Milana Jocic

Gastric Acid-Evoked c-fos Messenger RNA Expression in Rat Brainstem Is Signaled by Capsaicin-Resistant Vagal Afferents

BACKGROUND & AIMS Gastric acid is known to contribute to ulcer pain, but the mechanisms of gastric chemonociception are poorly understood. This study set out to investigate the pathways and mechanisms by which gastric acid challenge is signaled to the brain. METHODS Neuronal excitation in the rat brainstem and spinal cord after intragastric administration of HCl (0.35-0.7 mol/L) was examined by in situ hybridization autoradiography for the immediate early gene c-fos. RESULTS Gastric acid challenge did not induce c-fos transcription in the spinal cord but caused many neurons in the nucleus tractus solitarii and area postrema to express c-fos messenger RNA (mRNA). The HCl concentration-dependent excitation of medullary neurons was in part associated with behavioral manifestations of pain but not directly related to the acid-induced injury and contraction of the stomach. Subdiaphragmatic vagotomy suppressed the c-fos mRNA response to intragastric acid, and morphine inhibited it in a naloxone-reversible manner, whereas pretreatment of rats with capsaicin was without effect. CONCLUSIONS Gastric acid challenge is signaled to the brainstem, but not the spinal cord, through vagal afferents that are sensitive to acid but resistant to capsaicin. It is hypothesized that the gastric acid-induced c-fos transcription in the brainstem is related to gastric chemonociception.

Vagal afferent signaling of a gastric mucosal acid insult to medullary, pontine, thalamic, hypothalamic and limbic, but not cortical, nuclei of the rat brain

&NA; Although gastric acid is a factor in upper abdominal pain, the signaling and processing of a gastric mucosal acid insult within the brain are not known. This study examined which nuclei in the rat brain respond to challenge of the gastric mucosa by a noxious concentration of hydrochloric acid (HCl) and whether the central input is carried by vagal afferent neurons. Activation of neurons in the brain was mapped by in situ hybridization autoradiography of messenger ribonucleic acid (mRNA) for the immediate early gene c‐fos 45 min after intragastric administration of saline or HCl. Following intragastric HCl (0.5 M) challenge, many neurons in the nucleus tractus solitarii, lateral parabrachial nucleus, thalamic and hypothalamic paraventricular nucleus, supraoptic nucleus, central amygdala and medial/lateral habenula expressed c‐fos mRNA as compared to intragastric treatment with saline (0.15 M). However, c‐fos transcription in the insular cortex was not enhanced by the gastric acid insult. Hypertonic saline (0.5 M) caused only a minor expression of c‐fos mRNA in the hypothalamus and amygdala. The acid‐evoked c‐fos induction in subcortical nuclei was depressed by at least 80% five days after bilateral subdiaphragmatic vagotomy. Collectively, these observations indicate that vagal afferent input from the acid‐threatened gastric mucosa does not reach the insular cortex but leads to activation of subcortical brain nuclei that are involved in emotional, behavioral, neuroendocrine, autonomic and antinociceptive reactions to a noxious stimulus.

Cutaneous vasodilatation induced by nitric oxide-evoked stimulation of afferent nerves in the rat

1 The site of action at which nitric oxide (NO) may contribute to neurogenic vasodilatation in the hindpaw skin of urethane‐anaesthetized rats was examined by the use of NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of NO synthase. 2 Skin blood flow was measured by laser Doppler flowmetry, and neurogenic vasodilatation was evoked either by topical application of mustard oil (5%) or antidromic electrical stimulation of the saphenous nerve (antidromic vasodilatation). 3 l‐NAME (60 μmol kg−1, i.v.) attenuated the hyperaemia evoked by mustard oil in an enantiomer‐specific manner but failed to reduce antidromic vasodilatation and the vasodilatation due to i.v. injected calcitonin gene‐related peptide (CGRP) and substance P (0.1–1 nmol kg−1 each), two proposed mediators of neurogenic vasodilatation. 4 Pretreatment of rats with capsaicin (125 mg kg−1, s.c. 2 weeks beforehand), to defunctionalize afferent neurones, reduced the hyperaemic response to mustard oil and prevented l‐NAME from further decreasing the vasodilatation evoked by mustard oil. 5 Intraplantar infusion of sodium nitroprusside (SNP, 0.15 nmol in 1 min), a donor of NO, induced hyperaemia which was significantly diminished by the CGRP antagonist CGRP8–37 (50 nmol kg−1, i.v.) and by capsaicin pretreatment. The ability of CGRP8–37 to inhibit the vasodilator response to SNP was lost in capsaicin‐pretreated rats. 6 Taken together, these data indicate that NO does not play a vasorelaxant messenger role in neurogenic vasodilatation but can contribute to activation of, and/or transmitter release from, afferent nerve fibres in response to irritant chemicals.

