popularized in developing and developed countries, owing to its natural origin and lesser side effects. One of such medicinal plant is Ricinus communis (Euphorbiaceae), which is commonly known as Castor. It is a small tree which is found all over the India (Manpreet et al., 2012).There is a wide spectra of trees, plants and shrubs whose seeds, roots, barks and leaves are used by humans throughout the globe due to their nutritional or medicinal value (Abayomi, 1986). In the last few years, there has been an exponential growth in the field of herbal medicine and these drugs are gaining popularity both in the developing and developed countries because of their natural origin and less side effects (Manisha et al., 2007).However, these complementary components give the plant as a whole, the safety and efficiency much superior to that of its isolated and pure active components (Shariff,

2001). The World Health Organisation (WHO) report in 1993 showed that nearly 80 percent of world population is dependent on the traditional system of medication, that is the use of plants and their parts as medicine (Mathur et al.,2011).

It is true that without nature, it is impossible for human beings to survive. The food, clothes and shelter are the three basic necessities of human beings and the most important is good health, which is being provided by the plant kingdom. In traditional medicine, there are many natural crude drugs for different health purposes, one of such plants is Ricinus communis (Jitendra and Ashish, 2012).

1.1    Ricinus communis

Castor bean, Ricinus communis is a species of flowering plant in the spurge family, Euphorbiaceae (Momoh et al., 2012). It is an important drought-resistant shrub, also native to the Ethiopian region of the tropical Africa and has become naturalized in tropical and temperate regions throughout the world (Weiss, 2000). Ricinus communis (Euphorbiaceae family) is a soft wooden small tree, grown throughout the tropics and warm temperate regions of the world (Parkeh and Chanda, 2007). Its seed is the castor bean, which despite its name, is not a true bean. Castor is indigenous to the South Eastern Mediterranean basin, Eastern Africa and India, but its widespread throughout tropical regions and widely grown elsewhere as an ornamental plant (Philips and Martyn, 1999). Ricinus communis is a small wooden tree which grows to about 6 meters in height and found in South Africa, India, Brazil and Russia (Singh et al., 2010). The Euphorbiaceae is the fourth largest family of the angiosperms comprising over 300 genera and about 7500 species and are distributed widely in tropical Africa (Gill, 1988). The Euphorbiaceae plants are shrubs, trees, herbs or rarely lianas (Pandey, 2006). The family provides food and varied medicinal properties used in ethnobotany (Etukudo, 2003).

The plant has many common names such as castor plant, castor oil plant, castor bean plant, wonderboom, dhatura, eranda and palma Christi. Locally, the plant  is known in Nigeria as “Zurman” (Hausa), “Laraa” (Yoruba), “Ogili isi” (Igbo), “Kpamfinigulu” (Nupe), “Jongo” (Tiv) and Era ogi (Bini) (Sani and Sule, 2007). The castor plant is considered by most authorities to be native of the Tropical Africa and may have originated in Abyssinia, Ethiopia. The plant is a native of India with about 17 species that have been grouped into two: as shrubs and trees that produce large seeds or as annual herbs that produce smaller seeds (Weiss, 1971).

1.1.1   Morphology and classification of Ricinus communis seeds

It consists of several branches, each terminated by a spike. The mature spike is fifteen to 30cm long and each spike bears 15 to 80 capsules (Oplinger et al.,1990). The leaves  are alternate, curved, cylindrical, purplish petioles, sub peltate, drooping, stipules large, ovate, yellowish, united into a cap enclosing the buds, deciduous, blade 6-8 inches across, palmately cut for three quarters of its depth into 7-11 lanceolate, acute, coarsely serrate segments, smooth blue green, paler beneath, red and shinning when young (Manpreet et al., 2012).

The flowers are monoecious and about 30-60cm long. The fruit is a three-celled thorny capsule. The capsule of fruit covered with soft spins like processes and dehiscing into three 2-valved cocci. The seeds are somewhat compressed, oval, 8-18mm long and 4-12mm broad. The testa is very smooth, thin and brittle (Jitendra and Ashish, 2012). The male flowers are yellowish-green with prominent creamy stamens and are carried in ovoid spikes up to 15 centimeters (5.9inches) long; the female flowers, borne at the tips of the spikes, have prominent red stigmas. The fruit is a spiny, greenish (to reddish-purple) capsule containing large, oval, shiny, bean-like, highly poisonous seeds with variable brownish mottling. Castor seeds have a warty appendage called the caruncle, which is a type of elaiosome. The caruncle promotes the dispersal of the seed by ants (Myrrmecochony) (Brickell, 1996).

