Réjean Couture

Kinin receptors in pain and inflammation

Kinins are among the most potent autacoids involved in inflammatory, vascular and pain processes. These short-lived peptides, including bradykinin, kallidin and T-kinin, are generated during tissue injury and noxious stimulation. However, emerging evidence also suggests that kinins are stored in neuronal elements of the central nervous system (CNS) where they are thought to play a role as neuromediators in various cerebral functions, particularly in the control of nociceptive information. Kinins exert their biological effects through the activation of two transmembrane G-protein-coupled receptors, denoted bradykinin B(1) and B(2). Whereas the B(2) receptor is constitutive and activated by the parent molecules, the B(1) receptor is generally underexpressed in normal tissues and is activated by kinins deprived of the C-terminal Arg (des-Arg(9)-kinins). The induction and increased expression of B(1) receptor occur following tissue injury or after treatment with bacterial endotoxins or cytokines such as interleukin-1 beta and tumor necrosis factor-alpha. This review summarizes the most recent data from various animal models which convey support for a role of B(2) receptors in the acute phase of the inflammatory and pain response, and for a role of B(1) receptors in the chronic phase of the response. The B(1) receptor may exert a strategic role in inflammatory diseases with an immune component (diabetes, asthma, rheumatoid arthritis and multiple sclerosis). New information is provided regarding the role of sensory mechanisms subserving spinal hyperalgesia and intrapleural neutrophil migration that occur upon B(1) receptor activation in streptozotocin-treated rats, a model of insulin-dependent diabetes mellitus in which the B(1) receptor seems to be rapidly overexpressed. Although it is widely accepted that the blockade of kinin receptors with specific antagonists could be of benefit in the treatment of somatic and visceral inflammation and pain, recent molecular and functional evidence suggests that the activation of B(1) receptors with an agonist may afford a novel therapeutic approach in the CNS inflammatory demyelinating disorder encountered in multiple sclerosis by reducing immune cell infiltration (T-lymphocytes) into the brain. Hence, the B(1) receptor may exert either a protective or detrimental effect depending on the inflammatory disease. This dual function of the B(1) receptor deserves to be investigated further.

New selective agonists for neurokinin receptors: pharmacological tools for receptor characterization

Abstract The neurokinin system is replete with a multiplicity of endogenous ligands (substance P, neurokinin A, neurokinin B) and their receptors (NK 1 , NK 2 and NK 3 ). Neurokinin receptors have previously been studied almost exclusively with natural — but non-selective — agonists. However, new selective agonists have now been developed . Domenico Regoli and colleagues describe how such compounds have been successfully used in vitro to characterize the responses of peripheral organs to neurokinins, and in vivo to elucidate possible physiological roles of the neurokinin system .

Nuclear Retention of MBP mRNAs in the Quaking Viable Mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.

Characterization of the effects produced by neurokinins and three agonists selective for neurokinin receptor subtypes in a spinal nociceptive reflex of the rat.

In the awake restrained rat the intrathecal (i.th.) administration of 6.5 pmol-40 nmol of substance P (SP), neurokinin A (NKA) or one of two selective NK-1 receptor agonists [Pro9, Met(O2)11]SP, denoted ana1 and [beta-Ala4, Sar9, Met(O2)11]SP , denoted ana2 decreased reaction time (RT) to a noxious radiant heat stimulus in a dose-related manner. The following rank order of potency was observed in relation to this response: ana1 = ana2 greater than SP much greater than NKA. The decrement of tail-flick latency was greatest at 1 min and RT returned to the basal level within 6-11 min post-administration. However, in some rats SP produced a small increase in RT (anti-nociception) at 6-11 min post-administration. The i.th. administration of neurokinin B (NKB) or a selective NK-3 receptor agonist [beta-Asp4, MePhe7]NKB), denoted ana3 induced an antinociceptive effect which was greatest at 1 min and lasted less than 11 min after NKB or more than 30 min after ana3 administration. The magnitude of the increase in RT produced by 65 pmol-40 nmol doses of these peptides is ana3 much greater than NKB much greater than SP. The effect of NKB (8.0 nmol) was significantly blocked (P less than 0.005) by prior i.th. administration of naloxone (opioid antagonist) but not by idazoxan (alpha 2-adrenoceptor antagonist), [Thi5,8, D-Phe7]BK (kinin antagonist), or following bilateral adrenalectomy. From these results, we conclude that NKB-induced antinociception is mediated by the spinal release of an opioid and not through a BK or NA mechanism. The results also suggest that the nociceptive and antinociceptive effects of neuro-kinins are mediated by the activation of NK-1 and NK-3 receptor subtypes respectively, in the rat spinal cord.

