Diana Gallego

Background: Colonic samples from asymptomatic diverticulosis (DS) patients presented enhanced electrical field stimulation (EFS)‐contractions, in an earlier study of ours, suggesting increased endogenous responses. The aim of this study was to explore changes in excitatory neuromuscular transmission and to assess the pharmacodynamics of spasmolytic agents in DS.Methods: Circular muscle strips from sigmoid colon of DS patients (n = 30; 69.5 ± 14.8 years) and controls (n = 32; 64.7 ± 16.2 years) were studied using organ baths to evaluate the direct effect of excitatory agonists (carbachol, neurokinin A [NKA] and substance P [SP]), and the effect of antagonists (atropine and NK₂ antagonist GR94800) and spasmolytic drugs (otilonium bromide [OB] and N‐butyl‐hyoscine) on the contractions induced by EFS‐stimulation of excitatory motorneurons. qRT‐PCR was also performed to compare mRNA expression of M₂, M₃, NK₂ receptors and L‐type calcium channels.Key Results: Contractions to carbachol (E_max: 663.7 ± 305.6% control vs 2698.0 ± 439.5% DS; p < 0.0005) and NKA (E_max: 387.8 ± 35.6% vs 1102.0 ± 190.1%; p < 0.0005) were higher in DS group, without differences for SP. Higher potency for DS patients was observed in the concentration‐response curves for atropine (pIC₅₀ = 8.56 ± 0.15 control vs pIC₅₀ = 9.95 ± 0.18 DS group; p < 0.005) and slightly higher for GR94800 (pIC₅₀ = 7.21 ± 0.18 control vs pIC₅₀ = 7.97 ± 0.32 group; p < 0.0001). Lower efficacy (E_max) and potency (pIC₅₀) was observed for spasmolytic drugs in DS, whereas no differences were found regarding the relative expression of the receptors evaluated between groups.Conclusions & Inferences: The greater response to cholinergic and tachykinergic agonists and greater potency for muscarinic and NK₂ antagonists observed in DS might play a role in the spasticity found in diverticular disease.

Enteric motor pattern generators involve both myogenic and neurogenic mechanisms in the human colon

Aim: ATP and nitric oxide (NO) are released from enteric inhibitory motor neurones and are responsible for colonic smooth muscle relaxation. However, how frequency of neural stimulation affects this cotransmission process and the post‐junctional responses has not been systematically characterized in the human colon.Methods: The dynamics of inhibitory cotransmission were studied using different protocols of electrical field stimulation (EFS) to characterize the inhibitory junction potentials (IJP) and the corresponding relaxation in colonic strips obtained from 36 patients.Results: Single pulses elicited a fast IJP (IJPf_MAX = −27.6 ± 1.6 mV), sensitive to the P2Y₁ antagonist MRS2500 1 μm, that ran down with frequency increase leaving a residual hyperpolarization at high frequencies (IJPf_∞ = −3.7 ± 0.6 mV). Accordingly, low frequencies of EFS caused purinergic transient relaxations that cannot be maintained at high frequencies. Addition of the P2Y₁ agonist MRS2365 10 μm during the purinergic rundown did not cause any hyperpolarization. Protein kinase C (PKC), a putative P2Y₁ desensitizator, was able to reduce the amplitude of the IJPf when activated, but the rundown was not modified by PKC inhibitors. Frequencies higher than 0.60 ± 0.15 Hz were needed to evoke a sustained nitrergic hyperpolarization that progressively increased reaching IJPs_∞ = −13 ± 0.4 mV at high frequencies and leading to a sustained inhibition of spontaneous motility.Conclusion: Changes in frequency of stimulation possibly mimicking neuronal firing will post‐junctionally determine purinergic vs. nitrergic responses underlying different functional roles. NO will be responsible for sustained relaxations needed in physiological processes such as storage, while purinergic neurotransmission evoking sharp transient relaxations will be dominant in processes such as propulsion.

Nerve‐mediated relaxation is necessary for the correct accomplishment of gastrointestinal (GI) motility. In the GI tract, NO and a purine are probably released by the same inhibitory motor neuron as inhibitory co‐transmitters. TheP2Y₁receptor has been recently identified as the receptor responsible for purinergic smooth muscle hyperpolarization and relaxation in the human gut. This finding has been confirmed inP2Y₁‐deficient mice where purinergic neurotransmission is absent and transit time impaired. However, the mechanisms responsible for nerve‐mediated relaxation, including the identification of the purinergic neurotransmitter(s) itself, are still debatable. Possibly different mechanisms of nerve‐mediated relaxation are present in theGItract. Functional demonstration of purinergic neuromuscular transmission has not been correlated with structural studies. Labelling of purinergic neurons is still experimental and is not performed in routine pathology studies from human samples, even when possible neuromuscular impairment is suspected. Accordingly, the contribution of purinergic neurotransmission in neuromuscular diseases affectingGImotility is not known. In this review, we have focused on the physiological mechanisms responsible for nerve‐mediated purinergic relaxation providing the functional basis for possible future clinical and pharmacological studies onGImotility targeting purine receptors.