Nitric oxide-dependent and -independent hyperaemia due to calcitonin gene-related peptide in the rat stomach.

1 Calcitonin gene‐related peptide (CGRP) potently enhances mucosal blood flow in the rat stomach. The aim of this study was to examine whether CGRP also dilates extramural arteries supplying the stomach and whether the vasodilator action of CGRP involves nitric oxide (NO). 2 Rat CGRP‐α (0.03–1 nmol kg−1, i.v.) produced a dose‐dependent increase in blood flow through the left gastric artery (LGA) as determined by an ultrasonic transit time technique in urethane‐anaesthetized rats. Blockade of NO synthesis by NG‐nitro‐l‐arginine methyl ester (l‐NAME, 20 and 60 μmol kg−1, i.v.) significantly reduced basal blood flow (BF) in the LGA and attenuated the hyperaemic activity of CGRP by a factor of 2.8–4. d‐NAME tended to enhance basal BF in the LGA but had no influence on the dilator activity of CGRP. The ability of vasoactive intestinal polypeptide to increase left gastric arterial blood flow remained unaltered by l‐NAME. 3 l‐NAME (20 and 60 μmol kg−1, i.v.) evoked a prompt and sustained rise of mean arterial blood pressure (MAP) and caused a slight decrease in the hypotensive activity of CGRP. In contrast, d‐NAME induced a delayed and moderate increase in MAP and did not influence the hypotensive activity of CGRP. 4 Rat CGRP‐α dilated the isolated perfused bed of the rat LGA precontracted with methoxamine and was 3 times more potent in this respect than rat CGRP‐β. The dilator action of rat CGRP‐α in this preparation was not affected by l‐NAME or d‐NAME (40 μm). 5 L‐NAME (60 μmol kg−1, i.v.) reduced gastric mucosal blood flow as assessed by laser Doppler flowmetry and diminished the hyperaemic activity of rat CGRP‐α in the gastric mucosa by a factor of 4.5, whereas d‐NAME was without effect. 6 These data show that CGRP is a potent dilator of mucosal and extramural resistance vessels in the rat stomach. Its dilator action involves both NO‐dependent and NO‐independent mechanisms.

Vascular bed‐dependent roles of the peptide CGRP and nitric oxide in acid‐evoked hyperaemia of the rat stomach.

1. Acid back‐diffusion through a disrupted gastric mucosal barrier is known to increase gastric mucosal blood flow via a neural mechanism. The present study examined how the acid‐evoked change in the gastric microcirculation compares with blood flow changes in the left gastric artery, one of the major arteries supplying the stomach, and whether the dilator mediators in the left gastric artery are identical to those in the gastric mucosa. 2. The experiments were performed on rats anaesthetized with urethane. Blood flow in the left gastric artery was measured by the ultrasonic transit time shift technique, and blood flow in the gastric mucosa was assessed by the hydrogen gas clearance method. 3. Gastric acid back‐diffusion evoked by perfusion of the stomach with 15% ethanol in 0.15 M HCl increased blood flow in the left gastric artery by a factor of 4.7, which was significantly larger than the 2.9‐fold increase in blood flow through the gastric mucosa. Blood pressure and heart rate were not altered appreciably. 4. The acid‐evoked hyperaemia in the left gastric artery was left unaltered by atropine and the substance P receptor antagonist RP‐67580. 5. The calcitonin gene‐related peptide (CGRP) antagonist CGRP (8‐37) had no effect on gastric blood flow but prevented the dilator action of CGRP and inhibited the acid‐evoked hyperaemia in the gastric mucosa to a larger degree than the hyperaemia in the left gastric artery. 6. Blockade of nitric oxide synthesis by N omega‐nitro‐L‐arginine methyl ester (L‐NAME) caused constriction of the left gastric artery and the gastric mucosal microvessels. The acid‐evoked vasodilatation in the gastric mucosa was blocked by L‐NAME, whereas the dilator response in the left gastric artery was not significantly depressed. 7. The data show that the gastric hyperaemic response to acid back‐diffusion results from dilatation of mucosal microvessels and extramural arteries. The dilator mechanisms, however, differ between the two vascular beds. CGRP and nitric oxide are important vasodilator mediators in the gastric mucosa but are of less relevance in the left gastric artery.

