Anorexigenic effects of substance P in Coturnix japonica.

Substance P (SP) is an 11-amino acid tachykinin-related peptide that has anorexigenic effects in birds and mammals although the central mechanism is not well understood. Hence, the objective was to identify appetite-associated hypothalamic mechanisms in Japanese quail (Coturnix japonica). Seven days post-hatch, quail were intracerebroventricularly injected with 0, 0.25, 0.5 or 1.0 nmol of SP and monitored for 180 min. On a cumulative basis, quail that received 0.5 and 1.0 nmol of SP consumed less food for 90 min post-injection. On a non-cumulative basis, food intake was reduced in 0.5 nmol-injected birds at 30 min post-injection. Water intake was not affected. A comprehensive behavior analysis was performed, revealing that SP-injected chicks displayed less feeding pecks and reduced locomotion compared to vehicle-injected birds. To identify molecular mechanisms, the hypothalamus was isolated at 1 h post-injection and real-time PCR was performed to measure mRNA. Agouti-related peptide (AgRP) mRNA was reduced in SP-injected chicks. Immunohistochemistry was used to quantify c-Fos-expressing cells in appetite-associated hypothalamic nuclei. There were more reactive cells in the lateral hypothalamus (LH) and the paraventricular nucleus (PVN) of SP- than vehicle-injected chicks. The LH and PVN were collected for gene expression analysis. Corticotropin-releasing factor (CRF) and urotensin 2 (UTS2) mRNAs were greater in SP- than vehicle-injected chicks in the PVN. In the LH, CRF receptor sub-type 2 (CRFR2) mRNA was greater and kappa opioid receptor mRNA was reduced in SP- compared to vehicle-injected quail. Thus, SP induces a potent anorexia in quail that coincides with increased LH-specific CRFR2 mRNA and increased UTS2 mRNA in the PVN. Future studies will evaluate whether SP-induced anorexigenic effects are mediated through CRF receptors.

Incorporation of ROS-Responsive Substance P-Loaded Zeolite Imidazolate Framework-8 Nanoparticles into a Ca2+-Cross-Linked Alginate/Pectin Hydrogel for Wound Dressing Applications.

PURPOSE: Wound healing, especially of extensive full-thickness wounds, is one of the most difficult problems in clinical studies. In this study, we prepared a novel substance P (SP)-delivery system using zeolite imidazolate framework-8 (ZIF-8) nanoparticles. METHODS: We synthesized ZIF-8 nanoparticles using a modified biomimetic mineralization method. We then coated SP-loaded ZIF-8 nanoparticles (SP@ZIF-8) with polyethylene glycol-thioketal (PEG-TK) to fabricate SP@ZIF-8-PEG-TK nanoparticles, and encapsulated them in injectable hydrogel composed of sodium alginate and pectin and cross-linked using calcium chloride. The final hydrogel wound dressing containing SP@ZIF-8-PEG-TK nanoparticles was called SP@ZIF-8-PEG-TK@CA. RESULTS: The fabricated ZIF-8 nanoparticles had high SP-loading efficiency. SP-release assay showed that the SP@ZIF-8-PEG-TK nanoparticles maintained drug activity and showed responsive release under stimulation by reactive oxygen species. The SP@ZIF-8-PEG-TK nanoparticles promoted proliferation of human dermal fibroblasts, up-regulated expression levels of inflammation-related genes in macrophages, and exhibited favorable cytocompatibility in vitro. Full-thickness excision wound models in vivo confirmed that SP@ZIF-8-PEG-TK@CA dressings had excellent wound-healing efficacy by promoting an early inflammatory response and subsequent M2 macrophage polarization in the wound-healing process. CONCLUSION: In conclusion, these findings indicated that SP@ZIF-8-PEG-TK@CA dressings might be useful for wound dressing applications in the clinic.

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

The NK1 receptor antagonist aprepitant attenuates NK1 agonist-induced scratching behaviour in the gerbil after intra-dermal, topical or oral administration.

