Investigating the role of P2 receptors in uterine smooth muscle contraction has the potential to prove very useful in modern medicine. Knowing the involvement of these receptors in the control of up- and down- regulation of contractions and how they interact with agonists and antagonists will provide valuable information for dealing with the problems some women experience during and at the end of pregnancy. Hopefully with further study, adequate agonists and antagonists will be found that are suitable uterotonic drugs which could be used to either cease or induce uterine contractions, when necessary, in the safest possible way for both mother and child.
Smooth muscle is a specialized, non-striated tissue composed of contractile cells found in many vital organs throughout the body. It lines most of the hollow internal organs and is generally the supporting tissue found in arteries, veins, bladder, male and female reproductive organs, uterus, respiratory tract and the gastrointestinal tract .
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This type of muscle performs many diverse tasks throughout the different parts of the body. In blood vessels, smooth muscle helps regulate the blood flow around the body; it helps to regulate the movement of materials along internal passageways in the urinary and digestive tracts . As it contracts and relaxes in the respiratory passageways, the diameter is altered thus changing the resistance to airflow. Layers of smooth muscle can also help to move oocytes and possibly sperm along the reproductive tracts. The muscle in the walls of the uterus contracts and relaxes powerfully and rapidly during birth, expelling the foetus.
Mechanism of Contraction
The contraction of smooth muscle is involuntary. A Contraction can occur spontaneously and can be initiated by mechanical, electrical, and chemical stimuli. Contractions can either be described as phasic (rapid contraction and relaxation) or tonic (slow and sustained contraction).
Single-unit smooth muscle makes up the bulk of the muscle in most visceral organs. A high degree of electrical coupling between cells allows large regions of the tissue react as if they were a single cell. Gap junctions are the most effective structure in this type of coupling. In the uterus muscle, gap junctions are rare during pregnancy, the weak contractions seen during this period lack coordination, however there is a dramatic increase in both the number and size of gap junctions just prior to the onset of labour, and contractions appear stronger and more coordinated.
Uterine Smooth Muscle
Uterine contractility is generated by contractions of myometrial smooth muscle cells (SMCs) that compose most of the myometrial layer of the uterine wall . In the myometrium, as in other smooth muscle, contractile activity is initiated by a Ca2+ - calmodulin interaction which stimulates phosphorylation of the myosin light chain. RhoA/Rho kinase pathway signals a Ca2+ senzitisation of the contractile proteins to inhibit the dephosporylation of the light chain by myosin phosphatase, thus maintaining the generation of force. Stimulation of myosin phosphatase and Removal of Ca2+ from the cytosol, initiate the process of smooth muscle relaxation.
Studies have shown, when investigating the role of Calcium store in uterine contractility, the smooth muscle cells have an internal Calcium store i.e. the sarcoplasmic reticulum (SR). The SR has a Ca-ATPase, called SERCA, which allows it to take up Ca ions from the cytoplasm into the lumen of the SR at the expense of ATP. There are two known types of release channels in the SR membrane; IP3-gated and Ca-gated (ryanodine) receptors. Both receptors have been noted in the uterus smooth muscle, but the IP3 receptors are seen to be more important in its contractile function
It is also known, that in uterine smooth muscle, many pathways control the intracellular calcium concentrations and the contractile apparatus. One major pathway targets PLC activation, the release of intracellular calcium and stimulation of calcium entry. The production of diacylglycerol and inositol 1,4,5-trisphosphate (IP3) from phosphatidylinositide 4,5-bisphosphate (PIP2) is catalysed by PLC enzymes. Calcium is released from intracellular stores when IP3 interacts with receptors on the endoplasmic reticulum. The release of calcium from these intracellular stores is hugely important for sustained contractile activity and is also an important component of the action of uterine contractions.
Adenosine 5'-triphosphate: P2 Receptor Agonists
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As we know, the contraction of smooth muscle is caused by the sliding action of actin and myosin filaments over each other. Hydrolysis of ATP provides the energy the cells need for this mechanism to occur. ATP is widely accepted as a signalling molecule in the body, and is recognised by purinoreceptors. Extracellular ATP can stimulate contractile responses in visceral smooth muscle derived from organ types such as vas deferens, urinary bladder and myometrium. There is evidence that in the female reproductive tract, ATP, acting through P2 receptors, acts as a major non-adrenergic, non-cholinergic cotransmitter in mediating sympathetic regulation of contractile responses in smooth muscle.
