Profiling of how nociceptor neurons detect danger - new and old foes.

The host evolves redundant mechanisms to preserve physiological processing and homeostasis. These functions range from sensing internal and external threats, creating a memory of the insult and generating reflexes, which aim to resolve inflammation. Impairment in such functioning leads to chronic inflammatory diseases. By interacting through a common language of ligands and receptors, the immune and sensory nervous systems work in concert to accomplish such protective functions. Whilst this bidirectional communication helps to protect from danger, it can contribute to disease pathophysiology. Thus, the somatosensory nervous system is anatomically positioned within primary and secondary lymphoid tissues and mucosa to modulate immunity directly. Upstream of this interplay, neurons detect danger, which prompts the release of neuropeptides initiating (i) defensive reflexes (ranging from withdrawal response to coughing) and (ii) chemotaxis, adhesion and local infiltration of immune cells. The resulting outcome of such neuro-immune interplay is still ill-defined, but consensual findings start to emerge and support neuropeptides not only as blockers of TH 1-mediated immunity but also as drivers of TH 2 immune responses. However, the modalities detected by nociceptors revealed broader than mechanical pressure and temperature sensing and include signals as various as cytokines and pathogens to immunoglobulins and even microRNAs. Along these lines, we aggregated various dorsal root ganglion sensory neuron expression profiling datasets supporting such wide-ranging sensing capabilities to help identifying new danger detection modalities of these cells. Thus, revealing unexpected aspects of nociceptor neuron biology might prompt the identification of novel drivers of immunity, means to resolve inflammation and strategies to safeguard homeostasis.

A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Sorption of arsenite, arsenate, and thioarsenates to iron oxides and iron sulfides: a kinetic and spectroscopic investigation.

Sorption to iron (Fe) minerals determines the fate of the toxic metalloid arsenic (As) in many subsurface environments. Recently, thiolated As species have been shown to dominate aqueous As speciation under a range of environmentally relevant conditions, thus highlighting the need for a quantitative understanding of their sorption behavior. We conducted batch experiments to measure the time-dependent sorption of two S-substituted arsenate species, mono- and tetrathioarsenate, and compared it to the sorption of arsenite and arsenate, in suspensions containing 2-line ferrihydrite, goethite, mackinawite, or pyrite. All four As species strongly sorbed to ferrihydrite. For the other sorbents, binding of the thiolated As species was generally lower compared to arsenate and arsenite, with the exception of the near instantaneous and complete sorption of monothioarsenate to pyrite. Analysis of the X-ray absorption spectroscopy (XAS) spectra of sorbed complexes implied that monothioarsenate binds to Fe oxides as a monodentate, inner-sphere complex. In the presence of Fe sulfides, mono- and tetrathioarsenate were both unstable and partially reduced to arsenite. Adsorption of the thiolated As species to the Fe sulfide minerals also caused the substitution of surface sulfur (S) atoms by As and the formation of As-Fe bonds.

Blockade of kinin B(1) receptor reverses plasma fatty acids composition changes and body and tissue fat gain in a rat model of insulin resistance.

AIM: Kinin B(1) receptor (B(1) R) contributes to insulin resistance through a mechanism involving oxidative stress. This study examined the effect of B(1) R blockade on the changes in plasma fatty acids composition, body and tissue fat mass and adipose tissue inflammation that influence insulin resistance. METHODS: Sprague-Dawley rats were fed with 10% D-glucose or tap water (Control) for 13 weeks and during the last week, rats were administered the B(1) R antagonist SSR240612 (10 mg/kg/day, gavage) or vehicle. The following parameters were assessed: plasma fatty acids (by gas chromatography), body composition (by EchoMRI), metabolic hormone levels (by radioimmunoassay), expression of B(1) R and inflammatory markers in adipose tissue (by Western blot and qRT-PCR). RESULTS: Glucose feeding significantly increased plasma levels of glucose, insulin, leptin, palmitoleic acid (16:1n-7), oleic acid (18:1n-9), Δ6 and Δ9 desaturases while linoleic acid (18:2n-6), arachidonic acid (20:4n-6) and Δ5 desaturase were decreased. SSR240612 reduced plasma levels of insulin, glucose, the homeostasis model assessment index of insulin resistance, palmitoleic acid and n-7 family. Alterations of Δ5, Δ6 and Δ9 desaturases were normalized by SSR240612. The B(1) R antagonist also reversed the enhancing effect of glucose feeding on whole body and epididymal fat mass and on the expression of macrophage CD68, interleukin-1ß, tumour necrosis factor-α and inducible nitric oxide synthase in retroperitoneal adipose tissue. B(1) R protein and mRNA were not detected in retroperitoneal adipose tissue. CONCLUSION: Insulin resistance in glucose-fed rats is associated with low state inflammation in adipose tissue and plasma fatty acids changes which are reversed by B(1) R blockade. These beneficial effects may contribute to insulin sensitivity improvement and the prevention of obesity.

