Synergistic augmentation of rhythmic myogenic contractions of human stomach by arginine vasopressin and adrenaline: Implications for the induction of nausea.

BACKGROUND AND PURPOSE: Nausea is associated with the hormonal secretion of vasopressin and adrenaline, although their actions in inducing nausea is poorly understood. Here, we have investigated their actions on human stomach muscle. EXPERIMENTAL APPROACH: Muscle strips were suspended in tissue baths and neuronal-/non-neuronally-mediated contractions were measured. Custom software analysed eight motility parameters defining spontaneous phasic non-neuronally mediated contractions. Receptor distributions were assessed by qPCR and immunofluorescence. KEY RESULTS: V1A receptors and α1 -adrenoceptors were located on muscle as well as interstitial cells of Cajal (ICCs). Myogenic contractions of human proximal and distal stomach (respectively, 2.6 ± 0.1 and 2.7 ± 0.0 per minute; n = 44) were larger in the distal area (1.1 ± 0.1 and 5.0 ± 0.1 mN), developing relatively slowly (proximal) or rapidly (distal). Vasopressin caused tonic (proximal) or short-lived (distal) increases in muscle tone and increased myogenic contraction amplitude, frequency and rate (acting at V1A receptors; thresholds 10-11 -10-10  M); by contrast, cholinergically mediated contractions were unaffected. Oxytocin acted similarly to vasopressin but less potently, at OT receptors). Adrenaline increased (10-10 -10-5  M; α1 -adrenoceptors) and decreased (≥10-6  M; ß-adrenoceptors) muscle tone and enhanced/reduced myogenic contractions. Cholinergically mediated contractions were reduced (α2 -adrenoceptors). Combined, vasopressin (10-9  M) and adrenaline (10-8  M) increased muscle tone and phasic myogenic activity in a synergistic manner. CONCLUSIONS AND IMPLICATIONS: Vasopressin and adrenaline increased human gastric tone and myogenic contraction amplitude, rate of contraction and frequency. In combination, their actions were further increased in a synergistic manner. Such activity may promote nausea.

A multi-parameter approach to measurement of spontaneous myogenic contractions in human stomach: Utilization to assess potential modulators of myogenic contractions.

Electrical slow waves, generated by interstitial cells of Cajal (ICC), cause spontaneous contractions of human stomach. Software was developed to measure muscle tone and eleven different parameters defining these contractions in human stomach, displaying data as radar plots. A pilot study assessed the effects of potential modulators, selected from among compounds known to influence ICC activity; n = 4-7 each concentration tested/compound. Human distal stomach (corpus-antrum) muscle strips were suspended in tissue baths for measuring myogenic (non-neuronal) contractions in the presence of tetrodotoxin (10-6 M). Initial characterization: Contractions (amplitude 4 ± 0.4mN, frequency 3 ± 0.1 min-1, n = 49) were unchanged by ꭃ-conotoxin GVIA (10-7 M) or indomethacin (10-6 M) but abolished by nifedipine (10-4 M). Carbachol (10-7 M) increased contraction rate and amplitude; 10-6-10-5 M increased tone and caused large, irregular contractions. [Ca2+]imodulators: Ryanodine (10-5-10-4 M) increased muscle tone accompanied by inhibition of myogenic contractions. Xestospongin-C (10-6 M; IP3 channel inhibitor) had no effects. SERCA pump inhibitors, 2-APB and cycloplazonic acid (10-5-10-4 M) increased tone and myogenic contraction amplitude before abolishing contractions; thapsigargin was weakly active. CaCC blockers: MONNA and CaCCinh-A01 had little-or-no effects on tone but reduced myogenic contractions; MONNA (10-4 M) was more effective, reducing amplitude (77.8 ± 15.2%) and frequency. CaV3.1/3.2/3.3 channel block: Mibefradil reduced tone and myogenic contraction amplitude (pIC50 4.8 ± 0.9). Inward-rectifying K+-channel inhibitor: E-4031 (10-4 M) increased contraction duration (17.4 ± 5.8%). Conclusions: (1) Measurement of multiple parameters of myogenic contractions identified subtle differences between compounds, (2) only E-4031 and CaCC blockers influenced myogenic contractions, not muscle tone, (3) studies are needed with compounds with known and/or improved selectivity/potency for human targets affecting ICC functions.

Antispasmodic Effect of Bergamot Essential Oil on Rat Isolated Gut Tissues.

Preclinical data indicate that bergamot essential oil (BEO) can modulate the synaptic functions within the central nervous system (CNS). Particularly, several data shows that essential oil is endowed with reproducible analgesic and anxiolytic effects that may derived from the ability to modulate the excitatory and inhibitory neurotransmission in the CNS. Although there are differences in the functional complexity of the enteric nervous system (ENS), it is likely that the phytocomplex has biological properties in gut superimposable to those showed in the CNS. Accordingly, the aim of this study was to investigate ex-vivo the effect of bergamot essential oil and its main constituents on the contractile activity of rat isolated colon, jejunum and ileum induced by different muscle stimulants such as acetylcholine (10-6 M) and potassium chloride (80 mM). Our present data demonstrate that BEO inhibits cholinergically- and non cholinergically-mediated contractions in rat isolated gut and that linalool is the most active component. These results suggest that the phytocomplex might be useful in the treatment of spastic disorders in ENS mainly characterized by the presence of pain; incidentally, irritable bowel syndrome (IBS) is a painful condition in which a role for neurotransmitter dysfunction has been envisaged. More investigation is required for clinical translation of the present data.

