François Nantel

Distribution and regulation of cyclooxygenase-2 in carrageenan-induced inflammation.

We characterized the regulation of cyclooxygenase‐2 (COX‐2) at the mRNA, protein and mediator level in two rat models of acute inflammation, carrageenan‐induced paw ædema and mechanical hyperalgesia. Carrageenan was injected in the hind paw of rat at low (paw ædema) and high doses (hyperalgesia). COX‐2 and prostaglandin E2 (PGE2) levels were measured by RT–PCR and immunological assays. We also determined the distribution of COX‐2 by immunohistochemistry. The injection of carrageenan produced a significant and parallel induction of both COX‐2 and PGE2. This induction was significantly higher in hyperalgesia than in paw ædema. This was probably due to the 9 fold higher concentration of carrageenan used to provoke hyperalgesia. Immunohistochemical examination showed COX‐2 immunoreactivity in the epidermis, skeletal muscle and inflammatory cells of rats experiencing hyperalgesia. In paw ædema however, only the epidermis showed positive COX‐2 immunoreactivity. Pretreatment with indomethacin completely abolished the induction of COX‐2 in paw ædema but not in hyperalgesia. These results suggest that multiple mechanisms regulate COX‐2 induction especially in the more severe model. In carrageenan‐induced paw ædema, prostanoid production have been linked through the expression of the COX‐2 gene which suggest the presence of a positive feedback loop mechanism.

Relationship of EP1‐4 prostaglandin receptors with rat hypothalamic cell groups involved in lipopolysaccharide fever responses

The action of prostaglandin E2 (PGE2) in the preoptic area is thought to play an important role in producing fever. Pharmacologic evidence suggests that, among the four subtypes of E‐series prostaglandin (EP) receptors, i.e., EP1, EP2, EP3, and EP4, the EP1 receptor mediates fever responses. In contrast, evidence from mice with EP receptor gene deletions indicates that the EP3 receptor is required for the initial (<1 hour) fever after intravenous (i.v.) lipopolysaccharide (LPS). To investigate which subtypes of EP receptors mediate systemic infection‐induced fever, we assessed the coexpression of Fos‐like immunoreactivity (Fos‐IR) and EP1‐4 receptor mRNA in nuclei in the rat hypothalamus that have been shown to be involved in fever responses. Two hours after the administration of i.v. LPS (5 μg/kg), Fos‐IR was observed in the ventromedial preoptic nucleus, the median preoptic nucleus, and the paraventricular hypothalamic nucleus. In these nuclei, EP4 receptor mRNA was strongly expressed and the Fos‐IR intensely colocalized with EP4 receptor mRNA. Strong EP3 receptor mRNA expression was only seen within the median preoptic nucleus but Fos‐IR showed little coexpression with EP3 receptor mRNA. EP2 receptor mRNA was not seen in the PGE2 sensitive parts of the preoptic area. Although approximately half of the Fos‐immunoreactive neurons also expressed EP1 receptor mRNA, EP1 mRNA expression was weak and its distribution was so diffuse in the preoptic area that it did not represent a specific relationship. In the paraventricular nucleus, EP4 mRNA was found in most Fos‐immunoreactive neurons and levels of EP4 receptor expression increased after i.v. LPS. Our findings indicate that neurons expressing EP4 receptor are activated during LPS‐induced fever and suggest the involvement of EP4 receptors in the production of fever. J. Comp. Neurol. 428:20–32, 2000.

Immunolocalization of cyclooxygenase-2 in the macula densa of human elderly

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX‐2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX‐1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX‐2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX‐2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.

A novel biological role for prostaglandin D2 is suggested by distribution studies of the rat DP prostanoid receptor.

We report the cloning, functional expression and cell-specific localization of the rat homologue of the prostaglandin D2 receptor (DP). In situ hybridization, utilizing multiple digoxigenin-labelled riboprobes and their complementary sense controls, was performed to determine the detailed distribution of DP receptor mRNA in the central nervous system and the gastrointestinal tract. Within the brain, the leptomeninges and choroid plexus expressed DP receptor mRNA. Transcripts detected in the spinal cord were localized to the sensory and motor neurons of the dorsal and ventral horns, respectively, suggesting a role for the DP receptor in the modulation of central nervous system processes, including pain transmission. Within the gastrointestinal tract (stomach, duodenum, ileum and colon) signals were highly localized to the mucous-secreting goblet cells and the columnar epithelium. These findings suggest a novel biological role for prostaglandin D2-mediated activity at the DP receptor, namely mucous secretion. In addition, radioligand binding assays (saturation analyses and equilibrium competition assays) and functional assays (measuring cAMP accumulation) were performed to characterize the recombinant rat DP receptor expressed in human embryonic kidney (HEK) 293(EBNA) cells. A single site of binding (K(D) = 14 nM, Bmax = 115 fmol/mg protein) was measured for prostaglandin D2-specific binding to the rat DP receptor. Prostaglandin D2 proved to be a potent agonist at the rat DP receptor (EC50 = 5 nM). The rank order of efficacy for DP receptor specific agonists [prostaglandin D2 = prostaglandin J2 = BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)) > L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)] reflected the affinity with which the ligands bound to the receptor.

