Irmgard Th. Lippe

The effect of omega conotoxin GVIA, a peptide modulator of the N-type voltage sensitive calcium channels, on motor responses produced by activation of efferent and sensory nerves in mammalian smooth muscle.

Summary1.The effect of omega-conotoxin (CTX) GVIA, a peptide which blocks neuronal calcium channels, were investigated on nerve-mediated motor responses in a variety of isolated smooth muscle preparations from rats and guinea-pigs.2.In the rat or guinea-pig isolated vas deferens CTX (1 nM − 1 μM) produced a concentration and time-related inhibition of the response to field stimulation, while the responses to KCI, noradrenaline or adenosine triphosphate were unaffected. In the presence of CTX a series of tetrodotoxin-resistant contractions could be elicited by field stimulation by increasing pulse width and/or voltage.3.In the rat or guinea-pig isolated urinary bladder, CTX produced a concentration and time-dependent inhibition of twitch responses to field stimulation without affecting the response to exogenous acetylcholine. In the rat bladder the maximal effect did not exceed 25% inhibition while a much larger fraction of the response (about 70%) was inhibited in the guinea-pig bladder. The CTX-resistant response was abolished, in both tissues, by tetrodotoxin.4.The effects of CTX in the rat bladder were also studied with a whole range of frequencies of field stimulation (0.1–50 Hz). Maximal inhibition was observed toward contractions elicited at frequencies of 2–5 Hz. At low frequencies the inhibitory effects of CTX and atropine were almost additive while at high frequencies of stimulation a large component of the atropine-sensitive response was CTX-resistant.5.In the rat isolated proximal duodenum, field stimulation in the- presence of atropine and guanethidine produced a primary relaxation followed by a rebound contraction. Both responses were abolished by tetrodotoxin, indicating the activation of intramural nonadrenergic noncholinergic nerves. The primary relaxation was totally CTX resistant while the rebound contraction was slightly inhibited.6.The motor responses produced by capsaicin (1 μM) in the rat or guinea-pig bladder (contraction) and in the rat proximal duodenum (relaxation) were unaffected by CTX. Likewise, the release of substance P-like immunoreactivity from sensory nerves of the guinea-pig bladder muscle was unaffected by CTX.7.These findings indicate that CTX-sensitive calcium channels modulate transmitter release in autonomic nerve terminals of mammals, but noticeable species and organ related variations exist in sensitivity to this peptide, possibly reflecting the existence of a heterogenous population of voltage-sensitive calcium channels. CTX-sensitive calcium channels are apparently not involved in the excitatory action of capsaicin on sensory nerve terminals.

Participation of endothelium-derived nitric oxide but not prostacyclin in the gastric mucosal hyperaemia due to acid back-diffusion