Perceived psychosocial benefits associated with perceived restorative potential of wilderness river-rafting trips.

Analysis of the restorative experiences and psychosocial benefits of wilderness river rafting trips of varying difficulty with 186 Canadian participants of different ages supported the restorative potential of natural settings for all age groups as measured by the Perceived Restorativeness Scale. The two-factor structure (General Restorativeness and Coherence) was confirmed. Significant associations were found between scores on the General Restorative subscale and perceived psychosocial benefits (relaxation, nature appreciation or kinship, and physical fitness or achievement) and positive affect. However, the findings associated with the Coherence subscale were not conclusive.

Retinal plasma extravasation in streptozotocin-diabetic rats mediated by kinin B(1) and B(2) receptors.

BACKGROUND AND PURPOSE: We investigated whether or not kinin receptors play a role in diabetic blood-retinal barrier breakdown, which is a leading cause of vision loss. EXPERIMENTAL APPROACH: Blood-retinal barrier breakdown was quantified using Evans blue, and expression of kinin B(1) receptor mRNA was measured using quantitative reverse transcrition-PCR. Diabetic rats (streptozotocin (STZ), 65 mg kg(-1)) received a single intraocular injection of bradykinin (BK) or des-Arg(9)-BK, alone, or in combination with antagonists for B(1) (des-Arg(10)-Hoe140, R-715) and/or B(2) (Hoe140) receptors, given intraocularly or intravenously (i.v.). KEY RESULTS: In control rats, BK (0.1-10 nmol) dose-dependently increased plasma extravasation, which was inhibited by Hoe140 (0.2 nmol), whereas des-Arg(9)-BK (0.1 and 1 nmol) was without effect. B(1) receptor mRNA was markedly increased in retinas of diabetic rats, and this was prevented by N-acetyl-L-cysteine (1 g kg(-1) day(-1) for 7 days). Plasma extravasation in retinas of STZ-diabetic rats was higher than in controls and enhanced by des-Arg(9)-BK. Response to des-Arg(9)-BK was inhibited by intraocular or i.v. injection of B(1) receptor antagonists. Diabetes-induced plasma extravasation was inhibited only by a combination of des-Arg(10)-Hoe140 and Hoe 140 (100 nmol kg(-1), i.v. 15 min earlier) or by R-715 (1 micromol kg(-1), i.v.) injected daily for 7 days. CONCLUSIONS AND IMPLICATIONS: Kinin B(1) receptors are upregulated in retinas of STZ-diabetic rats through a mechanism involving oxidative stress. Both kinin B(1) and B(2) receptors contribute to increased plasma extravasation in diabetic retinopathy. Chronic inhibition of both kinin receptors, possibly with antioxidant adjuvants, may be a novel therapeutic strategy for diabetic retinopathy.

Role of kinin B1 and B2 receptors in a rat model of neuropathic pain.

Kinin B1 and B2 receptor (R) gene expression (mRNA) is increased in the sensory system after peripheral nerve injury. This study measured the densities of B1R and B2R binding sites in the spinal cord and dorsal root ganglia (DRG) by quantitative autoradiography, and evaluated the effects of two selective non-peptide antagonists at B1R (LF22-0542) and B2R (LF16-0687) on pain behavior after partial ligation of the left sciatic nerve. Increases of B1R binding sites were seen in superficial laminae of the ipsi- and contralateral spinal cord at 2 and 14 days while B2R binding sites were increased on the ipsilateral side at 2 days and on both sides at 14 days. In DRG, B1R and B2R binding sites were significantly increased at 2 days (ipsilateral) and 14 days on both sides. Whereas tactile allodynia started to develop progressively from 2 to 25 days post-ligation, the occurrence of cold allodynia and thermal hyperalgesia became significant from day 8 and day 14 post-ligation, respectively. At day 21 after sciatic nerve ligation, thermal hyperalgesia was blocked by LF22-0542 (10 mg/kg, s.c.) and LF16-0687 (3 mg/kg, s.c.), yet both antagonists had no effect on tactile and cold allodynia. Data highlight the implication of both kinin receptors in thermal hyperalgesia but not in tactile and cold allodynia associated with peripheral nerve injury. Hence LF22-0542 and LF16-0687 present therapeutic potential for the treatment of some aspects of neuropathic pain.

