Hepta and octapeptide agonists of protease-activated receptor 2.

Protease-activated receptor 2 (PAR(2)) is a G protein-coupled cell surface receptor for trypsin-like enzymes. Proteolytic cleavage at a specific site in the extracellular N-terminus exposes a receptor-activating sequence, the 'tethered ligand', which binds intramolecularly to initiate receptor signalling. Peptide or small molecule agonists for PAR(2), devoid of the non-specific and proteolytic effects of enzyme activators, may be promising therapeutic agents for proliferative and inflammatory diseases reportedly mediated by PAR(2). Synthetic hexapeptides that correspond to the native tethered ligand of human or rodent PAR(2) (SLIGKV and SLIGRL, respectively) can activate the receptor independently of proteolytic cleavage; however, known peptide agonists have much lower potency compared to protease-mediated activation. Here, we investigated the agonist activity of 94 hepta and octapeptide derivatives of the human and rodent PAR(2)-tethered ligand sequences in human airway epithelial (A549) cells which endogenously express PAR(2). Thirty synthetic peptides were found to be as potent as or more potent than SLIGRL on the basis of intracellular Ca(2+) responses. The more active peptide agonists were also examined for agonist cross-reactivity at PAR(1) in Chinese Hamster Ovary (CHO) cells that endogenously express functional PAR(1) but not PAR(2). Two potent and PAR(2)-selective agonists were further examined for their capacity to relax phenylephrine-contracted rat aortic rings. Our findings reveal an important role for carboxyl extensions to native PAR(2) activating peptides in potentiating agonist activity.

Initial support for the hypothesis that PAR2 is involved in the immune response to Nippostrongylus brasiliensis in mice.

Protease-activated receptor 2 (PAR(2)) is a cell surface receptor that detects trypsin and trypsin-like enzymes. Although the precise pathophysiological roles of PAR(2) are yet to be determined, the receptor has been broadly implicated in inflammation and allergy. However, no studies have investigated the possible roles of PAR(2) in hosts infected by parasitic helminths. Therefore, in this preliminary investigation, we compared the infectivity of the nematode Nippostrongylus brasiliensis in mice lacking the PAR(2) gene (PAR2-/- ) and in their 'background-strain' controls (129SV). PAR2-/- mice displayed elevated fecal egg counts and decreased levels of total serum IgE, after a subcutaneous infection with 900 infective third-stage N. brasiliensis larvae compared with 129SV mice that were not susceptible to infection. In addition, in a separate study in BALB/c mice, two immunological hallmarks of parasite infection, IgE- and IL-10-expressing lymphocytes, were shown to be augmented after the coadministration of the classic antigen ovalbumin with the PAR(2)-activating peptide SLIGRL (single letter amino acid sequence) but not the inactive reverse peptide LRGILS. These findings provide initial support for the proposal that PAR(2) is a recognition receptor for nematode-derived proteases.

Regulation of serotonin 5-HT2C receptors by chronic ligand exposure.

The effect of ligand pretreatment on human 5-hydroxytryptamine2C (5-HT2C) receptors was examined in CHO cells expressing high (CHO-1C7; 67+/-3 pmol/mg) or low (CHO-1C19; 72+/-10 fmol/mg) levels of the receptor. Seventy-two hours pretreatment of CHO-1C7 cells with various ligands did not affect receptor expression. Pretreatment with inverse agonists enhanced 5-HT-mediated inositol phosphate accumulation with no change in constitutive receptor activity. The enhanced agonist responsiveness was inversely correlated with the intrinsic activity of the pretreatment ligand. Seventy-two hours of pretreatment with the weak agonist, 5-methoxygramine, caused an elevation in constitutive activity but no alteration in 5-HT-mediated signaling. In CHO-1C19 cells, 24 but not 72 h of pretreatment with the inverse agonist mianserin enhanced 5-HT-mediated signaling, with no effect on basal signaling; pretreatment with 5-methoxygramine had no significant effect. These findings highlight differences in the pattern of chronic regulation of 5HT2C receptor signaling between high and low receptor expression levels in a common cellular background.

Acute effects of L- and T-type calcium channel antagonists on cardiovascular reflexes in conscious rabbits.

1. The effects of the relatively selective T-type voltage- operated calcium channel (VOCC) antagonist mibefradil were compared with verapamil, an L-type VOCC antagonist, on a range of autonomic reflexes in conscious rabbits. 2. Mean arterial pressure (MAP), heart rate (HR), the baroreceptor-HR reflex, postural adaptation reflex (90 degrees head-up tilt), Bezold-Jarisch-like reflex and the vasoconstrictor component of the nasopharyngeal reflex were assessed before and during i.v. infusion of vehicle (saline), mibefradil or verapamil. Doses of mibefradil that gave low (M1; 0.45 +/- 0.02 microg/mL) and high (M2; 0.93 +/- 0.05 microg/mL) plasma concentrations, or verapamil (0.059 +/- 0.004 microg/mL; n = 6 each) were chosen to mimic clinically observed therapeutic levels. 3. At steady state infusion over 30-90 min, MAP was significantly lower in M2 (- 7 mmHg) and verapamil (- 6 mm Hg) treatments, but only verapamil caused a significant tachycardia (+ 31 b.p.m.) compared with vehicle. Mibefradil (M2) and verapamil decreased the HR range of the baroreflex by 27 and 29%, respectively, but neither treatment affected the vagal or sympathetic constrictor components of the Bezold-Jarisch-like and nasopharyngeal reflexes, respectively. 4. In response to 90 degrees tilt, vehicle- and verapamil-treated rabbits responded with small rises in MAP of 4 +/- 2 and 8 +/- 2 mm Hg, respectively, 5 s into the upright posture, while M1 and M2 caused falls in MAP of 6 +/- 4 and 9 +/- 3 mm Hg, respectively, at 5 s. 5. Thus, both L- and T-type VOCC antagonists, at plasma concentrations in the clinical range, lowered MAP in the conscious rabbit, but only mibefradil caused postural hypotension. We conclude that T-type VOCC may play an important role in the venoconstrictor reflex in response to tilt in the rabbit.

Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum.

Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat cerebellar membranes was investigated using radioligand binding. In competition against the CB1 antagonist, [3 H]SR141716A, the agonist, WIN 55,212-2 yielded a biphasic isotherm. The majority of binding was to a high-affinity state that was significantly reduced by the GTP analogue, Gpp(NH)p. Interestingly, 5-HT enhanced the high-affinity binding constant of WIN 55,212-2 while attenuating the proportion of high-affinity binding. 5-HT also significantly reduced the proportion of high-affinity binding of the cannabinoid agonist, HU 210, but had no effect on the agonist, CP 55,940. The effect of 5-HT on WIN 55,212-2 binding was inhibited by the 5-HT2 receptor antagonist ritanserin as well as Gpp(NH)p, suggesting a dependence on the 5-HT2 receptor and on G protein-receptor interactions, respectively. Subsequent [3 H]WIN 55,212-2 dissociation kinetic experiments revealed that 5-HT promoted a slower-dissociating species of radiolabelled agonist-receptor complex. Our findings support a membrane-delimited cross-talk between two G protein-coupled receptors that are co-localized in certain cells of the central nervous system. Intriguingly, the cannabinoid agonist dependence of the 5-HT modulatory effect suggests that agonist-specific conformations of the CB1 receptor may also be important in determining the extent of this cross-talk.