Acid challenge delays gastric pressure adaptation, blocks gastric emptying and stimulates gastric fluid secretion in the rat

Abstract Functional dyspepsia can be associated with impaired gastric relaxation in response to food intake and delayed gastric emptying. In this study, we investigated whether luminal hydrochloric acid (HCl) may reproduce these motor alterations in phenobarbital‐anaesthetized rats via activation of extrinsic neural pathways. Intragastric pressure (IGP) changes induced by a 2‐mL fluid bolus were recorded with an oesophageal catheter, and gastric emptying was determined via the fluid volume recovered from the stomach 30‐min post‐bolus. Experiments involving acute nerve transections or pharmacological blockade of nitric oxide synthesis revealed that the initial increase of IGP after a 0.35 mol L−1 HCl bolus is dampened by duodenogastric and gastrogastric relaxation reflexes depending on vagal and splanchnic pathways as well as nitric oxide. Compared with saline, HCl (0.15–0.5 mol L−1) delayed the subsequent decrease (adaptation) of IGP, inhibited gastric emptying and stimulated gastric fluid secretion as seen in stomachs with ligated pylorus. The acid‐evoked delay in IGP adaptation and inhibition of gastric emptying involved duodenogastric and duodenopyloric extrinsic nerve reflexes, whereas the gastric fluid secretion was independent of the extrinsic innervation. It is proposed that the gastropyloric motor changes induced by luminal acid challenge have a bearing on the motor disturbances underlying functional dyspepsia.

Cooperation of NMDA and tachykinin NK1 and NK2 receptors in the medullary transmission of vagal afferent input from the acid-threatened rat stomach

&NA; Noxious challenge of the rat gastric mucosa by hydrochloric acid (HCl) is signaled to the nucleus tractus solitarii (NTS) and area postrema (AP). This study examined the participation of glutamate and tachykinins in the medullary transmission process. Activation of neurons was visualized by in situ hybridization autoradiography of c‐fos messenger RNA (mRNA) 45 min after intragastric (IG) administration of 0.5 M HCl or saline. IG HCl caused many neurons in the NTS and some neurons in the AP to express c‐fos mRNA. The NMDA glutamate receptor antagonist MK‐801 (2 mg/kg), the NK1 tachykinin receptor antagonist GR‐205,171 (3 mg/kg) and the NK2 receptor antagonist SR‐144,190 (0.1 mg/kg) failed to significantly reduce the NTS response to IG HCl, whereas the triple combination of MK‐801, GR‐205,171 and SR‐144,190 inhibited it by 45–50%. Only in rats that had been preexposed IG to HCl 48 h before the experiment was MK‐801 alone able to depress the NTS response to IG HCl. In contrast, the c‐fos mRNA response in the AP was significantly augmented by MK‐801, an action that was prevented by coadministration of GR‐205,171 plus SR‐144,190. Inhibition of neuronal nitric oxide synthase with 7‐nitroindazole (45 mg/kg) was without effect on the IG HCl‐evoked c‐fos mRNA expression in the NTS and AP. Our data show that glutamate acting via NMDA receptors and tachykinins acting via NK1 and NK2 receptors cooperate in the vagal afferent input from the acid‐threatened stomach to the NTS and participate in the processing of afferent input to the AP in a different and complex manner. These opposing interactions in the AP and NTS and the increase in NMDA receptor function in the NTS after a gastric acid insult are likely to have a bearing on the neuropharmacology of dyspepsia.

Tachykinin inhibition of acid-induced gastric hyperaemia in the rat.

1 Primary afferent neurones releasing the vasodilator, calcitonin gene‐related peptide, mediate the gastric hyperaemic response to acid back‐diffusion. The tachykinins neurokinin A (NKA) and substance P (SP) are located in the same neurones and are co‐released with calcitonin gene‐related peptide. In this study we investigated the effect and possible role of tachykinins in the acid‐evoked gastric vasodilatation in urethane‐anaesthetized rats. 2 Gastric acid back‐diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 m HCl, increased gastric mucosal blood flow by 60–90%, as determined by the hydrogen clearance technique. NKA and SP (0.14‐3.78 nmol min−1 kg−1, infused intra‐aortically) inhibited the gastric mucosal hyperaemic response to acid back‐diffusion in a dose‐dependent manner, an effect that was accompanied by aggravation of ethanol/acid‐induced macroscopic haemorrhagic lesions. 3 The inhibitory effect of NKA (1.26 nmol min−1 kg−1) on the acid‐induced gastric mucosal vasodilatation was prevented by the tachykinin NK2 receptor antagonist, MEN 10,627 (200 nmol kg−1) but left unaltered by the NK1 receptor antagonist, SR 140,333 (300 nmol kg−1) and the mast‐cell stabilizer, ketotifen (4.6 μmol kg−1). 4 Under basal conditions, with 0.05 m HCl being perfused through the stomach, NKA (1.26 nmol min−1 kg−1) reduced gastric mucosal blood flow by about 25%, an effect that was abolished by SR 140,333 but not MEN 10,627 or ketotifen. 5 SR 140,333, MEN 10,627 or ketotifen had no significant effect on basal gastric mucosal blood flow nor did they modify the gastric mucosal hyperaemic reaction to acid back‐diffusion. 6 The effect of NKA (1.26 nmol min−1 kg−1) in causing vasoconstriction and inhibiting the vasodilator response to acid back‐diffusion was also seen when blood flow in the left gastric artery was measured with the ultrasonic transit time shift technique. 7 Arginine vasopressin (AVP, 0.1 nmol min−1 kg−1) induced gastric mucosal vasoconstriction under basal conditions but was unable to inhibit the dilator response to acid back‐diffusion. 8 These data show that NKA has two fundamentally different effects on the gastric circulation. Firstly, NKA reduces gastric blood flow by activation of NK1 receptors. Secondly, NKA inhibits the gastric hyperaemic response to acid back‐diffusion through an NK2 receptor‐mediated mechanism. These two tachykinin effects appear to take place independently of each other since they are mediated by different receptors. This concept is further supported by the inability of AVP to mimic tachykinin inhibition of the gastric vasodilator response to acid back‐diffusion.

