Mohammed Yaqoob

Anandamide-Evoked Activation of Vanilloid Receptor 1 Contributes to the Development of Bladder Hyperreflexia and Nociceptive Transmission to Spinal Dorsal Horn Neurons in Cystitis

The role of anandamide in the development of inflammatory hyperalgesia and visceral hyperreflexia was studied in the rat urinary bladder. Animals were given intraperitoneal cyclophosphamide injection, which evokes painful hemorrhagic cystitis accompanied by increased bladder reflex activity. The vanilloid receptor 1 [transient receptor potential vanilloid 1 (TRPV1)] antagonist capsazepine, applied onto the serosal surface of bladders, significantly reduced the hyperreflexia. Mass spectrometric analysis revealed that cyclophosphamide injection significantly and persistently increased the anandamide content of bladder tissues. The increase in the anandamide content paralleled the development of reflex hyperactivity. Anandamide (1-100 μm), applied onto the serosal surface of naive bladders, increased the reflex activity in a concentration-dependent manner. Repeated anandamide applications did not produce desensitization of the response. The anandamide-evoked effect was blocked by capsazepine or by instillation of resiniferatoxin, the ultrapotent TRPV1 agonist, into the bladders 24 hr before the anandamide challenge. The cannabinoid 1 receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] significantly increased the potency of anandamide in enhancing bladder reflex activity in naive but not in cyclophosphamide-injected animals. Application of the fatty acid amide hydrolyze inhibitor palmitoylisopropylamine onto the serosal surface of bladders also increased the reflex activity both in naive and cyclophosphamide-injected rats. This latter effect in naive animals was blocked by capsazepine and by resiniferatoxin pretreatment. Finally, intravesical instillation of anandamide (50 μm) increased c-fos expression in the spinal cord, which was reduced by capsazepine or by resiniferatoxin pretreatment. These results suggest that anandamide, through activating TRPV1, contributes to the development of hyperreflexia and hyperalgesia during cystitis.

Activation of capsaicin-sensitive primary sensory neurones induces anandamide production and release.

The inhibitory cannabinoid 1 receptor and the excitatory vanilloid receptor 1, both of which are responsive to the endogenous ligand anandamide, are co‐expressed on a subpopulation of primary sensory neurones. We report that activation of the cannabinoid 1 receptor/vanilloid receptor 1‐co‐expressing primary sensory neurones induces the production and release of anandamide. Application of capsaicin (3 nm−1 µm) to cultured primary sensory neurones evoked calcitonin gene‐related peptide release, which was significantly increased by the selective cannabinoid 1 receptor antagonist, SR141716A (200 nm). Mass spectrometric analyses of the extracellular solution revealed that exposure to capsaicin (10 nm or 100 nm) enhanced the anandamide concentration of the medium from less then 0.05 pmol/µL to more then 2 pmol/µL. Depolarization of the neurones with 50 mm KCl also enhanced the anandamide content of the buffer. Both the capsaicin‐ and KCl‐induced anandamide release depended on extracellular Ca2+. Prolonged treatment of the cultures with capsaicin (10 µm) reduced both the capsaicin‐ and KCl‐induced anandamide release. These findings indicate that activation of capsaicin‐sensitive primary sensory neurones evokes anandamide production and release, and that anandamide might be a key endogenous regulator of the excitability of these neurones.

Antihyperalgesic properties of the cannabinoid CT-3 in chronic neuropathic and inflammatory pain states in the rat.

&NA; CT‐3 (ajulemic acid) is a synthetic analogue of a metabolite of Δ9‐tetrahydrocannabinol that has reported analgesic efficacy in neuropathic pain states in man. Here we show that CT‐3 binds to human cannabinoid receptors in vitro, with high affinity at hCB1 (Ki 6 nM) and hCB2 (Ki 56 nM) receptors. In a functional GTP‐γ‐S assay CT‐3 was an agonist at both hCB1 and hCB2 receptors (EC50 11 and 13.4 nM, respectively). In behavioural models of chronic neuropathic and inflammatory pain in the rat, oral administration of CT‐3 (0.1–1 mg/kg) produced up to 60% reversal of mechanical hyperalgesia. In both models the antihyperalgesic activity was prevented by the CB1‐antagonist SR141716A but not the CB2‐antagonist SR144528. In the tetrad of tests for CNS activity, CT‐3 (1–10 mg/kg, po) produced dose‐related catalepsy, deficits in locomotor performance, hypothermia, and acute analgesia. Comparison of 50% maximal effects in the tetrad and chronic pain assays produced an approximate therapeutic index of 5–10. Pharmacokinetic analysis showed that CT‐3 exhibits significant but limited brain penetration, with a brain/plasma ratio of 0.4 measured following oral administration, compared to ratios of 1.0–1.9 measured following subcutaneous administration of WIN55,212‐2 or Δ9‐THC. These data show that CT‐3 is a cannabinoid receptor agonist and is efficacious in animal models of chronic pain by activation of the CB1 receptor. Whilst it shows significant cannabinoid‐like CNS activity, it exhibits a superior therapeutic index compared to other cannabinoid compounds, which may reflect a relatively reduced CNS penetration.

