The aim of this study was to determine whether nitric oxide induced relaxations in the circular smooth muscle of the colon were mediated by more than one pathway. Previous studies (De Man et al., 2007, Franck et al., 1997) have shown considerable evidence indicating that NO causes the activation of soluble guanylate cyclase through which cGMP eventually causes a relaxation. However, a study by (Van Crombruggen and Lefebvre, 2004) suggested that NO may activate a cGMP-independent pathway by which NO caused an inhibitory response. This cGMP-independent pathway has been postulated to involve apamin sensitive potassium channels.
In this study, the results demonstrated that the NO induced relaxations were only blocked when both ODQ and apamin were used together. This showed that both cGMP dependent and cGMP-independent pathways were involved in the response to NO (SNP).
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Preliminary experiments were carried out so that a better understanding of the preparation was obtained. This involved investigating the effects of atropine and propanolol so that NANC conditions could be established. NANC conditions were used to remove the direct effect of the autonomic nervous system in order to study the primary effects of the enteric nervous system.
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As previously seen (Mulè et al., 1999a, Benabdallah et al., 2008, Plujà et al., 2001), the preparations exhibited spontaneous activity. Much like the study by (Kato et al., 2009), the current experiment showed the presence to two rhythms in the circular smooth muscle of the colon. These two rhythms were large, slow phasic contractions and small, fast phasic contractions.
When atropine was added to the preparation, there was a significant reduction in amplitude of the large, slow phasic contractions. As well as reducing the amplitude of the large, slow oscillations, the rebound contraction exhibited immediately on cessation of stimulation with EFS was also decreased. This observation suggested that there may have been a high cholinergic tone already present in the tissue and may also account for the fact that addition of acetylcholine did not cause an additional contractile response. The atropine is most likely to be acting post-synaptically to inhibit the ACh released from excitatory motor neurons. Antagonising the muscarinic receptors of the circular smooth muscle using atropine also revealed an inhibitory junction potential which was not previously seen when stimulated using EFS.
To determine whether the inhibitory junction potential was due to adrenergic innervation and establish NANC conditions, propanolol was used. Adrenergic agonists such as adrenaline and isoprenaline confirmed the presence of adrenergic receptors on the tissue by causing an inhibitory response. When using EFS, propanolol abolished the relaxation previously seen in the control. This indicated that the relaxation seen using EFS was primarily due activation of Î² adrenoreceptors. Isoprenaline is a Î² adrenergic agonist which mimics the effect the sympathetic nervous system by causing an inhibitory effect through activation of intracellular messenger systems.
Under NANC conditions using ATR & PRO, the adrenergic and cholinergic responses of the tissue had been inhibited. There was a significant reduction in amplitude of both sets of rhythms and upon using electrical field stimulation, an inhibitory response was seen.
Investigating the effect of lidocaine on the spontaneous activity of the circular smooth muscle
In recent years, the mechanism of underlying spontaneous activity in the colon and the rest of the GI tract has been widely investigated. As seen in the present study, the spontaneous activity is not due to excitatory motor neurons of the enteric nervous system acting on muscarinic receptors on the smooth muscle cell as atropine does not stop the rhythmic, phasic contractions. Upon addition of a quaternary lidocaine derivative QX314, the spontaneous activity was abolished. This provides evidence to suggest that the spontaneous activity is predominantly neuronal. This has also been seen in studies by (Plujà et al., 2001, Kato et al., 2009, Yoneda et al., 2001)
Investigating the spontaneous activity in the smooth muscle of the colon
Previous studies (Benabdallah et al., 2008, Kato et al., 2009) have indicated that the spontaneous activity of the colon is due to the interstitial cells of Cajal (ICC). The ICC were discovered in the 1890's but their specific roles in generation of the spontaneous slow waves were not identified until the early 1990's almost a century later. The ICC are found in the walls of the GI tract and are considered to be pacemaker cells, which are involved in the generation of spontaneous activity. This discovery was further supported by a study by (Huizinga et al., 1995) in which mutant mice that lacked the c-kit gene did not express this spontaneous activity. The c-kit gene is protein found in ICC and is responsible for the development of the cells (Furness, 2006). The ICC are found in three parts of the enteric nervous system. The first group of cells are found in the myenteric plexus, the second group of cells are found in the intramuscular layer and the third group of cell are found in the submucosal layer of the ENS. ICC are not considered to be neurons as they do not contain vesicles containing neurotransmitters. They can be located between neurons and muscle cells of the plexuses mentioned above.