Visceral vasodilatation and somatic vasoconstriction evoked by acid challenge of the rat gastric mucosa: diversity of mechanisms.

1. Acid back‐diffusion through a disrupted gastric mucosal barrier increases blood flow to the stomach without any change in systemic blood pressure. This study was undertaken to examine the gastric acid‐evoked changes in blood flow in a number of visceral and somatic arterial beds and to elucidate the mechanisms which lead to the regionally diverse haemodynamic responses. 2. The gastric mucosa of urethane‐anaesthetized rats was challenged with acid by perfusing the stomach with ethanol (15%, to disrupt the gastric mucosal barrier) in 0.15 M HCl. Blood flow was estimated by laser Doppler flowmetry, the hydrogen clearance method or the ultrasonic transit time shift technique. 3. Gastric acid challenge increased blood flow in the gastric mucosa and left gastric artery while blood flow in the femoral artery and skin declined. 4. Afferent nerve stimulation by intragastric administration of capsaicin enhanced blood flow in the left gastric artery but did not diminish blood flow in the femoral artery when compared with the vehicle. 5. The gastric acid‐evoked dilatation of the left gastric artery was depressed by acute extrinsic denervation of the stomach, capsaicin‐induced ablation of afferent neurones or hexamethonium‐induced blockade of autonomic ganglionic transmission. 6. The gastric acid‐induced constriction of the femoral artery was attenuated by acute extrinsic denervation of the stomach but left unaltered by capsaicin, hexamethonium, guanethidine, indomethacin, telmisartan (an angiotensin II antagonist), [d(CH2)5(1), Tyr(Me)2, Arg8]‐vasopressin (a vasopressin antagonist), bosentan (an endothelin antagonist) and acute ligation of the blood vessels to the adrenal glands. 7. These data show that acid challenge of the gastric mucosa elicits visceral vasodilatation and somatic vasoconstriction via divergent mechanisms. The gastric hyperaemia is brought about by extrinsic vasodilator nerves, whereas the reduction of somatic blood flow seems to be mediated by non‐neural, probably humoral, vasoconstrictor messengers that remain to be identified.

Role of bradykinin in the hyperaemia following acid challenge of the rat gastric mucosa

1 This study examined whether the hyperaemia following acid challenge of the rat gastric mucosa involves bradykinin, a peptide formed in response to tissue injury. 2 Gastric mucosal blood flow in urethane‐anaesthetized rats was assessed by the hydrogen gas clearance method. Infusion of a bradykinin solution (10 μm) into the gastric wall augmented gastric mucosal blood flow by a factor of 2.3, an effect that was prevented by the bradykinin B2 antagonist Hoe‐140 (icatibant; 100 μmol kg−1, i.v.). 3 I.v. injection of bradykinin (20–60 nmol kg−1) caused a 2.3‐3.5 fold increase in blood flow through the left gastric artery as measured by the ultrasonic transit time shift technique. The hyperaemic effect of bradykinin in this gastric artery was also prevented by Hoe‐140 (100 μ mol kg−1, i.v.). 4 Gastric acid backdiffusion was evoked by perfusing the stomach with 15% ethanol, to break the gastric mucosal barrier, in the presence of luminal acid. Depending on the concentration of acid (0.05 and 0.15 M HC1), this procedure increased gastric mucosal blood flow by a factor of 1.6‐2.8 and caused formation of gross damage in 1.5‐3% of the glandular mucosa. Hoe‐140 (100 μmol kg−1, i.v.) failed to alter the moderate vasodilatation seen in the presence of 0.05 M HC1 but significantly (P < 0.05) attenuated the marked hyperaemia and enhanced the gross mucosal damage observed in the presence of 0.15 M HC1. 5 These data show that bradykinin is able to enhance gastric mucosal blood flow via activation of B2 receptors. It appears as if this kinin is formed during severe acid challenge of the rat gastric mucosa and participates in the hyperaemic reaction to gastric acid backdiffusion.