THIS ARTICLE HAS BEEN RETRACTED Activation of capsaicin‐sensitive primary sensory neurones induces anandamide production and release

The inhibitory cannabinoid 1 receptor and the excitatory vanilloid receptor 1, both of which are responsive to the endogenous ligand anandamide, are co‐expressed on a subpopulation of primary sensory neurones. We report that activation of the cannabinoid 1 receptor/vanilloid receptor 1‐co‐expressing primary sensory neurones induces the production and release of anandamide. Application of capsaicin (3 nm−1 µm) to cultured primary sensory neurones evoked calcitonin gene‐related peptide release, which was significantly increased by the selective cannabinoid 1 receptor antagonist, SR141716A (200 nm). Mass spectrometric analyses of the extracellular solution revealed that exposure to capsaicin (10 nm or 100 nm) enhanced the anandamide concentration of the medium from less then 0.05 pmol/µL to more then 2 pmol/µL. Depolarization of the neurones with 50 mm KCl also enhanced the anandamide content of the buffer. Both the capsaicin‐ and KCl‐induced anandamide release depended on extracellular Ca2+. Prolonged treatment of the cultures with capsaicin (10 µm) reduced both the capsaicin‐ and KCl‐induced anandamide release. These findings indicate that activation of capsaicin‐sensitive primary sensory neurones evokes anandamide production and release, and that anandamide might be a key endogenous regulator of the excitability of these neurones.

Regulation of bradykinin receptor gene expression in human lung fibroblasts.

In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3); interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.

Discovery of Orally Bioavailable Cathepsin S Inhibitors for the Reversal of Neuropathic Pain

Cathepsin S inhibitors are well-known to be an attractive target as immunological therapeutic agents. Recently, our gene expression analysis identified that cathepsin S inhibitors could also be effective for neuropathic pain. Herein, we describe the efficacy of selective cathepsin S inhibitors as antihyperalgesics in a model of neuropathic pain in rats after oral administration.

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2.

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.

Carbohydrate analysis of the B2 bradykinin receptor from rat uterus

Abstract: The B2 bradykinin receptor purified from rat uterus has an apparent molecular mass of 81 kDa. This is higher than the value of 42 kDa estimated from the sequence data of rat and human B2 receptors. Carbohydrate analysis of the rat B2 bradykinin receptor indicated that it was a sialoglycoprotein with three N‐linked complex oligosaccharide side chains. This was consistent with the sequence, which has three potential glycosylation sites. The receptor did not appear to possess O‐linked carbohydrate side chains. Removal of the N‐linked carbohydrates with endo‐β‐N‐acetylglucosaminidase yielded a core protein of 42–44 kDa. The presence of these N‐linked carbohydrates explains the discrepancy between the molecular size of the purified receptor protein and that estimated from the sequence. The sequence of the rat receptor suggests an isoelectric point of about pH 7.0, but the purified receptor had an isoelectric point of pH 4.5–4.7. Sialic acid residues on the N‐linked side chains are likely to be responsible for the acidic nature of the rat receptor. Carbohydrate does not appear to play a role in ligand‐receptor interactions, as deglycosylation did not alter the affinity of the B2 bradykinin receptor for bradykinin or the B2‐selective antagonist HOE‐140.

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts.

1 Binding of the Bj bradykinin receptor radioligand, [3H]‐des‐Arg10‐kallidin (‐KD) and the B2 receptor radioligand [3H]‐bradykinin (‐BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2 One‐site analysis of the saturation data for [3H]‐des‐Arg10‐KD gave an equilibrium dissociation constant (KD) value of 0.51 ± 0.12 nM and a maximum receptor density (Bmax) of 260 ± 49 fmol mg−1 of protein. [3H]‐des‐Arg10‐KD binding was displaced by ligands in the order: des‐Arg10‐KD > KD > > des‐Arg9[Leu8]‐BK > des‐Arg9‐BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3 One‐site analysis of the binding of [3H]‐BK to WI38 membranes indicated that it had a KD value of 0.25 ± 0.06 nM and a Bmax of 753 ± 98 fmol mg−1 of protein. The potencies for displacement of [3H]‐BK binding were: Hoe 140 > > BK = KD > > > des‐Arg10‐KD = des‐Arg9[Leu8]‐BK = des‐Arg9‐BK, which was consistent with binding to B2 receptors. 4 This is the first characterization of [3H]‐des‐Arg10‐KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]‐des‐Arg10‐KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.

Anandamide produced by Ca2+-insensitive enzymes induces excitation in primary sensory neurons

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca2+-sensitive and Ca2+-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca2+-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca2+. We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca2+-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca2+-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca2+-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.