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Much like cardiac muscle, ICC's form gap junctions. The gap junctions are formed amongst the ICC's themselves and with adjacent smooth muscle cells. These gap junctions allow the electrical signals to be carried through the cells in tandem. As the cells are located very close to a number of motor neurons; they also carry receptors for a number of neurotransmitters. The ICC themselves can be influenced by a number of neurotransmitters such as NO, VIP and Ach (Furness, 2006).
A recent study (Kato et al., 2009) provided evidence to suggest that the large, slow contractions of the spontaneous activity originated from the ICC in the myenteric plexus whereas the small, fast contractions originated from the ICC in the submucosal plexus. As with the current study, Kato el al also suggested that the frequency of the small contractions are greater than the frequency of the large contractions.
Studying the rebound contraction seen upon cessation of electrical field stimulation
Investigating the effect of Nitric oxide on the circular smooth muscle of the colon
In this experiment, sodium nitroprusside was used to determine whether NO would cause an inhibitory response. At 1ÂµM, 10ÂµM and 1mM of SNP a NO induced relaxation of the smooth muscle occurred. Addition of 10ÂµM SNP onto the circular smooth muscle caused a decrease in basal tone. Throughout this relaxation, the spontaneous activity remained with a constant amplitude and frequency of the various contractions. However at 100ÂµM, during the relaxation, the frequency and the amplitude of the spontaneous activity were reduced. This increase in dose may have caused the NO to act on the ICC as well as the muscle; thereby altering the frequency of the oscillations. Nevertheless, the tissue was able to recover fully. When 1mM of SNP was added, spontaneous activity was blocked during the relaxation period and upon recovery the frequency of the large, slow contractions had decreased. This indicated that at a larger dose, the NO remained on the ICC in the myenteric plexus causing an inhibitory effect on the frequency.
Investigation the effects of L-NAME and ODQ on the smooth muscle of the colon
Upon adding L-NAME to the preparation in NANC conditions, the inhibitory junction potential caused by EFS was completely abolished showing that the substance released was in fact NO. However, when ODQ was added to the organ bath the inhibitory junction potential elicited by EFS was not totally eliminated. This observation differs from that seen by De Man et al., (2007) where ODQ did abolish the IJP. Our finding suggested that the NO-induced IJP was being caused via a different pathway other than sGC-CGMP. Moreover, this was further supported by the fact that 10ÂµM of SNP was still able to cause a relaxation in the presence of ODQ. The spontaneous activity of the circular smooth muscle was also affected by ODQ. The spontaneous activity may have been under an inhibitory tone caused via NO released from enteric inhibitory motor neurons. In the presence of ODQ, NO released from these inhibitory neurons would no longer be able to bind post synaptically leading to a loss of inhibitory tone thereby causing a contractile tone.
Mechanism by which Nitric oxide causes relaxation
There are three distinct mechanisms by which NO can produce relaxation of smooth muscle cells. The first two mechanisms involve the binding of NO to soluble guanylate cyclase (sGC). Activation of sGC by NO initiates the production of cGMP which ultimately leads to the production of protein kinase G (PKG). It is hypothesised that PKG is responsible for the relaxation effect seen on smooth muscle cells (SMC). Firstly, PKG can cause a reduction in the levels of intracellular calcium concentrations and is achieved by phosphorylation of 'key target proteins, including ion channels, ion pumps, receptors and enzymes'(CARVAJAL et al., 2000).
The CaÂ²âº/ATP pump situated on the plasma membrane may be activated by PKG resulting in CaÂ²âº efflux and therefore SMC relaxation. This has been reported to occur by the phosphorylation of phosphatidyl inositol kinase by PKG producing phosphatidyl inositol-4 phosphate (PI-4P) which in turn activates the CaÂ²âº/ATP pump. Phosphorylation of a regulatory protein called phospholamban by PKG has also been postulated to cause SMC relaxation. This activates of the CaÂ²âº/ATP pump causing sequestration of calcium into the sarcoplasmic reticulum (SR), reducing intracellular calcium concentrations.
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PKG can also reduce levels of intracellular CaÂ²âºused for SMC contraction by phosphorylating serine 1755 on the IPâ‚ƒ receptor in the SR. This reduces the activity of the receptor thereby leading to less CaÂ²âº being released from intracellular stores.
The second mechanism by which NO elicits its' effects is via decreasing the sensitivity of the contractile system in response to CaÂ²âº. This means that even at high intracellular CaÂ²âº concentrations, a contraction will not be produced. This has been hypothesed to have been achieved by disrupting the equilibrium between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) in SMC. MLCK is responsible for causing a contraction in smooth muscle via phosphorylation and MLCP produces a relaxation by stopping MLCK (Colpaert et al., 2005). PKG is said to increase the activity of the MLCP enzyme through 'phosphorylation of the M110 regulatory subunit of MLCP'. This can either lead to an activation of the enzyme of an inactivation of the 'inhibitory protein'. (CARVAJAL et al., 2000).
