Nitric Oxide in Mediated Relaxations in CCK-stimulated Gallbladder Strips.
Response of gallbladder strips to CCK-8
The application of CCK-8 to gallbladder strips confirmed that CCK-8 initiates the contraction of gallbladder smooth muscle and that this process is concentration-dependent (Ryan 1981). All the doses of CCK-8, from10-0.05 nM, 100.5 nM, 101.0 nM, 101.5 nM, 102.0 nM to 102.5 nM elicited a contractile response. In each case CCK-8 will have caused the release of Ca2+ from intracellular stores through the action of IP3, and from extracellular stores via DG and ACh. ACh mediates the opening of voltage-gated Na+ channels causing depolarisation of the cells (Mawe 1987). This depolarisation by influx of extracellular Na+ resulted in the opening of voltage-gated Ca2+ channels and a subsequent influx of Ca2+. The maximum number of voltage-gated channels will have opened at this point as the cells had previously been sensitised by the application of 80 mM K+ solution (Wilson 1996). Free Ca2+ from these three sources will bind with the protein calmodulin and cause smooth muscle contraction.
The lowest dose of CCK-8, 10-0.05 nM, elicited the smallest contractile response. At this dose less IP3 and DG will have been formed and fewer voltage-gated Na+ channels will have opened, reducing the amount by which the cell was depolarised. L-type Ca2+ channels are high voltage-activated channels and their threshold activation occurs at -40 to – 10 mV (Yamakage 2002). If insufficient Na+ enters the cell, depolarisation will not reach the required levels to open the L-type Ca2+ channels. This may partly account for the low contractile response at low concentrations of CCK-8. As the dose of CCK-8 was increased so did the contractile response, which suggests that greater amounts of IP3 and DG are being produced and that the L-type Ca2+ channels are opening.
Effect of Sodium Nitroprusside on Pre-contracted Gallbladder Strips
The application of SNP to CCK-8-stimulated gallbladder strips confirmed that nitric oxide produces a concentration-dependent smooth muscle relaxation. Once SNP has been administered it donates nitric oxide to the tissue. The amount of nitric oxide which is released from SNP is concentration dependent. Each dose of SNP resulted in a percentage inhibition of the contractile response ranging from 4.55 at 10-7.5 M to 31 at 10-6 M (see figure 3).
At SNP concentrations of 10-7 M, 10-6.5 M and 10-6 M (see figure 4) the mean responses showed a linear increase in response that is statistically significant (p = 0.02). This confirms that as the concentration of SNP, and therefore nitric oxide, increases so did the inhibition of the contractile response and that this response is concentration dependent.
This indicates that PKG1 is being produced and that it is phosphorylating the IP3 receptors and the L-type calcium channels as well as activating ATPase Ca2+ pump on the sarcoplasmic reticulum. These actions will have reduced the concentration of free intracellular Ca2+.
Interestingly, when the concentration of SNP reached 10-5.5 M and 10-5 M, the percentage inhibition of the contractile response began to reduce to 13.8 ± 3.6 and 9.8 ± 1.8, respectively (see figure 4). At these two concentrations nitric oxide will have been released into the tissue in greater quantities than at 10-7 M, 10-6.5 M and 10-6 M without a corresponding increased inhibition of smooth muscle contraction. Studies have shown that SNP elicits a concentration-dependent contraction of resting tone in gallbladder smooth muscle which involves the phosphorylation of tyrosine (Alcon 2001). In this 2001 study there was no detectable change in gallbladder smooth muscle resting tone at lower concentrations of SNP (1 nM to 1 μM). A maximal contraction was produced at 1 mM SNP, which indicates that tyrosine phosphorylation occurs at this concentration. It may be the case that, in pre-contracted tissue, if the levels of nitric oxide are increased above a certain threshold, for example 10-6 M, that tyrosine is phosphorylated as well as cGMP/PKG-1 being produced. This would account for the reduction in effect of the SNP at concentrations of 10-5.5 M and 10-5 M.
Effect of Electric Field Stimulation on Pre-contracted Gallbladder Strips
An electrical stimulus that exceeds a certain threshold generates an action potential in a neurone. This change in membrane potential triggers the release of excitatory and inhibitory neurotransmitters. Excitatory neurotransmitters, such as ACh, open cation channels and cause membrane depolarisation. Inhibitory neurotransmitters, such as VIP and nitric oxide, open Cl- channels or K+ channels and therefore hyperpolarise the membrane.
In this study, the strips were subjected to cholinergic blockade by atropine and adrenergic blockade by guanethidine. As a result, the responses produced by EFS were nonadrenergic and noncholinergic (NANC). NANC nerves are inhibitory in nature and are present in gallbladder tissue. Neurogenic relaxations evoked by EFS at a frequency ≥ 10 Hz depend on a combination of nitric oxide and VIP release (Tonini et al 2000). At each frequency (10 Hz, 20 Hz, 40 Hz and 80 Hz) EFS resulted in an inhibitory response pre-contracted gallbladder smooth muscle (see figure 6). There was a linear increase in inhibition from 10 Hz to 40 Hz which, although not statistically significant, suggests that as the frequency increased so did the amount of VIP and nitric oxide that was produced. It is not possible to determine the quantities of neurotransmitters generated when using EFS in this manner; however, it is clear that gallbladder ganglia have neurones which are capable of synthesising nitric oxide (Talmage et al 1995). At 80 Hz, however, there was no further increase in inhibition (42.25 ± 9.54 at 40 Hz compared to 43.25 ± 11.46 at 40 Hz). As seen previously (see figures 4 and 5), as the concentration of SNP increased beyond 10-5.5 M the inhibition of the contractile response reduced. It may be that at 80 Hz, nitric oxide was being produced in such quantities that some of the neurotransmitter contributed to the contractile response, as described in section 4.2.