1 The possible participation of prostacyclin and nitric oxide (NO) in the gastric mucosal hyperaemic response to acid back‐diffusion through a disrupted gastric mucosal barrier was examined. The experiments were carried out on anaesthetized rats in which acid back‐diffusion was elicited by gastric perfusion with dilute ethanol in 0.15 m HCl and gastric mucosal blood flow (MBF) was measured by the hydrogen gas clearance technique. 2 Indomethacin (28 μmol kg−1, s.c.), an inhibitor of the formation of cyclo‐oxygenase products including prostacyclin, failed to alter mean arterial blood pressure (MAP), basal MBF and the hyperaemic response to acid back‐diffusion in urethane‐anaesthetized rats. 3 NG‐nitro‐l‐arginine methyl ester (l‐NAME; 13 and 43 μmol kg−1, i.v.), an inhibitor of endothelium‐derived NO formation, increased MAP in a dose‐dependent manner. Whilst basal MBF in urethane‐anaesthetized rats was not changed, the increase in MBF caused by gastric perfusion with dilute ethanol in acid was dose‐dependently depressed by l‐NAME. The loss of H+ ions from the gastric lumen, an indirect measure of acid back‐diffusion, was significantly enhanced by 43 μmol kg−1 l‐NAME. In contrast, d‐NAME (13 and 43 μmol kg−1) was without effect on MAP, basal and stimulated MBF, and acid back‐diffusion. 4 Unlike in urethane‐anaesthetized rats, l‐NAME led to a significant reduction of basal MBF in phenobarbitone‐anaesthetized rats. MAP in the phenobarbitone‐anaesthetized rats was significantly higher than in urethane‐anaesthetized rats, and the hypertensive effect of l‐NAME under phenobarbitone anaesthesia was significantly smaller than under urethane anaesthesia. 5 The rise in MBF brought about by acid back‐diffusion was blocked by l‐NAME administered to phenobarbitone‐anaesthetized rats. Infusion of l‐arginine (120 μmol kg−1 min−1, i.v.) led to a partial, but significant, reversal of the effects of l‐NAME on MAP and the hyperaemia due to acid back‐diffusion. 6 These findings indicate that endothelium‐derived NO plays an important mediator role in the gastric mucosal vasodilatation caused by back‐diffusion whilst vasodilator prostanoids such as prostacyclin are not involved.

Increased expression of TRPV1 receptor in dorsal root ganglia by acid insult of the rat gastric mucosa

It is still unknown which receptors of peripheral sensory pathways encode and integrate an acid‐induced nociceptive event in the gastric mucosa. The transient receptor potential vanilloid receptor 1 (TRPV1) and the acid‐sensing ion channel 3 (ASIC3) are two nociception‐related receptors. Here we investigated (i) to what extent these receptors are distributed in stomach‐innervating neurons of dorsal root and nodose ganglia, using immunohistochemistry and retrograde tracing, and (ii) whether their expression is altered in response to a noxious acid challenge of the stomach. We also explored the presence of TRPV1 in the gastric enteric nervous system because of its possible expression by intrinsic sensory neurons. Most stomach‐innervating neurons in nodose ganglia were immunoreactive for TRPV1 (80%) and ASIC3 (75%), these results being similar in the dorsal root ganglia (71 and 82%). RT‐PCR and Western blotting were performed up to 6 h after oral application of 0.5 m HCl to conscious rats. TRPV1 protein was increased in dorsal root but not in nodose ganglia whereas TRPV1 and ASIC3 mRNAs remained unchanged. TRPV1 mRNA was detected in longitudinal muscle–myenteric plexus preparations of control stomachs and was not altered by the acid challenge. Combined vagotomy and ganglionectomy abolished expression of TRPV1, indicating that it may derive from an extrinsic source. In summary, noxious acid challenge of the stomach increased TRPV1 protein in spinal but not vagal or intrinsic sensory afferents. The TRPV1 receptor may be a key molecule in the transduction of acid‐induced nociception of the gastric mucosa and a mediator of visceral hypersensitivity.

Intragastric capsaicin enhances rat gastric acid elimination and mucosal blood flow by afferent nerve stimulation