Hemodynamic and renal involvement of B1 and B2 kinin receptors during the acute phase of endotoxin shock in mice.

B1 kinin receptor (B1R) is up-regulated by endotoxins and thus may represent a therapeutic target in sepsis. We investigated the expression and role of B1R and B2R in the acute phase of lipopolysaccharide (LPS)-induced endotoxin shock in C57BL/6 mice (WT) and B1R and B2R knock out mice (B1KO, B2KO). B1R mRNA was enhanced from 6 to 48 h after LPS while B2R mRNA was further increased in B1KO. Maximal hypotension was found 24 h after LPS, and was more pronounced in B1KO, but was reduced in B2KO. Glomerular filtration rate was more reduced by LPS in B1KO than in WT and B2KO. Glycemia was reduced by LPS and particularly in B1KO and B2KO mice. Mortality was increased by LPS in B1KO. These data suggest that the up-regulated B1R plays, at least transiently, a significant beneficial role in acute LPS-induced hypotension. Conversely, supra activation of B2R could be also involved in the increased mortality observed in B1KO mice.

The kinin B1 receptor antagonist SSR240612 reverses tactile and cold allodynia in an experimental rat model of insulin resistance.

BACKGROUND AND PURPOSE: Diabetes causes sensory polyneuropathy with associated pain in the form of tactile allodynia and thermal hyperalgesia which are often intractable and resistant to current therapy. This study tested the beneficial effects of the non-peptide and orally active kinin B(1) receptor antagonist SSR240612 against tactile and cold allodynia in a rat model of insulin resistance. EXPERIMENTAL APPROACH: Rats were fed with 10% D-glucose for 12 weeks and effects of orally administered SSR240612 (0.3-30 mg kg(-1)) were determined on the development of tactile and cold allodynia. Possible interference of SSR240612 with vascular oxidative stress and pancreatic function was also addressed. KEY RESULTS: Glucose-fed rats exhibited tactile and cold allodynia, increases in systolic blood pressure and higher plasma levels of insulin and glucose, at 12 weeks. SSR240612 blocked tactile and cold allodynia at 3 h (ID(50)=5.5 and 7.1 mg kg(-1), respectively) in glucose-fed rats but had no effect in control rats. The antagonist (10 mg kg(-1)) had no effect on plasma glucose and insulin, insulin resistance (HOMA index) and aortic superoxide anion production in glucose-fed rats. CONCLUSIONS AND IMPLICATIONS: We provide the first evidence that the B(1) receptors are involved in allodynia in an experimental rat model of insulin resistance. Allodynia was alleviated by SSR240612 most likely through a direct inhibition of B(1) receptors affecting spinal cord and/or sensory nerve excitation. Thus, orally active non-peptide B(1) receptor antagonists should have clinical therapeutic potential in the treatment of sensory polyneuropathy.

Involvement of kinin B1 receptor and oxidative stress in sensory abnormalities and arterial hypertension in an experimental rat model of insulin resistance.

Diabetes Mellitus leads to pain neuropathy and cardiovascular complications which remain resistant to current therapies involving the control of glycaemia. This study aims at defining the contribution of kinin B(1) receptor (B(1)R) and the oxidative stress on sensory abnormalities and arterial hypertension in a rat model of insulin resistance. Rats were fed with 10% d-glucose for a chronic period of 12-14 weeks and the impact of a diet supplemented with alpha-lipoic acid, a potent antioxidant, was determined on tactile and cold allodynia, arterial hypertension and the expression of kinin B(1)R (real-time PCR and autoradiography) in several tissues. Acute effects of brain penetrant (LF22-0542) and peripherally acting (R-715) B(1)R antagonists were also assessed. Glucose-fed rats exhibited tactile and cold allodynia along with increases in systolic blood pressure between 4 and 12 weeks; these alterations were alleviated by alpha-lipoic acid. The latter regimen also decreased significantly increased plasma levels of insulin and glucose and insulin resistance (HOMA index) at 14 weeks. B(1)R mRNA was virtually absent in liver, aorta, lung, kidney and spinal cord isolated from control rats, yet B(1)R mRNA was markedly increased in all tissues in glucose-fed rats. Up-regulated B(1)R mRNA and B(1)R binding sites (spinal cord) were significantly reduced by alpha-lipoic acid in glucose-fed rats. LF22-0542 reduced tactile and cold allodynia (3h) and reversed arterial hypertension (3-48h) in glucose-fed rats. R-715 abolished tactile and cold allodynia but had not effect on blood pressure. Data suggest that the oxidative stress contributes to the induction and up-regulation of B(1)R in the model of insulin resistance induced by glucose feeding. The over expressed B(1)R contributes centrally to arterial hypertension and in the periphery to sensory abnormalities.