1.1.2 Taxonomical classification of Ricinus communis seeds

Castor bean plant (Ricinus communis) is a flowering plant that belongs to the Euphorbiaceae

family and is classified scientifically as

Kingdom                                       Plantae

Phylum                                         Magnoliophyta Class                                             Magnoliopsida Order                                            Malpighiales Family                                           Euphorbiaceae Sub family                                    Acalyphoideae Tribe                                             Acalypheae

Sub tribe                                       Ricininae

Genus                                            Ricinus

Species                                          R. communisSource: (Jitendra and Ashish, 2012)

Fig. 1: The fruit ofRicinus communis plant

(Jitendra and Ashish, 2012)

Fig.2: The whole plant of Ricinus communis

(Jitendra and Ashish, 2012)

Fig. 3: The seeds of Ricinus communis

(Sabina et al., 2009).

Fig. 4: The leaves of Ricinus communis

(Source; Rotblatt and Ziment, 2002).

1.1.3 Pharmacological Uses of Ricinus communis

Ricinus communis or castor plant  has high traditional and  medicinal value  for  maintaining disease free healthy life. Traditionally, the plant is used as laxative, purgative, fertilizer and fungicide. The plant also possess beneficial effects like anti-oxidant, antihistaminic, antinociceptive, antiashmatic, antiulcer, immunomodulatory, antidiabetic, hepatoprotective, anti- fertility, anti-inflammatory, antimicrobial, central nervous system stimulant, lipolytic, wound healing, insecticidal, larvicidal and many other medicinal properties (Jitendra and Ashish, 2012).

All the parts of the plants are used medicinally (Obumselu et al., 2011). All these uses are due to the presence of certain phytoconstituents in the plant. The major phytoconstituents in this plant are rutin, gentistic acid, quercetin, gallic acid, kaempferol-3-o beta-d-rutinoside, kaempferol-3-0 beta-d-xylopyranoid, tannins, ricin A, B and C, ricinus agglutinin, indole-3-acetic acid and an alkaloid ricinine (Manpreet et al., 2012). In the traditional system of medicines, Euphorbiaceae plants are used to treat various microbial diseases such as diarrhoea, dysentery, skin infections

and gonorrhoea (Ajibesin et al., 2008). In the Indian system of medicine, the leaf, root and seed oil  of Ricinus  communis  have  been  used  for  the  treatment  of the  inflammation and  liver disorders, hypoglycaemia and laxative (Kensa and Syhed, 2011). Ricinus communis of the family Euphorbiaceae,  is  traditionally  used  by  Traditional Birth  Attendants (TBAs)  in  Machakos district of Kenya to induce or augument labour, manage protracted labour, post partum haemorrhage (Kaingu et al., 2012). It has also being the practice that, in the Middle Belt of Nigeria,  traditional  healers  administer  to  women  three  seeds  of  the  variety  minor  as contraceptives for a duration of 12 months (Okwusaba et al.,1997). Castor oil has many uses in medicine and other applications. A water extract of the root bark showed analgesic activity in rats; antihistamine and anti-inflammatory properties were found in ethanol extract of Ricinus communis root bark (Lomash et al., 2010). It was also found out that the methanol extract of the ether  soluble  fraction  of Ricinus communis seed  possesses anti-ovulatory activity and  also distorts the oestrous cycle of adult cyclic rats (Ogunranti, 1997).

1.2.  Phytochemistry

Phytochemistry is the study of natural bioactive products found in plants that work with nutrients and dietary fibre to protect against diseases (Doughari et al., 2009). “Phyto” is a Greek word that means plant and phytochemicals are usually related to plant pigments. So fruits and vegetables that have bright colours – yellow, orange, red, green, blue and purple, generally contain more phytochemicals and more nutrients. Research suggests that phytochemicals, working together with nutrients found in fruits, vegetables and nuts, may help slow the ageing process and reduce the risk of many diseases including cancer, heart diseases, stroke, high blood pressure, cataracts, osteoporosis and urinary tract infections (Gao et al., 2001).