Development of digoxigenin-labeled peptide: Application to chemiluminoenzyme immunoassay of bradykinin in inflamed tissues

A new ultrasensitive chemiluminoenzyme immunoassay (CLEIA) using digoxigenin-labeled bradykinin (BK) as a tracer is proposed to quantify kinins in tissue samples. Rabbit polyclonal IgGs anti-BK directed against the C-terminal end were used for the immunoconcentration step along with dioxetane derivative for the revelation step. The sensitivity of the assay for BK was 0.1 fmol/ml with ED50 of 0.78 pmol/ml. This method was applied on extracts of normal and carrageenan-inflamed tissues. The edema produced by the injection of carrageenan in rat hindpaws was associated with a sevenfold increase of immunoreactive kinins in the inflamed paw extract (from 0.021 +/- 0.007 to 0.141 +/- 0.021 pmol/g tissue; p < 0.01), the immunoreactivity corresponded to BK, kallidin, and T-kinin after HPLC separation. When a mixture of inhibitors of kininase I (mergepta) and kininase II (captopril) was coinjected with carrageenan, the carrageenan-induced edema was unaffected but the kinin tissue content was significantly enhanced (0.207 +/- 0.003 pmol/g tissue; p < 0.01). However, the kinin tissue content and the edema response were unaltered by inhibitors given separately. Hence, this highly sensitive assay provides a biochemical evidence that kinins may act as proinflammatory mediators, and highlights a compensatory increase of kininase I and II activities in inflamed tissues.

Cardiovascular and behavioural effects of centrally administered tachykinins in the rat: characterization of receptors with selective antagonists

1 The effects of intracerebroventricular (i.c.v.) injection of selective and potent NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 486, [Trp7, β‐Ala8]NKA(4–10)) receptor antagonists were assessed on the cardiovascular and behavioural responses elicited by the i.c.v. injection of substance P (SP), neurokinin A (NKA) or [MePhe7]neurokinin B ([MePhe7]NKB) in the conscious freely moving rat. 2 SP, NKA and [MePhe7]NKB (5–650 pmol) evoked dose‐dependent increases in mean arterial blood pressure (MAP) and heart rate (HR) with the rank order of potency SP > NKA > [MePhe7]NKB. The cardiovascular responses were accompanied by excessive face washing, grooming and wet dog shakes. 3 The cardiovascular effects and face washing behaviour induced by SP (25 pmol) were significantly reduced by the pre‐injection (i.c.v., 5 min earlier) of RP 67580 (6.5 nmol). However, this antagonist failed to affect the central effects of 25 pmol NKA or [MePhe7]NKB. 4 The cardiovascular and behavioural responses (except for wet dog shakes) elicited by NKA (25 pmol) were significantly reduced by 6.5 nmol SR 48968. However, the latter antagonist had no effect on the SP or [MePhe7]NKB‐mediated responses. 5 Both cardiovascular and behavioural effects produced by either SP or NKA (25 pmol) were completely abolished when rats were pretreated with a combination of RP 67580 (6.5 nmol) and SR 48968 (6.5 nmol), yet this combination of antagonists failed to modify the central effects of [MePhe7]NKB. 6 R 486 (6.5 nmol) inhibited the cardiovascular effects as well as wet dog shakes produced by [MePhe7]NKB, but it was inactive against the responses induced by either SP or NKA. 7 None of the tachykinin receptor antagonists or agonists caused motor impairment or respiratory distress. All antagonists blocked in a reversible manner and were devoid of intrinsic activity except R 486 (6.5 nmol) which produced a transient increase of MAP and HR. 8 These results suggest that the central effects of SP, NKA and [MePhe7]NKB are primarily mediated by central NK1, NK2 and NK3 receptors, respectively. However, a minor activation of NK2 receptors by SP and NK1 receptors by NKA was seen during blockade of both receptors. This study therefore supports the existence of functional NK1, NK2 and NK3 receptors in the adult rat brain.

Key role for spinal dorsal horn microglial kinin B1 receptor in early diabetic pain neuropathy

BackgroundThe pro-nociceptive kinin B1 receptor (B1R) is upregulated on sensory C-fibres, astrocytes and microglia in the spinal cord of streptozotocin (STZ)-diabetic rat. This study aims at defining the role of microglial kinin B1R in diabetic pain neuropathy.MethodsSprague-Dawley rats were made diabetic with STZ (65 mg/kg, i.p.), and 4 days later, two specific inhibitors of microglial cells (fluorocitrate, 1 nmol, i.t.; minocycline, 10 mg/kg, i.p.) were administered to assess the impact on thermal hyperalgesia, allodynia and mRNA expression (qRT-PCR) of B1R and pro-inflammatory markers. Spinal B1R binding sites ((125I)-HPP-desArg10-Hoe 140) were also measured by quantitative autoradiography. Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1. Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations.ResultsSTZ-diabetic rats displayed significant tactile and cold allodynia compared with control rats. Intrathecal or peripheral blockade of B1R or inhibition of microglia reversed time-dependently tactile and cold allodynia in diabetic rats without affecting basal values in control rats. Microglia inhibition also abolished thermal hyperalgesia and the enhanced allodynia induced by intrathecal des-Arg9-BK without affecting hyperglycemia in STZ rats. The enhanced mRNA expression (B1R, IL-1β, TNF-α, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%.ConclusionThe upregulation of kinin B1R in spinal dorsal horn microglia by pro-inflammatory cytokines is proposed as a crucial mechanism in early pain neuropathy in STZ-diabetic rats.

