Peter McLean

Kinin B1 receptors and the cardiovascular system: regulation of expression and function

Kinins are important peptide mediators of a diverse range of physiological and pathological functions of the cardiovascular system. The kinin peptides exert their effects by selective activation of two distinct G-protein coupled receptors termed B(1) and B(2). The principal kinin peptides involved in the acute regulation of cardiovascular function during normal physiology are bradykinin (BK) and Lys-BK which produce their effects via activation of B(2) receptors. The B(1) receptor is activated by the des-Arg(9)kinin metabolites namely des-Arg(9)BK and Lys-des-Arg(9)BK, the synthesis of which are increased during inflammation. The B(1) receptor, which is not constitutively expressed, is induced in various pathologies relating to inflammation. Recent investigations into the molecular mechanisms of B(1) receptor induction and their distribution and function in the cardiovascular system have shown that following an inflammatory stimulus the B(1) receptor is induced and may play an important role in modulation of cardiovascular function. This review summarises recent studies on B(1) receptor expression and function in the cardiovascular system and discusses the role of these receptors in regulation of circulatory homeostasis and their potential as therapeutic targets.

Lipocortin 1 reduces myocardial ischemia-reperfusion injury by affecting local leukocyte recruitment

We assessed here the effect of the glucocorticoid‐regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25‐min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with ~50% inhibition at the highest dose tested of 50 per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose‐dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor α, and macrophage inflammatory protein‐1α. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 μg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte‐dependent myocardial damage associated with an ischemia‐reperfusion procedure.

Inducible expression of the kinin B1 receptor in the endotoxemic heart: mechanisms of des-Arg9bradykinin-induced coronary vasodilation.

We have investigated the role of kinin B1 receptor induction in the endotoxemic rat heart and elucidated the mechanisms underlying B1 receptor‐mediated coronary vasodilation. We also investigated the role of these receptors in endotoxin‐induced hypotension. Endotoxin treatment induced cardiac B1 receptor mRNA expression and promoted a coronary vasodilation response to des‐Arg9bradykinin (DABK; ED50=149.4 pmol, n=9) ex vivo peaking at 6 h. The B1 receptor antagonist des‐Arg9‐[Leu8]‐BK (DALBK, 30 nM) significantly (P<0.05) inhibited the DABK‐induced response (pA2=8.4, n=5) whilst HOE140 (B2 receptor antagonist, 10 nM) was inactive (n=4). Removal of the endothelium or infusion with indomethacin (5 μM), but not L‐NAME (300 μM) or ODQ (1 μM), inhibited (>85%, P<0.05, n=5) the DABK‐induced response. DABK caused a dose‐dependent release of the prostacyclin metabolite, 6‐keto‐PGF1a (Emax=0.3 ng ml−1, n=6). In vitro perfusion of hearts with endotoxin (1 μg ml−1, n=6) or interleukin‐1β (5 ng ml−1, n=6) induced B1 receptor mRNA expression and promoted a time‐dependent vasodilation response to DABK. Endotoxin treatment (6 h) in vivo promoted a hypotensive response to DABK (ED50=29.7 nmol kg−1, n=10) which was antagonised by DALBK (3–6 nmol kg−1 min−1, P<0.05, n=7). DALBK (3 nmol kg−1 min−1) and des‐Arg10HOE140 (B1 receptor antagonist, 30 nmol kg−1 min−1) produced a 5.3% (n=6, P<0.05) and 8.8% (n=5, P<0.05) reversal, respectively, of endotoxin‐induced hypotension. In summary, we have shown that in endotoxemia activation of B1 receptors causes coronary vasodilation via endothelial prostacyclin release. Additionally, B1 receptor antagonists partially reversed endotoxin‐induced hypotension. Therefore activation of B1 receptors may have a role to play in the vascular changes associated with endotoxemia.