Background: Neuro‐transmission impairment could be associated to motility changes observed in patients with diverticular disease. Therefore, the objective was to characterize the inhibitory neuro‐muscular transmission and gene expression changes of the enteric inhibitory pathways in patients with diverticulosis (DS).Methods: Circular muscle strips from sigmoid colon of patients with DS and controls were studied using the organ bath technique to evaluate spontaneous contractility and enteric motor neurons stimulated by electrical field and qRT‐PCR to assess the expression of nNOS, iNOS, P2Y₁R and PGP9.5.Key Results: Patients with DS presented decreased spontaneous rhythmic contractions (SRC) that were significantly enhanced after incubation with L‐NNA (1 mM) and TTX (1 μM), and unaffected by the P2Y₁ antagonist MRS2500 (1 μM). Stimulation on enteric motor neurons caused an increased duration of the latency of OFF‐contractions in DS group (p < 0.001), antagonized by L‐NNA and slightly affected by MRS2500 (1 μM). No differences in the IC₅₀ between controls and DS patients were observed on inhibition of SRC for the NO‐donor sodium nitroprusside (SNP) and the preferential P2Y agonist ADPβS. Moreover, nNOS relative expression was also up‐regulated 2.3‐fold in the DS group (p < 0.05) whereas there was no significant difference in relative expression of iNOS, P2Y₁R and the neuronal marker PGP9.5 between groups.Conclusions & Inferences: Patients with DS presented an over‐expression of nNOS with increased endogenously NO‐mediated responses suggesting enhanced NO‐release. Up‐regulation in the nitrergic pathway in early stages of the disease might play a role in colonic motor disorders associated to diverticular disease.

Interaction of different neuromyogenic mechanisms determines colonic motility. In rats, cyclic depolarizations and slow waves generate myogenic contractions of low frequency (LF) and high frequency (HF), respectively. Interstitial cells of Cajal (ICC) located near the submuscular plexus (SMP) generate slow waves. Inhibitory junction potential (IJP) consists on a purinergic fast (IJPf) followed by a nitrergic slow (IJPs) component leading to relaxation. In the present study, we characterized (1) the dynamics of purinergic-nitrergic inhibitory co-transmission and (2) its contribution on prolonged inhibition of myogenic activity. Different protocols of electrical field stimulation (EFS) under different pharmacological conditions were performed to characterize electrophysiological and mechanical responses. Smooth muscle cells (SMCs) in tissue devoid of ICC-SMP had a resting membrane potential (RMP) of −40.7 ± 0.7 mV. Single pulse protocols increased purinergic and nitrergic IJP amplitude in a voltage-dependent manner (IJPf_MAX = −26.4 ± 0.6 mV, IJPs_MAX = −6.7 ± 0.3 mV). Trains at increasing frequencies enhanced nitrergic (k = 0.8 ± 0.2 s, IJPs_∞ = −15 ± 0.5 mV) whereas they attenuated purinergic responses (k = 3.4 ± 0.6 s,IJPf_∞ = −8.9 ± 0.6 mV). In tissues with intact ICC-SMP, the RMP was −50.0 ± 0.9 mV and nifedipine insensitive slow waves (10.1 ± 2.0 mV, 10.3 ± 0.5 cpm) were recorded. In these cells, (1) nitrergic and purinergic responses were reduced and (2) slow waves maintained their intrinsic frequency and increased their amplitude under nerve-mediated hyperpolarization. Based on the co-transmission process and consistent with the expected results on RMP, prolonged EFS caused a progressive reduction of LF contractions whereas HF contractions were partially insensitive. In conclusion, inhibitory neurons modulate colonic spontaneous motility and the principles determining post-junctional responses are (1) the frequency of firing that determines the neurotransmitter/receptor involved, (2) the transwall gradient and (3) the origin and nature of each myogenic activity.

Otilonium bromide (OB) is a spasmolytic compound of the family of quaternary ammonium derivatives and has been successfully used in the treatment of patients with irritable bowel syndrome (IBS) due to its specific pharmacodynamic effects on motility patterns in the human colon and the contractility of colonic smooth muscle cells. This article examines how. OB inhibits the main patterns of human sigmoid motility in vitro, which are spontaneous rhythmic phasic contractions, smooth muscle tone, contractions induced by stimulation of excitatory motor neurons and contractions induced by direct effect of excitatory neurotransmitters. It does this mainly by blocking calcium influx through L-type calcium channels and interfering with mobilization of cellular calcium required for smooth muscle contraction, thereby limiting excessive intestinal contractility and abdominal cramping. OB also inhibits T-type calcium channels and muscarinic responses. Finally, OB inhibits tachykinin receptors on smooth muscle and primary afferent neurons which may have the joint effect of reducing motility and abdominal pain. All these mechanisms mediate the therapeutic effects of OB in patients with IBS and might be useful in patients with other spastic colonic motility disorders such as diverticular disease.