Effect of Sodium Nitroprusside and EFS on DIDS/CCK-8-stimulated gallbladder strips
DIDS inhibits the spasmolytic effect nitric oxide has on CCK-stimulated gallbladder contractions. When nitric oxide is applied exogenously (SNP) the effect DIDS had was dependent on the concentration of SNP (see figure 5). In the presence of DIDS, 10-7 M SNP caused a percentage inhibition of 19.2 ± 3.2, whereas without DIDS the same concentration of SNP caused a significant inhibition of 27.1 ± 3.7. In the presence of DIDS, 10-6.5 M SNP caused a percentage inhibition of 24.3 ± 3.6, whereas without DIDS the same concentration of SNP caused a significant inhibition of 32.7 ± 5.2. When nitric oxide is applied endogenously (EFS) the effect of DIDS is significant over a frequency range of 10 to 80 Hz (see figure 6).
This indicates that chloride channels may play a role in gallbladder smooth muscle relaxation. Chloride-conducting ion channels (CIC) are α-helical membrane proteins which form a pathway through which chloride ions can flow down their concentration gradient (Mindell and Maduke 2001). In mammals, the CIC family has nine known members. Both the amino- and carboxy-terminal domains are cytoplasmic. All of the CIC pores studied so far are gated by transmembrane voltage. There are also extracellular ligand-gated chloride channels (ELG) which have four transmembrane domains in each subunit and nucleotide sensitive chloride channels. Glycine and GABA are both ligands for chloride channels.
Once chloride enters the cell, the plasma membrane becomes hyperpolarised, which causes the voltage-gated Ca2+ channels to close. This reduces the levels of free intracellular Ca2+ available to bind to calmodulin. This study shows that there is a statistically significant difference in the percentage inhibition mediated by SNP at 10-7 M and 10-6.5 M and EFS from 10 to 80 Hz in the absence of DIDS as compared to the percentage inhibition in the presence of DIDS and exposed to the same levels of nitric oxide.
Based on the evidence presented it can therefore be surmised that the mechanism of action of nitric oxide could involve the opening of chloride channels. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter like nitric oxide that inhibits presynaptic transmission in the central nervous system and the retina. It works to evoke a release of ACh from post-ganglionic cholinergic neurones (Saito, Taniyama and Tanaka 1985). GABA has receptors of two types; ionotropic which are actual ion channels and metabotropic which are G-protein coupled. It has been shown that this causes chloride channels to open, creating an influx of chloride ions resulting in hyperpolarisation. Nitric oxide could work in such a manner to open chloride channels.
GABA is an amino acid neurotransmitter derived from glutamate and as such requires a receptor on the cell surface. Nitric oxide is a gas and can cross the cell membrane. Its main receptor is soluble guanylyl cyclase (Bellamy and Garthwaite 2002) which catalyses the synthesis of cGMP. cGMP modulates the activity of cGMP-dependent kinases and cGMP-regulated phosphodiesterases which act to phosphorylate the proteins of L-type calcium channels and the IP3 receptor channels. In this way, the levels of free intracellular Ca2+ reduce, and smooth muscle relaxation occurs.
Nitric oxide could mediate the opening of cyclic nucleotide-gated (CNG) ion channels via the synthesis of cGMP. CNG channels are opened by the direct binding of cGMP and cAMP (Kaupp and Seifert 2002). They belong to the voltage-gated channel superfamily, despite showing minimal voltage dependence, and have a binding domain for nucleoside 3’,5’-cyclic monophosphates (cNMPs) in their COOH-terminal region. They form heterotetrameric complexes which have two or three different types of subunits. Ligand sensitivity and selectivity, ion permeation, and gating are determined by the composition of the channels. CNG channels are activated when cyclic nucleotides bind to a specific site on the channel protein. This activation depends on the concentration of the ligand and studies show that several ligand molecules are needed to open each channel completely.
CNG channels have been identified in retinal photoreceptors. Light stimulation causes an increase in the concentration of intracellular cGMP. Molecules of cGMP bind to the CNG K+ channels which results in K+ entering and depolarising the membrane. Molecular cloning of CNG channels has shown that they are also expressed in non-neuronal tissue (Kaupp and Seifert 2002). It could be the case that a rise in concentration of cGMP in gallbladder smooth muscle cells causes CNG-chloride channels to open. This increase in levels of intracellular Cl- will result in hyperpolarisation of the membrane. Voltage-gated Ca2+ channels will then be closed, reducing the level of intracellular Ca2+. It has also been reported that nitric oxide can act directly on olfactory sensory neurone CNG channels. Nitric oxide donators such as S-nitrosocysteine (SNC) have produced single-channels events and it has been suggested that in these cases nitric oxide is working through S-nitrolysation.
If the tissue is subjected to EFS, both nitric oxide and VIP are produced. VIP induces relaxation by stimulating the release of nitric oxide from smooth muscle cells by the action of nitric synthase. Nitric acts by diffusing across the junctional cleft to nerve terminals to presynaptically enhance the release of VIP (Grider et al., 1992). It is possible therefore that when SNP was applied to the pre-contracted tissue the nitric oxide causes the release of VIP. VIP receptors in guinea pig gastric smooth muscle are G-coupled proteins which stimulate membrane-bound adenylate cyclase which synthesises cyclic adenosine monophosphate (cAMP). As cAMP is a cyclic nucleotide it can be suggested that VIP itself may stimulate the opening of chloride channels via CNG channels.
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