Phytochemicals protect health. They can have complementary and overlapping mechanisms of action in the body including antioxidant effects, modulation of detoxification enzymes, stimulation of the immune system, modulation of hormone mechanisms and antibacterial and antiviral effects (Conn, 1995). Medicinal plants are of great importance to the health of individuals and communities. The medicinal value of these plants lies in some chemical substances that produce definite physiological actions on the human body (Hill, 1952). Many medicinal plants are used as spices and food plants. They are also sometimes added to foods

meant for pregnant and nursing mothers for medicinal purposes (Okwu, 2001). Medicinal herbs are significant sources of synthetic and herbal drugs. Medicinal plants have active ingredients which are responsible for most of the biological activities they exhibit (Fukumoto and Mazza,

2000).

1.2.1 Phytochemical constituents of plants

Phytochemicals are a heterogeneous group of chemical compounds with numerous biologically active plant compounds that have potential disease inhibiting capabilities (Akinmoladun et al.,

2007).  Phytochemicals (plant  chemicals)  are  bioactive  substances of plants that  have  been associated with the protection of human health against chronic degenerative diseases (Fukumoto and Mazza, 2000). The term ‘phytochemicals’ according to the American Cancer Society refers to a wide variety of compounds produced by plants and can be found in fruits, vegetables, beans, grains. They are chemical compounds formed during the plant normal metabolic processes. These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1998; Okwu, 2004). More than 900 different phytochemicals have been found in plant foods and more probably will be discovered (Polk,

1996). These protective plant compounds are an emerging area of nutrition and health, with new research reported every day. Some examples of phytochemicals in fruits and vegetables include – carotenoids, β-carotene, lutein, lycopene, zeaxanthin, flavonoids, anthocyanin, limonene, indoles and allium compounds. Phytochemicals are present in a variety of plants utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables (Okwu, 2005).

According to the World Health Organization, a medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semi-synthesis. Such a plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of a disease condition and therefore contains chemical components that are medically active. These non-nutrient plant chemical compounds or bioactive components are often referred to as phytochemicals (‘phyto-‘ from Greek – phyto meaning ‘plant’) or phytoconstituents and are

responsible for protecting the plant against microbial infections or infestations by pests (Abo et al., 1991; Liu et al., 2004; Nweze et al., 2004; Doughari et al., 2009).

1.2.1.1  Alkaloids

Alkaloids are  natural products that  contains heterocyclic  nitrogen atoms; they are  basic  in character. The name of alkaloids derives from the “alkaline” and it was used to describe any nitrogen-containing base (Mueller-Harvey and McAllan, 1992). These are the largest group of secondary chemical constituents; they are made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1°, 2° or 3° amines, contribute to the basicity of the alkaloid (Firn, 2010).

Alkaloids generally exert pharmacological activity particularly in mammals such as humans. Many of our most commonly used drugs are alkaloids from natural sources and new alkaloidal drugs are still being developed for clinical use (Roberts and Winks, 1998). Most alkaloids with biological activity in humans affect the nervous system, particularly the action of neural transmitters, example, acetylcholine, adrenaline, noradrenaline, gamma-aminobutyric acid (GABA), dopamine and serotonin (Schmeller and Wink, 1998). They react with acidsto form crystalline salts without the production of water (Firn, 2010). The majority of alkaloidsexist in the solid state such as atropine, some as liquids containing carbon, hydrogen, and nitrogen.Most alkaloids are readily soluble in alcohol. Though they are sparingly soluble in water,their salts of are usually soluble. The solutions of alkaloids are intensely bitter. Thesenitrogenous compounds function in the defence of plants against herbivores and pathogens,and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to theirpotent biological activities (Schmeller and Wink, 1998).

Fig. 5    Basic structures of some pharmacologically important plant derived alkaloids (Source; Madziga et al., 2010).

1.2.1.2  Flavonoids

Flavonoids are an important group of polyphenols widely distributed among the plant flora. Structurally, a flavonoid ismade of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. Flavonoids are also referred to as bioflavonoids. They are organic compounds that have no direct involvement with the growth or development of plants, they are plant  nutrients that  when consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the human body. Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than

4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960).

Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids, flavanones, flavonols and anthocyanidines (Nijveldt et al.,2001; Kuhnan, 1976). Flavones are characterized by a planar structure because of a double bond  in the central aromatic ring.

Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli and berries. Flavonones are mainly found in citrus fruit, an example is narigin.  Flavonoid  is  involved  in  scavenging  of  oxygen  derived  free  radicals  (Nijveldt  et al.,2001). It has been identified as a potent hypolipidemic agents in a number of studies (Harnafi and Amrani, 2007; Narender et al.,2006). It  has also been established that flavonoids from medicinal plants possess a high antioxidant potential due to their hydroxyl groups and protect more efficiently against free radical related diseases like arteriosclerosis (Vaya et al.,2003; Kris- Etherton  et  al.,2002).  Experimental  studies  showed  that  flavonoids  enhance  vaso-relaxant process (Bernatova et al.,2002) and prevent platelet activity-related thrombosis (Wang et al.,2002).

Fig. 6:Basic structures of some pharmacologically important plant derived flavonoids (Source; Kar, 2007).

1.2.1.3 Glycosides

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with  a  host  of different  varieties of organic  hydroxyl (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-

soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007). Alcohol, glycerol or phenol represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action (Firn, 2010).

1.2.1.4 Tannins

Tannins are polymerized phenols with defensive properties. Their name comes from their use in tanning, rawhides to produce leather. In tanning, collagen proteins are bound together with phenolic groups to increase the hide’s resistance to water, microbes and heat (Heldt and Heldt,

2005). Two categories of tannins that are of importance are the condensed and hydrolysable tannins. The polymerization of flavonoid molecules produces condensed tannins, which are commonly found in woody plants. Hydrolysable tannins are polymers, but they are a more heterogeneous mixture of phenolic acids (especially gallic acid) and simple sugars. Though widely distributed, their highest concentration is in the bark and galls of oaks (Heldt and Heldt,

2005).  These  are  widely  distributed  in  plant  flora.  They are  phenolic  compounds of  high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007). They form complexes with proteins, carbohydrates, gelatin and alkaloids.

Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins and various other organic compounds including amino acids and alkaloids (Petridis, 2010). The astringency from tanninsis what causes the dry and pucker feeling in the mouth following the consumption of unripened fruit or red wine (Serafini et al.,1994). Many human physiological activities, such as stimulation of phagocytic cells, host-mediated tumour activity, and a wide range of anti-infective actions, have been assigned to tannins (Haslam, 1996). One of their biological actions is to complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic interactions, as well as by covalent bond formation (Haslam, 1996,

Stern et al.,1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope, transport proteins etc

Fig. 7 Basic structures of some pharmacologically important plant derived tannins (Source; Heldt and Heldt, 2005).

1.2.1.5 Saponins

Saponins are glycosides of triterpenes and steroids which are characterized by bitter or astringent taste, foaming properties (Okigbo et al., 2009), haemolytic effect on red blood cells and cholesterol binding properties (Okwu, 2005). Saponins increase the permeability of intestinal mucosa cells, inhibit active nutrient transport and facilitate the uptake of substances to which the gut would normally be impermeable (Gee et al., 1997). It  has also been shown to possess beneficial effects such as cholesterol lowering properties and exhibits structure dependent biological activity (Harborne, 1998).

Saponins, being both fat and water soluble, have surfactant and detergent activity. Thus they would be expected to influence emulsification of fat-soluble substances in the gut, including the formation of mixed micelles containing bile salts, fatty acids and fat-soluble vitamin (Okwu,

2005).

1.2.1.6 Steroids

Sterols are triterpenes which are based on the cyclopentane hydrophenanthrene ring system (Harborne, 1998). Sterols were at one time considered to be animal substances (similar to sex hormones, bile acids, etc) but in recent years, an increasing number of such compounds have been detected in plant tissues. Sterols have essential functions in all eukaryotes. For example, free sterols are integral components of the membrane lipid bilayer where they play an important role  in  the  regulation of  membrane  fluidity  and  permeability  (Corey et  al.,  1993).  While cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants, with sitosterol usually predominating. Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol (formerly known as β-sitosterol), stigmasterol and campesterol (Harborne, 1998). These common sterols occur both free and as simple glucosides. Certain sterols are confined to lower plants; one of which is ergosterol, found in yeast and many fungi. Others occur mainly in lower plants but also appear occasionally in higher plants, e.g fucosterol, the main steroid of many brown algae and also detected in coconut (Harborne, 1998).