Evidence for tetrodotoxin-resistant spontaneous myogenic contractions of mouse isolated stomach that are dependent on acetylcholine.

BACKGROUND AND PURPOSE: Gastric pacemaker cells, interstitial cells of Cajal (ICC), are believed to initiate myogenic (non-neuronal) contractions. These become damaged in gastroparesis, associated with dysrhythmic electrical activity and nausea. We utilised mouse isolated stomach to model myogenic contractions and investigate their origin and actions of interstitial cells of Cajal modulators. EXPERIMENTAL APPROACH: Intraluminal pressure was recorded following distension with a physiological volume; tone, contraction amplitude and frequency were quantified. Compounds were bath applied. KEY RESULTS: The stomach exhibited regular large amplitude contractions (median amplitude 9.0 [4.7-14.8] cmH2 O, frequency 2.9 [2.5-3.4] c.p.m; n = 20), appearing to progress aborally. Tetrodotoxin (TTX, 10-6 M) had no effect on tone, frequency or amplitude but blocked responses to nerve stimulation. ω-conotoxin GVIA (10-7 M) ± TTX was without effect on baseline motility. In the presence of TTX, (1) atropine (10-10 -10-6 M) reduced contraction amplitude and frequency in a concentration-related manner (pIC50 7.5 ± 0.3 M for amplitude), (2) CaCC channel (previously ANO1) inhibitors MONNA and CaCCinh-A01 reduced contraction amplitude (significant at 10-5 , 10-4 M respectively) and frequency (significant at 10-5 M), and (3), neostigmine (10-5 M) evoked a large, variable, increase in contraction amplitude, reduced by atropine (10-8 -10-6 M) but unaffected (exploratory study) by the H1 receptor antagonist mepyramine (10-6 M). CONCLUSIONS AND IMPLICATIONS: The distended mouse stomach exhibited myogenic contractions, resistant to blockade of neural activity by TTX. In the presence of TTX, these contractions were prevented or reduced by compounds blocking interstitial cells of Cajal activity or by atropine and enhanced by neostigmine (antagonised by atropine), suggesting involvement of non-neuronal ACh in their regulation.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

Inhibition of Neuromuscular Contractions of Human and Rat Colon by Bergamot Essential Oil and Linalool: Evidence to Support a Therapeutic Action.

Bergamot essential oil (BEO) added to food and drink promotes a citrus flavour. Folklore suggests benefits on gastrointestinal functions but with little supporting evidence. BEO and major constituents (linalool, limonene, linalyl acetate) were therefore examined for any ability to influence neuromuscular contractions of human and rat colon. Circular muscle strips (macroscopically-normal human colon obtained following ethical approval at cancer surgery; Sprague-Dawley rats) were suspended in baths (Krebs solution; 37 °C; 5% CO2 in O2) for measurement of neuronally-mediated contractions (prevented by tetrodotoxin or atropine) evoked by electrical field stimulation (5 Hz, 0.5 ms pulse width, 10s/minute, maximally-effective voltage), or contractions evoked by KCl (submaximally-effective concentrations). BEO and each constituent concentration dependently inhibited neuronally-mediated and KCl-induced contractions. In human: apparent pIC50 for BEO (volume/volume Krebs), respectively, 3.8 ± 0.3 and 4.4 ± 0.3; Imax 55.8% ± 4.2% and 37.5% ± 4.2%. For the constituents, the rank order of potency differed in human (linalool > limonene >> linalyl-acetate) and rat colon (linalyl-acetate > limonene = linalool), but rank order of efficacy was similar (linalool >> (BEO) = linalyl-acetate >> limonene). Thus, linalool had high efficacy but greater potency in human colon (Imax 76.8% ± 6.9%; pIC50 6.7 ± 0.2; n = 4) compared with rat colon (Imax 75.3% ± 1.9%; pIC50 5.8 ± 0.1; n = 4). The ability of BEO and linalool to inhibit human colon neuromuscular contractility provides a mechanism for use as complementary treatments of intestinal disorders.

Anxiolytic-Like Effects of Bergamot Essential Oil Are Insensitive to Flumazenil in Rats.

Anxiety disorders are one of the most common mental disorders, and benzodiazepines (BDZs), acting on gamma-aminobutyric acid type A (GABA-A) receptor complex, represent the most common antianxiety medications in the world. However, chronic BDZ use elicits several adverse reactions. Reportedly, aromatherapy is safer for the management of anxiety. Bergamot essential oil (BEO) extracted from Citrus bergamia Risso et Poiteau fruit, like other essential oils, is widely used in aromatherapy to relieve symptoms of stress-induced anxiety. Interestingly, preclinical data indicate that BEO induces anxiolytic-like/relaxant effects in animal behavioural tasks not superimposable to those of benzodiazepine diazepam. To better elucidate the involvement of GABAergic transmission, the present study examines the effects of pretreatment with flumazenil (FLZ), a benzodiazepine site antagonist, on BEO effects using open-field task (OFT) in rats. The data yielded show that FLZ does not significantly affect behavioural effects of the phytocomplex. These results demonstrate the lack of overlapping between BEO and BDZ behavioural effects, contributing to the characterization of the neurobiological profile of the essential oil for its rational use in aromatherapy.