Patch-clamp electrophysiological studies have verified that ATP can activate receptors that act as ligand-gated ion channels in smooth muscle cells that have been isolated from tissues such as pregnant rat myometrium.
The contractile activity of ATP in the uterus was highlighted in a study undertaken by Michelle Bardini in 1999. Using immunohistochemistry techniques, it was discovered that there was an abundance of P2Xâ‚‚ receptors present in the smooth muscle of the ovary and uterus as well as in blood vessels. ATP is a potent agonist of the P2X receptor family.
Receptors for ATP and adenosine are widely distributed throughout the body in many different organs and tissues including the uterine smooth muscle. These surface receptors for extracellular nucleotides are known as purinoreceptors. Two types of purinorecptors, P1 and P2 (for adenosine and ATP/ADP, respectively) were distinguished in 1978; two years after Purinergic receptors were first defined.
The P1/adenosine receptor family is subdivided, according to much biochemical and pharmacological evidence into the four subtypes; A1, A2A, A2B, and A3, all of which couple to G proteins.
P2 receptors have been found on the cell membranes of numerous cell types, and there activation is the cause of many different types of physiological response. Abbracchio and Burnstock divided these P2 receptors into two main classes, the P2X and P2Y families. This was based on whether they are ligand-gated ion channels (P2X) or are G protein-coupled receptors (P2Y) . There are currently seven P2X receptor subtypes and eight P2Y receptor subtypes.
The P2X family ranges from P2X1 to P2X7, the subunit topology consists of intracellular N- and C- termini possessing consensus binding motifs for protein kinases; two transmembrane spanning regions (TM1 and TM2), the first involved with channel gating and the second lining the ion pore; a large extracellular loop, with 10 conserved cysteine residues forming a series of disulphide bridges; a hydrophobic H5 region close to the pore vestibule, for possible receptor/channel modulation by cations; and an ATP-binding site, which may involve regions of the extracellular loop adjacent to TM1 and TM2. It has been thought that the disulphide bridges may form the structural constraints needed to couple the ATP-binding site to the ion pore. P2X receptors increase intracellular levels of Ca2+ by creating an ion-selective channel to the extracellular fluid. In 2002 Ziganshin hypothesised that the P2 receptors in pregnant human uterus are members of the P2X family.
The P2Y receptor topology differs somewhat to that of the P2X receptors. They are characterised by an extracellular N-terminus and intracellular C-terminus, the latter possessing consensus binding motifs for protein kinases; seven transmembrane spanning regions which help to form the ligand docking pocket. Each P2Y receptor binds to a single heterotrimeric G protein. The receptors in the P2Y family are P2Yâ‚, P2Yâ‚‚, P2Yâ‚„, P2Yâ‚† and P2Yâ‚â‚. The P2Y receptors increase intracellular calcium levels by causing the release of Ca2+ from the non-mitochondrial, receptor-operated, intracellular calcium stores in the sarcoplasmic reticulum.
Role of P2 Receptors in Smooth Muscle contraction
We are aware of the importance of Ca2+ regulation in the control of smooth muscle throughout the body, and also of the role P2 receptors play in it's up and down regulation. Many studies have been done to try and fully understand the activity of these receptors in the smooth muscle cells (SMCs) along with the effects their agonists and antagonists have on them and ultimately, on the contraction of smooth muscle. The diversity of the results shown in some of the following studies, enlighten us with some knowledge on the role of P2 receptors in smooth muscle, and how their interactions with different agonists and antagonists can greatly affect the contraction of various tissues in the body.
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Ziganshin investigated P2 receptor characteristics in Human Uterus in 2002. Samples of both pregnant and non-pregnant myometrium were obtained and tested on. It was found that ATP produced dose-dependent contractions of the pregnant myometrium in concentrations of 10â»âµM and higher, but no such result was seen in the non-pregnant tissue, even at the maximum dose. Other agonists of P2 receptors were also tested i.e. Î±,Î²-meATP and UTP. It was found that the amplitude of induced contractions was lowest in ATP. His study also showed that PPADS (P2 receptor antagonist) highly decreased the amplitude of contractions produced by agonists, especially those induced by Î±,Î²-meATP. Ziganshin suggested that 'ATP could not be used as a uterotonic drug due to its short half-life after parenteral administration and rapid degradation with extracellular ATPases'.