1.3  Depolarization

Depolarization  is  a  positive-going change  in  a  cell’s  membrane  potential,  making  it  more positive, or less negative, and thereby removing the polarity that arises from the accumulation of negative charges on the inner membrane and positive charges on the outer membrane of the cell. In  neurons and some other cells, a large enough depolarization may result in an  action potential. Hyperpolarization is the opposite of depolarization, and inhibits the rise of an action potential (Yellen, 2002).

1.3.1 Hyperpolarization

Hyperpolarization is a change in a  cell’smembrane potential that makes it more negative. It is the opposite of a  depolarization. It inhibits action potentials by increasing the stimulus required to move the  membrane  potential to  the  action potential threshold (Goldin,  2007). Hyperpolarization is often caused by efflux of K+ (a  cation) through K+ channels, or influx of Cl– (an  anion)  through  Cl–   channels.  On  the  other  hand,  influx  of  cations,  e.g.  Na+   through

Na+channels  or  Ca2+    through  Ca2+   channels,  inhibits  hyperpolarization  (MacDonald  and Rorsman,  2006). If a cell has Na+ or Ca2+ currents at rest, then inhibition of those currents will also result in a hyperpolarization. Because hyperpolarization is a change in membrane  voltage,

electrophysiologists measure it using  current clamp techniques. In  voltage clamp, the membrane currents giving rise to hyperpolarization are either an increase in outward current or a decrease in inward current (Yellen, 2002).

1.3.2 Excitation-contraction coupling

Excitation–contraction couplingis a term coined in 1952 to describe the physiological process of converting an electrical stimulus to a mechanical response (Sandow, 1952). Excitation- contraction coupling refers to the sequence of events by which an action potential in the plasma membrane of a muscle fibre leads to cross-bridge activity by the mechanisms just described (Widmaier  et  al.,  2010).  A  smooth  muscle  is  excited  by  external  stimuli,  which  causes contraction. It may contract spontaneously (via ionic channel dynamics) or as in the gut, special pacemakers cells  interstitial cells of Cajal produce rhythmic contractions. Also, contraction, as well as relaxation, can be induced by a number of physiochemical agents (e.g., hormones, drugs, neurotransmitters – particularly from the  autonomic nervous system). Smooth muscle in various regions of the vascular tree, the airway and lungs, kidneys and vagina is different in their expression of ionic channels, hormone receptors, cell-signaling pathways, and other proteins that determine function (Aguilar et al., 2010).

This process is fundamental to muscle physiology, whereby the electrical stimulus is usually an action potential and the mechanical response is contraction. EC coupling can be dysregulated in many diseases. Though E-C coupling has been known for over half a century, it is still an active area of biomedical research. The general scheme is that an action potential arrives to depolarize the cell membrane. By mechanisms specific to the muscle type, this depolarization results in an increase in cytosolic  calcium that is called a calcium transient. This increase in calcium activates calcium-sensitive contractile proteins that then use  ATP to cause cell shortening (Crespo, 1990).

It is important to note that contraction of smooth muscle need not require neural input that is, it can function without an action potential. It does so by integrating a huge number of other stimuli

such as humoral/paracrine (e.g. Epinephrine, Angiotensin II, AVP, Endothelin), metabolic (e.g. oxygen, carbon dioxide, adenosine, potassium ions, hydrogen ions), or physical stimuli (e.g. stretch receptors, shear stress). This integrative character of smooth muscle allows it to function in the tissues in which it exists, such as being the controller of local blood flow to tissues undergoing metabolic changes. In these excitation-free contractions, then, there of course is no excitation-contraction coupling (Fabiato, 1983).