Protease-activated receptor 2-mediated vasodilatation in humans in vivo: Role of nitric oxide and prostanoids

Background—Systemic hypotension as a consequence of vascular dysfunction is a well-recognized and important feature of critical illness. Although serine protease activation has been implicated as a cause of vascular dysfunction in systemic inflammation, the mechanism is unknown. Recently, a class of receptors with an entirely novel mechanism of action, protease-activated receptors (PARs), has been identified that would explain the link between protease activation and systemic hypotension. Our aim was to test the hypothesis that in vivo activation of protease-activated receptor 2 (PAR-2) in humans would mediate vasodilatation. Methods and Results—For these first-in-human studies, an activating peptide for the human PAR-2 receptor was synthesized and administered to healthy volunteers. Using both the dorsal hand vein technique and forearm plethysmography, we studied the effects of PAR-2 activation in human blood vessels and investigated the mechanism of vasodilation. Activation of PAR-2 receptors in vivo dilated human blood vessels in a dose-dependent manner, and the effects were reduced by inhibition of both nitric oxide and prostanoid synthesis Conclusions—These findings demonstrate that serine protease activity can cause human vasodilation and provide a possible explanation of why serine protease activation in critical illness is associated with vascular dysfunction.

Targeting the microbiota-gut-brain axis to modulate behavior: which bacterial strain will translate best to humans?

We have read with great interest the paper by Bravo et al. (1), which showed that Lactobacillus rhamnosus (Lr JB-1) can modulate behavior and CNS biochemistry in healthy mice via the vagus nerve. This is a very well conducted study on an emerging topic in neurogastroenterology: the role of the microbiota–gut–brain axis in modulation of behavior and mood. We have previously shown that the probiotic Bifidobacterium longum (Bl NCC3001) normalizes behavior and CNS biochemistry (2–4) in mice with mild colitis, an effect also mediated via the vagus nerve (3, 4). Interestingly, both bacteria modulate enteric neuron excitability (3–5), suggesting that enteric …

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission

Significance G protein-coupled receptors (GPCRs) have long been considered to function primarily at the plasma membrane. Consequently, most drugs are designed to target GPCRs at the cell surface. Ligand-bound GPCRs undergo clathrin- and dynamin-dependent endocytosis. It is uncertain whether GPCRs in endosomes control complex pathophysiological processes in vivo and are a viable therapeutic target. We report that the CGRP receptor signals from endosomes to regulate activity of pain-transmitting neurons in the spinal cord. Lipid-conjugated CGRP receptor antagonists accumulate in endosomes, selectively inhibit endosomal signals, and block sustained excitation of spinal neurons and persistent nociception. The results suggest that GPCRs in endosomes, in addition to those at the cell surface, control ongoing pathophysiological processes in vivo and identify GPCRs in endosomes as a new target for therapy. G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8–37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8–37–cholestanol, but not unconjugated CGRP8–37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8–37–cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund’s adjuvant more effectively than unconjugated CGRP8–37. Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Impaired vascular sensitivity to nitric oxide in the coronary microvasculature after endotoxaemia

The effects of endotoxaemia on coronary vasodilator responses to bradykinin (BK), sodium nitroprusside (SNP) and nicardipine were investigated in the rat isolated heart perfused at constant flow ex vivo. Dose‐dependent reductions in coronary perfusion pressure reaching a maximum of 56±3 and 57±5 mmHg were observed for BK and SNP respectively. The BK response was biphasic, consisting of a rapid dilator response that was insensitive to NGnitro‐L‐arginine methyl ester (L‐NAME, 0.1 mM) and a second slower component whose duration was attenuated by L‐NAME. Hearts obtained from rats treated with endotoxin (2.5 mg kg−1, i.p.) for 2 or 6 h had increased basal coronary perfusion pressure and reduced vasodilator responses to BK or SNP. Dilator responses to nicardipine were not affected by endotoxin treatment. In vitro perfusion of hearts from endotoxin‐treated rats with L‐NAME (0.1 mM) restored SNP responses to control values. Treatment with dexamethasone (1 mg kg−1), 1 h before endotoxin did not alter the endotoxin‐induced impairment of dilator responses to BK or SNP. These results show that coronary microvascular responses are altered following endotoxin exposure. Endotoxin results in increased coronary microvascular tone despite induction of NO synthase and inhibits the dilator response to BK and SNP, vasodilators that act via the release of NO. Responses to SNP in endotoxin‐treated hearts were restored to control values in the presence of L‐NAME suggesting that enhanced endogenous NO synthesis might saturate guanylate cyclase resulting in reduced response to NO donors. The reduced response to vasodilators and increased coronary resistance might be important in determining the response of the coronary circulation to systemic inflammation and infection.