Impact of pioglitazone and bradykinin type 1 receptor antagonist on type 2 diabetes in high-fat diet-fed C57BL/6J mice.

AIM: Type 2 diabetes (T2D) is a major complication of obesity and a leading cause of morbidity and mortality. Antagonizing bradykinin type 1 receptor (B1R) improved body and tissue fat mass and reversed vascular and adipose tissue inflammation in a rat model of insulin resistance. This study aimed at evaluating further the role of B1R in a mouse model of T2D by comparing the antidiabetic and anti-inflammatory effects of the B1R antagonist SSR240612 (SSR) in adipose tissue with those of pioglitazone (TZD), an activator of peroxisome proliferator-activated receptor gamma. METHODS: C57BL/6J mice were fed with high-fat diet (HFD) or standard diet (control) for 20 weeks. Yet, during the last 4 weeks, HFD-fed mice were administered SSR and TZD (10 mg kg-1 d-1 each) as monotherapy or combined therapy subcutaneously. The impact of treatments was measured on metabolic hormones levels (ELISA), adipose tissue inflammatory status and the expression of candidate genes involved in T2D (quantitative real-time polymerase chain reaction and western blot). RESULTS: SSR240612 and TZD treatments improved hyperglycaemia, hyperinsulinaemia, insulin resistance, adipose tissue inflammation (expression of B1R, chemokine ligand 2, F4/80 and tumour necrosis factor) and modulated adipogenesis (peroxisome proliferator-activated receptor gamma, adipocytes' protein 2 and CD40 expressions) in HFD-fed mice. Yet, SSR was more effective than TZD to reduce visceral fat mass and resistin. TZD/SSR combined treatment had an additive effect to improve insulin sensitivity and glucose intolerance. CONCLUSION: Bradykinin type 1 receptor antagonism could represent a promising therapeutic tool in combination with TZD for the treatment of T2D, obesity and insulin resistance.

Hepoxilin A3 (HxA3) is formed by the rat aorta and is metabolized into HxA3-C, a glutathione conjugate.

In this paper we describe the release of hepoxilin A3 (HxA3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A3 (TrXA3), and a second pathway involves conjugation of HxA3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A3-C (HxA3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA3 and HxA3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA3 and to a lesser extent HxA3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

Targeting kinin receptors for the treatment of neurological diseases.

Kinins (bradykinin, kallidin and their active metabolites) are peptide autacoids with established functions in cardiovascular homeostasis, contraction and relaxation of smooth muscles, inflammation and nociception. They are believed to play a role in disease states like asthma, allergies, rheumatoid arthritis, cancer, diabetes, endotoxic and pancreatic shock, and to contribute to the therapeutic effects of ACE inhibitors in cardiovascular diseases. Although kinins are also neuromediators in the central nervous system, their involvement in neurological diseases has not been intensively investigated thus far. This review analyzes the potential of central kinin receptors as therapeutic targets for neurological disorders. Initial data highlight potential roles for B(1) receptor antagonists as antiepileptic agents, and for B(2) receptor antagonists (and/or B(1) agonists) in the treatment of stroke. Functional B(1) receptors located on T-lymphocytes and on the blood brain-barrier are also putative targets for the management of multiple sclerosis. However, successful elucidation of the therapeutic value of these new pharmacological approaches will require refinement of our knowledge on the physiology and cellular localization of central kinin receptors.

Kinin B1 receptor expression and function on human brain endothelial cells.