Experiments were conducted to develop a model to study the effect of oral and topical administration of the NK1 receptor antagonist aprepitant, on scratching behaviour in gerbils. The gerbil was selected due to its relevance for human NK1 receptor pharmacology. Intradermal injection of a specific NK1 receptor agonist GR73632 (100 nmol/100 µl) at the rostral back of gerbils produced scratching of the injection site. This could be attenuated by intradermal co-administration of a selective NK1 receptor antagonist aprepitant (30-100-300 nmol), demonstrating the role of dermal NK1 receptor in elicitation of scratching behaviour. Likewise, scratching was attenuated by oral (0.3-3-30 mg/kg) or topical application (0.01-0.1-1% w/v) of aprepitant and pharmacokinetic analysis of aprepitant levels in brain, blood and skin supported that efficacy of topically applied aprepitant was due to dermal rather than central target engagement. In conclusion, we showed that NK1 agonist-induced scratching in the gerbil can be reversed by systemic and topical administration of aprepitant. This test system may provide a useful model for the in vivo assessment of putative antipruritic agents.

Characterization of intrathecally administered hemokinin-1-induced nociceptive behaviors in mice.

Hemokinin-1 is a novel mammalian tachykinin cloned from mouse bone marrow. At present, pharmacological profile and physiological role of hemokinin-1 are still unclear. In the present study, we found that intrathecal (i.t.) administration of hemokinin-1 (0.00625-1.6 nmol) induced nociceptive responses consisting of scratching, biting and licking, which resemble substance P-induced behavioral responses in mice. The behaviors evoked by low-dose of hemokinin-1 (0.0125 nmol) were dose-dependently inhibited by i.t. co-administration of CP-99,994, a non-peptidic tachykinin NK(1) receptor antagonist, whereas high-dose of hemokinin-1 (0.1 nmol)-induced behaviors were not affected. Moreover, sendide, a peptidic tachykinin NK(1) receptor antagonist, failed to reduce the behavioral responses of both low- and high-dose of hemokinin-1. In contrast, substance P-induced behaviors were completely suppressed by both CP-99,994 and sendide. These results suggest that hemokinin-1 plays an important role in pain transmission at spinal cord. Moreover, the mechanism of hemokinin-1-induced nociceptive behaviors may be dose-dependent, and distinct from substance P-induced nociceptive behaviors.

Substance P neurotransmission and violent aggression: the role of tachykinin NK(1) receptors in the hypothalamic attack area.

Substance P and its tachykinin NK(1) receptors are highly expressed in brain regions involved in emotional control. We recently showed that NK(1)-mediated substance P neurotransmission is deeply involved in the control of aggressiveness. To get further insights into the NK(1) receptor/aggression relationship, we studied the role of NK(1) receptor-expressing neurons of the hypothalamic attack area, the only brain region in rats from which biting attacks can reliably be elicited by both electrical and neurochemical stimulation. We show here that the hypothalamic attack area preferentially expresses the NK(1) type of tachykinin receptors. When such neurons were lesioned by substance P-conjugated saporin (SP-sap) infused into the hypothalamic attack area, violent attacks were dramatically reduced, whereas milder forms of aggression (soft bites and offensive threats) remained unaltered. The lesions were neuron type-specific as SP-sap lesions markedly reduced NK(1) staining without significantly affecting total cell counts. NK(1) staining in the neighboring lateral hypothalamus was not affected, which confirms the spatial specificity of the lesion. Surprisingly, the lesions also reduced anxiety-like behavior in the elevated plus-maze. This effect is likely explained by the extensive connections of the hypothalamic attack area with brain regions involved in the control of anxiety. The present findings suggest that violent and milder forms of attack are differentially controlled. NK(1) receptor-expressing neurons of the hypothalamic attack area are tightly and specifically involved in the former but not in the latter. Our data also raise the possibility of a coordinated control of violent attacks and anxiety by the same NK(1)-expressing neurons.

Substance P is involved in the effect of histamine H3 receptor agonist, Sch 50971 on nasal allergic symptoms in mice.