In 2007 Airat U. Ziganshin, again studied the 'contrasting effects of P2 receptor agonists on spontaneous contractility of human fallopian tubes with and without acute inflammation'. Two groups of women were used to compare these effects, one with acute purulent inflammation of the fallopian tubes (study group) and the other without (control group). Spontaneous contractions of the isolated tubes were recorded before and after incubation of the tissues with different agonists of P2 receptors; ATP included. In the control group the agonist didn't produce any significant effect on fallopian tube contractility. In the study group, it was found that ATP significantly increased the spontaneous contractility of the isolated tubes. It was suggested that the higher activity of P2 receptor agonists in the uterine tubes of the study group was due to expression of several subtypes of P2 receptors during inflammation. Also it was discovered that ATP at concentrations of 10â»â¶- 10â»â´M had a minimal if any effect on spontaneous contractility of the uterine tubes without inflammation. At the same range of ATP concentrations, an increase of spontaneous contractility in the uterine tissue with acute purulent inflammation was seen. From this study it was seen that P2 receptor agonists like ATP can have an effect on the spontaneous activity of isolated human uterine tubes. Most of the tested agonists of P2 receptors, to a greater or lesser extent, increased the contractile activity.
A study undertaken by Miyuki Nagaoka in 2009, examined the regulation of adenosine 5-triphosphate (ATP)-gated P2X4 receptors on tracheal smooth muscle cells was investigated. It showed the effect of P2 receptors in the smooth muscle contraction of tracheal tissue. It was seen that the receptor and its agonist work in a similar way to the mechanism of contraction regulation in the uterine smooth muscle. The effects of extracellular ATP on single airway smooth muscle were examined and this resulted in ATP causing a sustained inward current after stimulation of P2Xâ‚„ receptors in porcine ASM cells. It was found in this study that direct Ca2+ entry through P2X4 receptors caused the contractile response.
PPADS and MRS2159: P2 Receptor Antagonists
There is a vast range of P2 receptor agonists and antagonists that are very well established today. As I have already discussed, ATP is a potent agonist of the P2X receptor family. The lesser known pyridoxal-5'-phosphate 6-azophenyl 2', 4'-disulfonate, also known as PPADS is an effective and selective antagonist to this same group. There are two antagonists of P2X receptors that I will focus on. Both of which I have used in my study to test the role of P2 receptor activation in uterine smooth muscle. These are PPADS and MRS2159.
PPADS, like any receptor antagonist, doesn't show any biological response itself in the tissue but dampens or even blocks the agonist-mediated responses on contraction. In 2005, Airat U. Ziganshin studied the 'Term-dependency of P2 receptor-mediated contractile responses of isolated human pregnant uterus'. Myometrial samples were tested that were obtained from women undergoing caesarean section at 28-30 weeks of pregnancy (group 1), 32-34 weeks of pregnancy (group 2) and 38-41 weeks of pregnancy (group 3). Results showed that PPADS did not have an effect on the contraction of the full term pregnant uterus (group 3). In the ATP curve of contraction-response, none of the corresponding points before and after incubation of PPADS were significantly different. Results obtained were not markedly different from those seen in groups 1 and 2. Overall this experiment showed that P2X receptor-mediated contractions of human pregnant uterus to Î±,Î²-methyleneATP are increased with the pregnancy progression, but not to ATP.
MRS2159 (have not found literature on this as of yet)
The role P2 receptors play in uterine smooth muscle contraction is obvious, and it is also clear that there are numerous different agonists and antagonists that can interact with these receptors there by altering the regulation of the muscle contractions.
Uterotonic drugs stimulate uterine contractions. It is known that drugs such as oxytocin and ergometrine have strong uterotonic properties and are regularly used to prevent or treat uterine atony and reduce the quantity of blood lost during and after childbirth. These drugs have also been widely used for the induction of labour and in the prevention of postpartum haemorrhage. The introduction of ATP as a suitable uterotonic drug would be interesting to investigate?
1. Webb, R., Smooth muscle contraction and relaxation. Advances in physiology education, 2003. 27(4): p. 201.
2. Somlyo, A. and A. Somlyo, Signal transduction and regulation in smooth muscle. 1994.
3. Nagaoka, M., et al., Regulation of adenosine 5 -triphosphate (ATP)-gated P2X4 receptors on tracheal smooth muscle cells. Respiratory Physiology & Neurobiology, 2009. 166(1): p. 61-67.
4. Bursztyn, L., et al., Mathematical model of excitation-contraction in a uterine smooth muscle cell. American Journal of Physiology- Cell Physiology, 2007. 292(5): p. C1816.