Expression of prostaglandin D synthase and the prostaglandin D2 receptors DP and CRTH2 in human nasal mucosa

Abstract Background : Prostaglandin D 2 (PGD 2 ) is released from mast cells during the allergic response. Objective : Since PGD 2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic prostaglandin D synthase (PGDS) and the two PGD 2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. Methods : DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression were revealed by immunohistochemistry. Results : In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: (1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, (2) both DP mRNA and CRTH2 were detected in eosinophils and (3) CRTH2 was detected on a subset of infiltrating T cells. Although DP mRNA could not be detected in the T cells invading the nasal mucosa, it was found to be expressed in the T cells present in the lymph node and the thymus from normal individuals. Conclusion : This study indicates that cells capable of producing PGD 2 are present in the nasal mucosa and that both PGD 2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.

Localization of phosphodiesterase-4 isoforms in the medulla and nodose ganglion of the squirrel monkey

Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.

Cellular distribution of prostanoid EP receptors mRNA in the rat gastrointestinal tract.

The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.

Enhanced dermal and retinal vascular permeability in streptozotocin-induced type 1 diabetes in Wistar rats: blockade with a selective bradykinin B1 receptor antagonist.

The vascular complications associated with type 1 diabetes are to some extent related to the dysfunction of the endothelium leading to an increased vascular permeability and plasma extravasation in the surrounding tissues. The various micro- and macro-vascular complications of diabetes develop over time, leading to nephropathy, retinopathy and neuropathy and cardiomyopathy. In the present study, the effect of a novel selective bradykinin B1 receptor (BKB1-R) antagonist, R-954, was investigated on the changes of vascular permeability in the skin and retina of streptozotocin (STZ)-induced type 1 diabetic rats. Plasma extravasation increased in the skin and retina of STZ-diabetic rats after 1 week and persisted over 4 weeks following STZ injection. Acute treatment with R-954 (2 mg/kg, bolus s.c.) highly reduced the elevated vascular permeability in both 1- and 4-week STZ-diabetic rats. These results showed that the inducible BKB1-R subtype modulates the vascular permeability of the skin and retina of type 1 diabetic rats and suggests that BKB1-R antagonists could have a beneficial role in diabetic neuropathy and retinopathy.

The kinin system mediates hyperalgesia through the inducible bradykinin B1 receptor subtype: evidence in various experimental animal models of type 1 and type 2 diabetic neuropathy.

Abstract Both insulin-dependent (type 1) and insulin-independent (type 2) diabetes are complex disorders characterized by symptomatic glucose intolerance due to either defective insulin secretion, insulin action or both. Unchecked hyperglycemia leads to a series of complications among which is painful diabetic neuropathy, for which the kinin system has been implicated. Here, we review and compare the profile of several experimental models of type 1 and 2 diabetes (chemically induced versus gene-prone) and the incidence of diabetic neuropathy upon aging. We discuss the efficacy of selective antagonists of the inducible bradykinin B1 receptor (BKB1-R) subtype against hyperalgesia assessed by various nociceptive tests. In either gene-prone models of type 1 and 2 diabetes, the incidence of hyperalgesia mostly precedes the development of hyperglycemia. The administration of insulin, achieving euglycemia, does not reverse hyperalgesia. Treatment with a selective BKB1-R antagonist does not affect basal nociception in most normal control rats, whereas it induces a significant time- and dose-dependent attenuation of hyperalgesia, or even restores nociceptive responses, in experimental diabetic neuropathy models. Diabetic hyperalgesia is absent in streptozotocin-induced type 1 diabetic BKB1-R knockout mice. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of painful diabetic neuropathy.

Inhibition of Type 1 Diabetic Hyperalgesia in Streptozotocin-Induced Wistar versus Spontaneous Gene-Prone BB/Worchester Rats: Efficacy of a Selective Bradykinin B1 Receptor Antagonist

Insulin-dependent type 1 diabetes (T1D) is linked to a series of complications, including painful diabetic neuropathy (PDN). Several neurovascular systems are activated in T1D, including the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype. We assessed and compared the efficacy profile of a selective BKB1-R antagonist on hyperalgesia in 2 models of T1D: streptozotocin (STZ) chemically induced diabetic Wistar rats and spontaneous BioBreeding/Worchester diabetic-prone (BB/Wor-DP) rats. Nociception was measured using the hot plate test to determine thermal hyperalgesia. STZ diabetic rats developed maximal hyperalgesia (35% decrease in their hot plate reaction time) within a week and remained in such condition and degree for up to 4 weeks postinjection. BB/Wor-DP rats also developed hyperalgesia over time that preceded hyperglycemia, starting at the age of 6 weeks (9% decrease in the hot plate reaction time) and stabilizing over the age of 16 to 24 weeks to a maximum (60% decrease in the hot plate reaction time). Single, acute subcutaneous administration of the selective BKB1-R antagonist induced significant time- and dose-dependent attenuation of hyperalgesia in both STZ diabetic and BB/Wor-DP rats. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of PDN.