1 This study investigated the effects of intragastric capsaicin on acid output, clearance of aniline, potential difference, and morphology of the mucosa in the rat stomach. The experiments were carried out on rats anaesthetized with urethane in which the stomachs were continuously perfused with saline. 2 When the stomach was perfused with normal saline (pH ∼6), intragastric capsaicin (32–640 μm) had no effect on the output of titratable acid. In contrast, when acid output was stimulated by pentagastrin or when the stomach was perfused with acid saline (pH 3), capsaicin reduced acid output. Acid loss which occurred during perfusion with saline of pH 2 was not significantly increased by capsaicin. This suggests that capsaicin does not enhance acid back‐diffusion but facilitates acid elimination by other means. 3 The gastric clearance of [14C]‐aniline, which is an indirect index of gastric mucosal blood flow, was estimated while the stomach was perfused with saline of pH 3. The clearance of aniline rose by 50–60% following intragastric administration of capsaicin (160 μm) whereas the mean arterial blood pressure was increased by about 2.5 mmHg only. Combined pretreatment of the rats with atropine, phentolamine, and propranolol did not alter the effect of capsaicin on the gastric clearance of aniline. 4 The gastric potential difference was not altered by capsaicin (160 μm) administered together with saline of pH 3. This and the finding that there were no signs of mucosal damage by light and scanning electron microscopy indicate that intragastric capsaicin does not irritate the gastric mucosa. 5 The effects of intragastric capsaicin on gastric acid output and aniline clearance and on blood pressure were absent in rats in which capsaicin‐sensitive afferent neurones had been ablated by neonatal treatment with a neurotoxic dose of capasicin, which indicates that they result from stimulation of afferent nerve endings in the stomach. It is concluded that facilitation of acid elimination and mucosal blood flow may contribute to the previously reported protective action of capsaicin on the gastric mucosa.

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.

Tachykinin NK1 and NK2 receptor-mediated control of peristaltic propulsion in the guinea-pig small intestine in vitro

The tachykinins substance P and neurokinin A are excitatory cotransmitters of cholinergic enteric neurons, their actions being mediated by NK1, NK2 and NK3 receptors. This study examined which of these receptors are part of the neural circuitry of peristalsis. Peristaltic propulsion in luminally perfused segments of the guinea-pig isolated ileum was elicited by a rise of the intraluminal pressure. The pressure threshold at which peristaltic contractions were triggered was used to quantify drug effects on peristalsis, inhibition of peristalsis being reflected by an increase in the pressure threshold. The NK1, NK2 and NK3 receptor antagonists SR-140333, SR-48968 and SR-142 801 (each at 0.1 microM), respectively, had little effect on peristaltic activity as long as cholinergic transmission was left intact. However, both the NK1 and NK2 receptor antagonist (each at 0.1 microM) abolished peristalsis after cholinergic transmission via muscarinic receptors had been blocked by atropine (1 microM) and peristalsis rescued by naloxone (0.5 microM). When cholinergic transmission via nicotinic receptors was suppressed by hexamethonium (100 microM) and peristalsis restored by naloxone (0.5 microM), only the NK2 receptor antagonist (0.1 microM) was able to attenuate peristaltic performance as deduced from a rise of the peristaltic pressure threshold by 106%. The NK3 receptor antagonist (0.1 microM) lacked a major influence on peristalsis under any experimental condition. It is concluded that tachykinins acting via NK1 and NK2 receptors sustain intestinal peristalsis when cholinergic neuroneuronal and neuromuscular transmission via muscarinic receptors has been suppressed. NK2 receptors help maintaining peristalsis once cholinergic neuroneuronal transmission via nicotinic receptors has been blocked, whereas NK3 receptors play little role in the neural pathways of peristalsis.

Participation of nitric oxide in the mustard oil-induced neurogenic i inflammation of the rat paw skin

The possibility of nitric oxide (NO) participating in the neurogenic inflammatory reaction of the rat hindpaw skin to topical application of mustard oil was examined in anesthetized rats. Vasodilation was measured by contactless infrared emission thermography, plasma protein exudation was measured by the Evans blue leakage technique, and formation of oedema was determined by measurement of the paw volume. Topical administration of mustard oil increased the temperature of the paw skin by 2-3 degrees C, a response that was accompanied by plasma protein extravasation and followed by development of slight oedema amounting to a 7-8% increase in paw volume. That the mustard oil-induced increase in paw temperature and oedema formation was neurogenic was shown by the finding that both responses were absent in adult rats treated with a neurotoxic dose of capsaicin (0.16 mmol kg-1 s.c.) as neonates. NG-Nitro-L-arginine methyl ester (L-NAME, 43 mumol kg-1 i.v.) an inhibitor of NO formation, caused a significant increase in mean arterial blood pressure and a moderate decrease in cutaneous blood flow, whereas the same dose of the inactive enantiomer, D-NAME, was without effect. L-NAME, but not D-NAME, reduced the cutaneous hyperaemia caused by topical administration of mustard oil by about 50% but did not significantly diminish the exudative reaction to mustard oil. These findings indicate that endothelium-derived NO plays a mediator role in the vasodilator component of mustard oil-induced neurogenic inflammation in the rat paw skin whereas the increase in vascular permeability does not seem to involve NO directly as a mediator.