The aim of this study was to clarify the mechanisms of the inhibitory effect of a histamine H3 receptor agonist, Sch 50971, on nasal signs in an allergic rhinitis model in mice. The severity of allergic rhinitis was assessed by determining the extent of two markers of allergic symptoms (sneezing and nasal rubbing). The topical application of a histamine H3 receptor antagonist, clobenpropit, into the nasal cavities resulted in a dose-dependent increase in sneezing and nasal rubbing, and both Sch 50971 and a tachykinin NK1 receptor antagonist, L-733,060, inhibited these reactions in non-sensitized mice. Sch 50971 caused no inhibition of histamine- and substance P-induced nasal symptoms; however, the reactions induced by capsaicin were significantly decreased by Sch 50971 in non-sensitized mice. Sch 50971 and cetirizine inhibited antigen-induced sneezing and nasal rubbing in sensitized mice. On the other hand, cetirizine inhibited nasal symptoms induced by antigen in capsaicin-pretreated mice, whereas no effect was observed in Sch 50971. From these results, we concluded that Sch 50971 blocked nasal symptoms by the inhibition of substance P release via histamine H3 receptors located on peri]pheral sensory nerve endings.

Anti-inflammatory evaluation of Solidago chilensis Meyen in a murine model of pleurisy.

UNLABELLED: The aim of this study was to investigate the anti-inflammatory effects and the mechanism of action of the aqueous extracts obtained from rhizomes, leaves and inflorescences of Solidago chilensis in the mouse model of pleurisy. The extracts were prepared by infusion and were lyophilized. RESULTS: The aqueous extracts of rhizomes, leaves or inflorescences inhibited leukocytes, neutrophils and exudation (P<0.05) in the inflammation induced by carrageenan. The rhizomes aqueous extract, butanolic and aqueous residual fractions inhibited leukocytes, neutrophils, myeloperoxidase, adenosine-deaminase, and tumor necrosis factor alpha levels in the inflammation induced by carrageenan (P<0.05). The rhizome aqueous extract and butanolic fraction also inhibited exudation, nitric oxide, and interleukin-1 beta levels (P<0.05). The rhizomes aqueous extract and its two derived fractions reduced leukocytes and mononuclears in the pleurisy induced by bradykinin, histamine, or substance P (P<0.05) and neutrophils in the pleurisy induced by histamine or substance P (P<0.05). Only aqueous residual fraction inhibited neutrophils induced by bradykinin (P<0.05). CONCLUSION: Solidago chilensis aqueous extracts from leaves, inflorescences and rhizomes demonstrated an important anti-inflammatory effect, inhibiting cells in the inflammation caused by carrageenan. In addition, the rhizomes aqueous extract and its derived fractions also decreased pro-inflammatory mediators release into the site of the inflammatory process. The rhizomes aqueous extract and the butanolic fraction showed more evident anti-inflammatory actions.

Intrathecal substance P (1-7) prevents morphine-evoked spontaneous pain behavior via spinal NMDA-NO cascade.

Previous research has shown that injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits an excitatory behavioral syndrome indicative of severe vocalization and agitation. Substance P N-terminal fragments are known to inhibit nociceptive responses when injected i.t. into animals. In this study, we investigated the effect of i.t. substance P (1-7) on both the nociceptive response and the extracellular concentrations of glutamate and nitric oxide (NO) metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine (500 nmol). The induced behavioral responses were attenuated dose-dependently by i.t. pretreatment with the substance P N-terminal fragment substance P (1-7) (100-400 pmol). The inhibitory effect of substance P (1-7) was reversed significantly by pretreatment with [d-Pro2, d-Phe7]substance P (1-7) (20 and 40 nmol), a d-isomer and antagonist of substance P (1-7). In vivo microdialysis analysis showed a significant elevation of extracellular glutamate and NO metabolites in the spinal cord after i.t. injection of high-dose morphine (500 nmol). Pretreatment with substance P (1-7) (400 pmol) produced a significant reduction on the elevated concentrations of glutamate and NO metabolites evoked by i.t. morphine. The reduced levels of glutamate and NO metabolites were significantly reversed by the substance P (1-7) antagonist (40 nmol). The present results suggest that i.t. substance P (1-7) may attenuate the excitatory behavior (vocalization and agitation) of high-dose i.t. morphine by inhibiting the presynaptic release of glutamate, and reducing NO production in the dorsal spinal cord.

In vitro and in vivo evaluation of topical formulations of spantide II.