Some stimuli for smooth muscle contraction, however, are neural. All neural input is autonomic (involuntary). In these the mechanism of excitation-contraction coupling is as follows: parasympathetic  input  uses  the  neurotransmitter  acetylcholine.  Acetylcholine  receptors  on smooth muscle are of the  muscarinic receptor type; as such they are metabotropic, or G-protein / second messenger coupled. Sympathetic input uses different neurotransmitters; the primary one is  norepinephrine. All adrenergic receptors are also  metabotropic. The exact  effects on the smooth muscle depend on the specific characteristics of the receptor activated—both parasympathetic input and sympathetic input can be either excitatory (contractile) or inhibitory (relaxing)  (Cannell,  1994).  The  main  mechanism  for  actual coupling  involves  varying  the calcium-sensitivity of specific cellular machinery. However it  occurs, increased intracellular calcium binds calmodulin, which activates myosin light chain kinase (MLCK). MLCK phosphorylates the regulatory light chains of the myosin heads.  Phosphorylated myosin heads are able to cross bridge-cycle. Thus, the degree to and velocity of which a whole smooth muscle contracts depends on the level of phosphorylation of myosin heads. Myosin light chain phosphatase removes the phosphate groups from the myosin heads, thus ending cycling (and leaving the muscle in latch-state) (Sandow, 1952).

1.3.3 Action potential of cell membranes

In physiology, an action potential is a short-lasting event in which the electrical membrane potential of a  cell rapidly rises and falls, following a consistent trajectory. Action potentials are generated by special types of  voltage-gated ion channels embedded in a cell’s  plasma membrane (Barnett and Larkman, 2007). These channels are shut when the membrane potential is near the resting potential of the cell, but they rapidly begin to open if the membrane potential increases to a precisely defined threshold value. When the channels open (by detecting the  depolarization in

transmembrane voltage (Barnett and Larkman, 2007). Action potentials occur in several types of animal cells, called  excitable cells, which include  neurons,  muscle cells, and  endocrine cells, as well as in some plant cells. In neurons, they play a central role in cell-to-cell communication (Goldin, 2007). In other types of cells, their main function is to activate intracellular processes. In muscle cells, for example, an action potential is the first step in the chain of events leading to contraction. In beta cells of the pancreas, they provoke release of insulin (MacDonald and Rorsman, 2006). Action potentials in neurons are also known as “nerve impulses” or “spikes”, and the temporal sequence of action potentials generated by a neuron is called its “spike train”. In animal cells, there are two primary types of action potentials, one type generated by voltage- gated sodium channels, the other by voltage-gated calcium channels (Yellen, 2002). Sodium- based action potentials usually last for under one millisecond, whereas calcium-based action potentials may last for 100 milliseconds or longer. In some types of neurons, slow calcium spikes provide the driving force for a long burst of rapidly emitted sodium spikes. In cardiac muscle cells, on the other hand, an initial fast sodium spike provides a “primer” to provoke the rapid onset of a calcium spike, which then produces muscle contraction (Doyle et al., 1998).

1.4 Muscles

The muscular systemis the biological system of humans that produces movement (Widmaier et al.,  2004). The  muscular system,  in  vertebrates, is  controlled through the  nervous system, although  some  muscles,  like  cardiac  muscle,  can  be  completely  autonomous.  Muscleis contractile tissue and is derived from the mesodermal layer of embryonic germ cells. Its function is to produce force and cause motion, either locomotion or movement within internal organs (Widmaier, et al., 2004). The term ‘muscle’ refers to a number of muscle fibers bound together by connective tissue. The relationship between a single muscle fiber and a muscle is analogous to that between a single neuron and a nerve, which is composed of the axons of many neurons. Muscles are usually linked to bones by bundles of collagen fibers known as tendons (Widmaier et al., 2004). A muscle causing an action when it contracts is called an agonist, a muscle working in opposition to the agonist moving structure in the opposite direction, is an antagonist. Most muscle function as members of a functional group to accomplish specific movements (Seeley et al.,   2004).   Muscles  are   categorized   into   smooth,   cardiac   and   skeletal   muscles.   This categorization is based on the structural and functional properties of these muscles (Ashlesha,

2011).Muscles generate force and movements used in the regulation of the internal environment, and they also produce movements in the external environment. In humans, the ability to communicate, whether by speech, writing or artistic expression also depends on muscle contractions. Indeed, it  is only by controlling the activity of muscles that the human mind ultimately expresses itself (Widmaier, et al., 2004). There are three general types of muscle tissues; Skeletal muscle responsible for movement, Cardiac muscle responsible for pumping blood and Smooth muscle responsible for sustained contraction in the blood vessels, gastrointestinal tract, uterus and other areas in the body (Gordon et al.,1966).

Fig. 8: The three types of muscle

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