Close arterial infusion of calcitonin gene-related peptide into the rat stomach inhibits aspirin- and ethanol-induced hemorrhagic damage

Afferent neuron-mediated gastric mucosal protection has been suggested to result from the local release of vasodilator peptides such as calcitonin gene-related peptide (CGRP) from afferent nerve endings within the stomach. The present study, therefore, examined whether rat alpha-CGRP, administered via different routes, is able to protect against mucosal injury induced by gastric perfusion with 25% ethanol or acidified aspirin (25 mM, pH 1.5) in urethane-anesthetized rats. Close arterial infusion of CGRP (15 pmol/min) to the stomach, via a catheter placed in the abdominal aorta proximal to the celiac artery, significantly reduced gross mucosal damage caused by ethanol and aspirin whereas mean arterial blood pressure (BP) was not altered. Intravenous infusion of CGRP (50 pmol/min) did not affect aspirin-induced mucosal injury but significantly enhanced ethanol-induced lesion formation. Intravenous CGRP (50 pmol/min) also lowered BP and increased the gastric clearance of [14C]aminopyrine, an indirect measure of gastric mucosal blood flow while basal gastric output of acid and bicarbonate was not altered. Intragastric administration of CGRP (260 nM) significantly inhibited aspirin-induced mucosal damage but did not influence damage in response to ethanol. BP, gastric clearance of [14C]aminopyrine, and gastric output of acid and bicarbonate remained unaltered by intragastric CGRP. These data indicate that only close arterial administration of CGRP to the rat stomach, at doses devoid of a systemic hypotensive effect, is able to protect against both ethanol- and aspirin-induced mucosal damage. As this route of administration closely resembles local release of the peptide in the stomach, CGRP may be considered as a candidate mediator of afferent nerve-induced gastric mucosal protection.

The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.

The Prostaglandin E2 Receptor EP4 Is Expressed by Human Platelets and Potently Inhibits Platelet Aggregation and Thrombus Formation

Objective—Low concentrations of prostaglandin (PG) E2 enhance platelet aggregation, whereas high concentrations inhibit it. The effects of PGE2 are mediated through 4 G protein-coupled receptors, termed E-type prostaglindin (EP) receptor EP1, EP2, EP3, and EP4. The platelet-stimulating effect of PGE2 has been suggested to involve EP3 receptors. Here we analyzed the receptor usage relating to the inhibitory effect of PGE2. Methods and Results—Using flow cytometry, we found that human platelets expressed EP4 receptor protein. A selective EP4 agonist (ONO AE1-329) potently inhibited the platelet aggregation as induced by ADP or collagen. This effect could be completely reversed by an EP4 antagonist, but not by PGI2, PGD2, and thromboxane receptor antagonists or cyclooxygenase inhibition. Moreover, an EP4 antagonist enhanced the PGE2-induced stimulation of platelet aggregation, indicating a physiological antiaggregatory activity of EP4 receptors. The inhibitory effect of the EP4 agonist was accompanied by attenuated Ca2+ flux, inhibition of glycoprotein IIb/IIIa, and downregulation of P-selectin. Most importantly, adhesion of platelets to fibrinogen under flow and in vitro thrombus formation were effectively prevented by the EP4 agonist. In this respect, the EP4 agonist synergized with acetylsalicylic acid. Conclusion—These results are suggestive of EP4 receptor activation as a novel antithrombotic strategy.