Kinin B1 receptors as novel anti-inflammatory targets

The kinin peptides exert their pro-inflammatory effects by the selective activation of two distinct G-protein coupled receptors, termed B1 and B2. The principle kinin peptides involved in the acute inflammatory response are bradykinin (BK) and Lys-BK, which produce their effects via the selective activation of constitutively expressed B2 receptors. The B1 receptor in contrast is not normally expressed, but is induced selectively by certain inflammatory stimuli. Additionally, the B1 receptor is selectively activated by the des-Arg9kinin metabolites, namely des-Arg9BK and Lys-des-Arg9BK, the synthesis of which are also increased during inflammation. Recent investigations into the molecular mechanisms of B1 receptor induction and their distribution and function have shown that, following an inflammatory stimulus, the B1 receptor is induced and may play an important role in the progression of an acute inflammatory response to a more sustained chronic inflammatory state characterised by leukocyte recruitment. This review summarises recent studies on B1 receptor expression and function, and discusses the role of these receptors in regulation of inflammation and their potential as therapeutic targets.

Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2

Diverse proteases cleave protease‐activated receptor‐2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N‐terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin‐S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists.

Tu1797 The Adoptive Transfer of Anxiety and Gut Dysfunction From IBS Patients to Axenic Mice Through Microbiota Transplantation

Studies of the effect of esophageal sensory stimulation on brain functional connectivity using magnetic resonance imaging (fc-MRI) requires insertion of perfusion or balloon catheters. The contribution of the presence of these catheters themselves on observed brain connectivity changes has not been systematically studied. Aim: To determine the effect of the presence of a catheter within the esophagus on a priori selected regional brain connectivity in healthy and patient volunteers. Methods: In 14 controls (7 female, age 27±4 years) and 12 nonerosive reflux disease (NERD) patients (6 female, age 46±5 years), fc-MRI data of amygdala connectivity to the rest of the brain were gathered during two 9-minute scans, one scan without transnasal esophageal intubation and one scan with esophageal intubation. Functional connectivity was quantified by the correlation coefficient (CC) between average fcMRI time series (seeds) in two spherical volumes centered in each of the right and left amygdala and time series of all fc-MRI volume elements (voxels) across the entire brain. The amygdala seeds were centered at the locations identified by the MNI atlas. Group analysis was performed using unpaired t-tests (with cluster size Monte Carlo simulation to correct for multiple comparison) of correlation coefficients to test amygdala-cortical connectivity differences across NERD patients and healthy subjects. Results: Amygdala connectivity was characterized by strong connections to the right and left superior temporal gyrus. The presence of esophageal catheter in the healthy controls resulted in significant differences in both left and right amygdala connectivity to the left superior temporal gyrus (Figure 1, p<0.001, corrected). No significant catheter effect was observed in the NERD patients. Presence of the catheter also effected the between group comparisons showing significant differences in amygdala connectivity. In the resting state without the catheter present, significant differences in both right and left amygdala connectivity to the right superior temporal gyrus were seen between the two groups (p<0,001, corrected). In the presence of catheter, significant differences were limited to the right amygdala connectivity to the right superior temporal gyrus (p<0,001, corrected). Conclusions: Pharyngo-esophageal stimulation by the presence of a catheter alters the functional connectivity of the amygdala in healthy controls. This response is absent in NERD patients which may represent a state of pre-existing maximum neural activity and associated BOLD signal alterations (ceiling effect). Furthermore, the presence of an esophageal catheter can affect the findings of comparative analyses between groups. This possibility needs to be taken into account when interpreting resting and non-resting state connectivity data involving esophageal stimulation.