The kinin B1 receptor is an inducible receptor expressed in response to inflammatory mediators. We sought to determine whether kinin B1 receptor can be expressed on human brain endothelial cells (HBECs) in vitro and whether signaling via this receptor can regulate permeability and chemokine production properties of these cells. Multiplex RT-PCR amplification and western blot techniques were used to evaluate B1 receptor expression by HBECs. Although B1 receptor mRNA and protein could not be detected on resting HBECs, interferon-gamma induced a dose- and time-dependent up-regulation of B1 receptor mRNA and protein on HBECs. Stimulation of interferon-gamma-treated HBECs with the selective B1 agonist R-838 (Sar [D-Phe8] des Arg9-BK) induced a dose- and time-dependent increase in the production of inositol 3,4,5 tri-phosphate and nitric oxide. Permeability of the HBECs monolayer, as measured by BSA diffusion, was significantly increased by application of the B1 agonist. This biological effect of R-838 could be prevented by R-715, a B1 receptor antagonist and by L-NAME, a nitric oxide synthase blocker. R-838 also inhibited interleukin-8 release from HBECs. We demonstrate that B1 receptors can be up regulated on the surface of HBECs by molecules released during inflammatory response and that signaling via this receptor can regulate BBB permeability and chemokine production in vitro.

Bradykinin B1 receptor expression and function on T lymphocytes in active multiple sclerosis.

BACKGROUND: Lesion development in MS is initiated by migration of inflammatory cells into the central nervous system, a process dependent on endothelial cell-lymphocyte interaction. Bradykinin B1 receptor is a membrane-bound G protein-coupled receptor shown to be upregulated on the surface of various cells types during inflammation. OBJECTIVE: To assess the expression and function of the bradykinin B1 receptor on T lymphocytes from MS patients. METHODS: The authors used multiplex polymerase chain reaction amplification and Western blot techniques to demonstrate B1 receptor expression by T cells. A modified Boyden chamber assay also was used to assess the effect of B1 agonist and antagonist on T cell migration. RESULTS: The authors demonstrated that the expression of B1 receptor was upregulated on T cells derived from peripheral blood of MS patients. Expression of this receptor was upregulated on T cells from patients with secondary progressive MS and relapsing-remitting patients in active relapse. Expression was lower in relapsing remitting patients in remission and least in control subjects, including patients with epilepsy, chronic inflammatory demyelinating polyneuritis, and systemic lupus erythematosus. In vitro treatment of cells from healthy control subjects with tumor necrosis factor-alpha and interferon-gamma also induced the expression of B1 receptors. The authors also found that the significantly higher rate of migration of MS T lymphocytes, compared with control subjects in the Boyden chamber assay, could be prevented by the addition of the selective and stable B1 agonist Sar (D-Phe8) desArg9-BK. CONCLUSION: The authors demonstrate that B1 receptors are upregulated by T lymphocytes during the course of MS and that signaling through this receptor with a B1 agonist can negatively regulate T-cell migration in vitro.

Synthesis of peptides by the solid-phase method. 7. Substance P and analogues.

Substance P and 21 related peptides containing isosteric or isofunctional groups were prepared by the solid-phase method. After purification by gel filtration and ion-exchange chromatography, the compounds were characterized by thin-layer chromatography, paper electrophoresis, and amino acid and elemental analysis. The biological activities of the peptides were evaluated in vitro on the guinea pig ileum, the rabbit mesenteric vein, and the dog common carotid artery and in vivo on the rat blood pressure. It is shown that the replacement of some residues in the undecapeptide substance P causes variable losses of apparent affinity with a little or no change in the intrinsic activity. All the analogues used in the present study were found to be inactive as antagonists.

Synthesis and biological activities of photoaffinity labeling analogues of substance P.