The third and final mechanism by which NO exerts its' effects is by direct activation of Kâº channels. Activation of these channels initiates a negative feedback effect whereby hypolarisation of the cell membrane occurs, inhibiting CaÂ²âº influx through voltage-gated channels. (CARVAJAL et al., 2000). However, activation of Kâº channels can also occur through the cGMP-PKG pathway. PKG is hypothesised to phosphorylate the Kâº channel or a regulatory protein associated with the channel leading to its activation.
Investigating the effect of apamin on the circular smooth muscle of the rat colon
The cGMP - independent pathway of NO relaxation that is thought be involved is through direct activation of apamin sensitive potassium channels. In the present study, addition of apamin caused an immediate contraction which lead to the basal tone of the spontaneous activity being raised. This suggests that apamin sensitive Kâº channels may be involved in the control of the spontaneous activity exhibited by the smooth muscle. In fact, (Mulè et al., 1999b) had very similar findings and also suggested that the apamin sensitive Kâº channels modulated the spontaneous mechanical activity of the rat small intestine (Mule et al,1992; Serio et al.,1996; Lefebvre & Bartho, 1997). (Serio et al., 2003, Mulè et al., 1999b). (Spencer et al., 1998)) preposed that apamin may raise the basaline tone of the spontaneous activity by 'act(ing) presynaptically to depress inhibitory neurotransmitter output'. The apamin could be affecting the inhibitory motor neurons directly or indirectly by acting on interneurons in the ENS.
EFS did not produce an IJP in the presence of apamin. This observation indicates that the relaxation is due to activation of apamin sensitive Kâº channels. As mentioned earlier, ODQ did not stop the IJP elicited by EFS and therefore as apamin did block the IJP; it can be suggested that NO is having a direct effect on the apamin sensitive Kâº channels.(Vanneste et al., 2004) However, when SNP was added, the preparation was still able to relax to a certain extent. This implied that the sGC-cGMP mediated pathway of NO-induced relaxation may also be occurring. To establish whether this event was taking place, ODQ was added on top of the apamin. Upon further addition of SNP, a relaxation did not occur. It seems as though the additive effects of both ODQ and apamin blocked the exogenous NO-induced relaxation thereby implying that both systems existed within the circular smooth muscle of the rat colon. (Serio et al., 2003) supports this finding in that the combination of ODQ and apamin prevent the relaxation effect induced by NO.
Apamin-sensitive Kâº Channels
Calcium activated potassium channels are found throughout the body including erythrocytes and hepatocytes and are involved with a variety of physicolocial processes such as smooth muscle tone and neurosecretion (Vergara et al., 1998, Malik-Hall et al., 2000). A study by Gardos, 1958 (Weatherall et al., 2010) was the first to demonstrate the hyperpolarising effect of the calcium activated Kâº current by looking at cell skrinkage of red blood cells. This finding eventually led to the discovery that the calcium activated K âºcurrent is responsible for the after hyperpolarising potential (AHP) exhibited in action potentials (Alger and Nicoll, 1980).
To date, three main types of calcium activated Kâº channels have been recognised. These include Big conductance (BK), intermediate conductance (IK) and small conductance channels (Strobak et al., 2000). In recent years all of these channels have been cloned and their subtypes identified. SK channels are potassium selective and have a crucial role in slow afterhyperpolarization potentials (sAHP) of the neurons. After hyperpolarisation is an essential part of the cell's activity as it prevents tetanus occurring in the muscle. There are two pharmacologically different types of SK channels that conduct sAHP's. These two channels are known as apamin-sensitive channels and apamin-insensitive channels. In this study, the effect of NO on apamin-sensitive channels has been investigated. (Vergara et al., 1998). Apamin is a selective inhibitor of SK channels. It is an octadecapeptide and is a toxin found in honey bee venom. SK channels have three subtypes known as SK1, SK2 and SK3. SK2 channels are apamin-sensitive whereas SK1 channels are apamin-insensitive. SK3 channels are an intermediate but are also affected by apamin. Apamin causes inhibition of the SK2 and SK3 channels by allosterically binding to the extracellular loop and outer pore region of the channel. Upon binding to the channel, apamin causes a conformational change in the structure thereby altering the 'channel's selectivity filter' so that the channel can no longer cause hyperpolarisation (Weatherall et al., 2010).