Effects of captopril and Icatibant on bradykinin (BK) and des [Arg9] BK in carrageenan-induced edema.

The effects of captopril, an angiotensin-converting enzyme inhibitor (ACEI), and a selective B2 kinin receptor antagonist (Icatibant) were examined on the paw edema and tissue contents of bradykinin (BK) and des[Arg9]BK following the intraplantar injection of carrageenan in rats. To this end, BK-like immunoreactivity (BK-LI) and des[Arg9]BK-LI were measured with highly sensitive and specific chemiluminescent enzyme immunoassays. Because pentobarbital significantly reduced the carrageenan-induced edema between 3 and 8 h, experiments were conducted in conscious rats. Icatibant (32.5 nmol/paw; intraplantar) significantly reduced carrageenan-induced paw edema between 3 and 8 h in captopril-untreated rats and at 1 and 3 h in captopril-treated rats (0.2 mg/kg x 5 days, per os). The paw content of BK-LI was increased 10-fold in captopril-untreated and 29-fold in captopril-treated rats 1 h after carrageenan injection. In parallel, des[Arg9]BK-LI was increased 8-fold in captopril-untreated and 24-fold in captopril-treated rats. Icatibant prevented the maximal increases in BK-LI and des[Arg9]BK-LI induced by carrageenan. It is concluded that inhibition of ACE by captopril enhanced the early production of endogenous BK and the edema formation induced by carrageenan through a B2 receptor-mediated mechanism. However, the B2 receptor does not appear to be involved in the late phase of the inflammatory response (from 5 to 24 h) to carrageenan in rats pretreated with ACEI. Although the concentrations of des[Arg9]BK were greater than those of BK, it is unlikely that B1 receptors play a significant role in this model of carrageenan-induced edema.

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs

1 Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des‐Arg9‐BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]‐des‐Arg9‐BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2 Des‐Arg9‐BK (0.05–3.3 nmol kg−1) produced dose‐dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg−1. The vasodepressor effects evoked by des‐Arg9‐BK (0.6 nmol kg−1) and BK (0.2 nmol kg−1) were greater after i.v. and i.a. injections, respectively. 3 The vasodepressor response to BK (0.6 nmol kg−1) but not to des‐Arg9‐BK (0.6 nmol kg−1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4 The vasodepressor response to des‐Arg9‐BK (0.6 nmol kg−1) but not to BK (0.6 nmol kg−1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]‐des‐Arg9‐BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5 Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg−1, i.v.) but not that to BK or des‐Arg9‐BK (0.6 nmol kg−1). 6 These results suggest, firstly, that the vasodepressor responses to i.v. BK and des‐Arg9‐BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins. These findings provide pharmacological evidence for the existence of functionally active B1 receptors in canine cardiovascular homeostasis.

Use of non-peptide tachykinin receptor antagonists to substantiate the involvement of NK1 and NK2 receptors in a spinal nociceptive reflex in the rat

Non-peptide antagonists of the NK1 and NK2 receptors were tested as inhibitors of the reaction time in the rat tail-flick and on the decrease of reaction time induced by the intrathecal injection of the NK1 receptor selective agonist [Sar9,Met(O2)11]SP or of the NK2 selective agonist NKA-(4-10). The decrease in reaction time produced by the NK1 agonist lasted less than 11 min while that evoked by the NK2 agonist persisted 26 min after injection. When given intrathecally, CP-96,345 and its chloro analog, Cl-CP, blocked dose-dependently both the behavioral responses and the decreases of reaction time induced by 6.5 nmol of [Sar9,Met(O2)11]SP while they failed to modify the hyperalgesic response to 6.5 nmol NKA-(4-10); CP-96,345 was found more potent than Cl-CP and was also active as an antagonist when given intravenously. In contrast, SR 48968 (6.5 and 65 nmol) blocked the NKA-(4-10)-induced decreases in reaction time and was inactive against the hyperalgesic effect of [Sar9,Met(O2)11]SP. The three antagonists blocked in a reversible manner, were inactive on their own on reaction time and non-toxic. The results indicate that the non-peptide CP-96,345 readily crosses the blood brain barrier and acts as a selective antagonist on spinal NK1 receptors, while SR 48968 is selective on NK2 receptors in the rat spinal cord. Hence, CP-96,345 and SR 48968 highlight a functional role of NK1 and NK2 receptors in spinal sensory neurotransmission.