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, SP) is an undecapeptide with important properties as a neurotransmitter and with other functions. No specific antagonists and no long-acting analogues of this peptide hormone are known to date. In order to reach these goals, analogues of SP have been prepared which contain potential affinity, as well as photoaffinity labeling functions, suitable for irreversible attachment to SP receptors. We report here the synthesis of SP analogues which have the Phe residues in positions 7 or 8 replaced with (4'-NO2)Phe, (4'-NH2)Phe, (4'-N2+)Phe, and (4'-N3)Phe. Some of these peptides are used for photoaffinity labeling studies using various bioassays. The synthesis of the (NO2)Phe-containing peptide was carried out on solid phase using Nle instead of Met and the Boc strategy up to residue 4; the remaining amino acids were added using an Fmoc strategy. The protected undecapetide was cleaved by ammonolysis, purified by chromatography on silica gel with chloroform/methanol and deprotected afterwards. The amino, diazonium, and azido peptides were obtained in this sequence by chemical modification of the nitro peptides. On guinea pig ileum the modified peptides in position 8 had close to maximal activity, whereas modifications in position 7 produced some reduced activity, especially the nitro modification. No diazonium peptide produced any irreversible effects on guinea pig ileum. Photoinactivation studies were carried out on strips of guniea pig trachea, but no irreversible effects have been observed, neither permanent stimulation nor permanent inactivation. The biological activities and effects are discussed in view of the molecular properties of the synthesized analogues.

Structure-activity studies on the C-terminal amide of substance P.

Twelve C-terminal heptapeptide analogues of substance P have been synthesized by solid phase and by the classical solution method. The modifications concerned all the C-terminal primary amide of SP and should therefore help to understand the biological significance of this carboxamide, as evaluated by in vivo and in vitro bioassays. From the results it can be seen that not the slightest change of the two amide protons is tolerated without an important loss of activity: replacement of one or two amide protons with alkyl groups, extension of the amide to the hydrazide and its alkyl analogues, and exchange of the amide with an ester or a carboxylic acid all reduce the relative activity/affinity at least by 2-fold. It is not clear for what reason all these modifications produce such a drastic activity reduction.

Tetrapeptide tachykinin antagonists: synthesis and modulation of the physicochemical and pharmacological properties of a new series of partially cyclic analogs.

We report on the synthesis and the pharmacological properties of a new series of tachykinin antagonists based on the pseudopeptide pharmacophore cyclo[-Abo-Asp(D-Trp-Phe-N(Me)Bzl)-] which contains the 2-azabicyclo[2.2.2]octane-3(S)-carboxylic acid (Abo) residue. Variation of the substituents on the tryptophan indole nitrogen was shown to modulate water solubility and transport properties of the analogs as well as potency in classical in vitro response and binding assays. One water-soluble compound, 16, in which the substituent was 3-carbonylpropionate, strongly prolonged the reaction time in the mouse hot-plate test both after iv or oral administration and was devoid of degranulating activity in rat peritoneal mast cells.

Localization of bradykinin-like immunoreactivity in the rat spinal cord: effects of capsaicin, melittin, dorsal rhizotomy and peripheral axotomy.

A putative role for bradykinin has been proposed in the processing of sensory information at the level of the spinal cord. Autoradiographic studies have demonstrated the presence of B2 kinin receptor binding sites in superficial laminae of the dorsal horn and a down-regulation of those receptors in rat models of pain injury. In this study, classical immunocytochemistry and confocal microscopy immunofluorescence were used first to localize bradykinin-like immunoreactivity in all major spinal cord segments of naive rats; second, to assess bradykinin-like immunoreactivity changes that occur in animals subjected to various chemical treatments and surgical lesions. High densities of bradykinin-like immunoreactivity were observed in motoneuron of the ventral horn, deeper laminae and nucleus dorsalis of the dorsal horn. Higher magnification of ventral horn showed strong immunostaining of motoneuron perikaryas and their proximal processes. Two types of bradykinin-like immunoreactivity immunostained cellular bodies were observed in deeper laminae of the dorsal horn. These interneurons, morphologically corresponding to islets and antenna-type cells project dendrites to adjacent laminae. Furthermore, numerous strongly marked dendrites, transversally cut, suggest the presence of projection neurons to higher cervical centres. Following unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesion of the sciatic nerve, important increases of bradykinin-like immunoreactivity were found in laminae III and IV of the ipsilateral dorsal horn. In contrast, significant decreases of immunodeposits were observed in both cell bodies and numerous dendrites of motoneuron surrounding neuropil. Specific destructions of sensory afferent fibres with capsaicin or selective activation of kallikreins with melittin caused increases of bradykinin-like immunoreactivity in both the dorsal and ventral horns of the spinal cord. These results which demonstrate the cellular localization of bradykinin-like immunoreactivity in both dorsal and ventral horns of the rat spinal cord, further reveal the plasticity of this non-sensory peptidergic system following various chemical and surgical treatments. Hence, these anatomical findings along with earlier functional and receptor autoradiographic studies reinforce the putative role of bradykinin in sensory function.