The purpose of this study was to develop and evaluate topical formulations of Spantide II, a neurokinin-1 receptor (NK-1R) antagonist, for the treatment of inflammatory skin disorders. Spantide II lotion and gel was formulated with and without n-methyl-2-pyrrolidone (NMP) as a penetration enhancer. The release of Spantide II from gels was evaluated using microporous polyethylene and polypropylene membranes in a Franz Diffusion cell setup. In vitro percutaneous absorption of Spantide II from lotion and gel formulations was evaluated using the above setup by replacing the membranes with hairless rat skin. The in vivo anti-inflammatory activity of Spantide II formulations was evaluated in an allergic contact dermatitis (ACD) mouse model. Among different gels studied, PF127 gel showed highest (70-fold) release of Spantide II compared with hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) gels. Lotion and gel formulations with or without NMP showed no detectable levels of Spantide II in the receiver compartment of the Franz diffusion cell until 24 hours. However, Spantide II showed significant retention in epidermis and dermis from lotion and gel formulations at 24 hours. The dermal levels increased approximately 3.5- and 2-fold when the lotion and gel formulations contained NMP as compared with the formulation with no NMP (P < .05). The in vivo studies indicated that Spantide II formulations with NMP were effective in significantly reducing ACD response, similar to dexamethasone (0.5 mM). In conclusion, Spantide II was stable as a topical formulation and delivered to target skin tissue (epidermis and dermis) for the treatment of ACD. In addition this study supports the role of cutaneous neurosensory system in modulating inflammatory responses in the skin.

Restoration of corneal epithelial barrier function and wound healing by substance P and IGF-1 in rats with capsaicin-induced neurotrophic keratopathy.

PURPOSE: To investigate the effects of topical application of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and epithelial wound closure in rats with capsaicin-induced neurotrophic keratopathy. METHODS: Neonatal rats were injected subcutaneously with a single dose of capsaicin to induce neurotrophic keratopathy. Corneal epithelial barrier function was evaluated with an anterior fluorophotometer. Tear fluid secretion was measured by the Schirmer test. Corneal epithelial wound healing was determined by measurement of the size of the epithelial defect after debridement of the entire epithelium. The combination of SP (1 mM) and IGF-1 (1 micro g/mL) in phosphate-buffered saline was administered in eye drops six times daily. RESULTS: Corneal epithelial barrier function was impaired and corneal epithelial wound healing was delayed in rats injected with capsaicin. The application of eye drops containing the combination of SP and IGF-1 to capsaicin-injected rats resulted in a significant improvement in corneal epithelial barrier function compared with that apparent in capsaicin-injected animals that received eye drops containing vehicle alone. Such treatment with SP and IGF-1 also significantly increased the rate of corneal epithelial wound closure in capsaicin-injected animals. CONCLUSIONS: Topical application of the combination of SP and IGF-1 improved both corneal epithelial barrier function and epithelial wound healing in an animal model of neurotrophic keratopathy.

Histological demonstration of increased vascular permeability in the dura mater of the rat.

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.

L-arginine and substance P reverse the pulmonary endothelial dysfunction caused by congenital heart surgery.

BACKGROUND: The increase in pulmonary vascular resistance (PVR) seen in children after cardiopulmonary bypass has been attributed to transient pulmonary endothelial dysfunction (PED). We therefore examined PED in children with congenital heart disease by assessing the L-arginine-nitric oxide (NO) pathway in terms of substrate supplementation (L-arginine [L-Arg]), stimulation of endogenous NO release (substance P [Sub-P]), and end-product provision (inhaled NO) before and after open heart surgery. METHODS AND RESULTS: Ten patients (aged 0.62+/-0.27 years) with pulmonary hypertension undergoing cardiac catheterization who had not had surgery and 10 patients (aged 0.65+/-0.73 years) who had recently undergone cardiopulmonary bypass were examined. All were sedated and paralyzed and received positive-pressure ventilation. Blood samples and pressure measurements were taken from catheters in the pulmonary artery and the pulmonary vein or left atrium. Respiratory mass spectrometry was used to measure oxygen uptake, and cardiac output was determined by the direct Fick method. PVR was calculated during steady state at ventilation with room air, during FIO(2) of 0.65, then during additional intravenous infusion of L-Arg (15 mg. kg(-1). min(-1)) and Sub-P (1 pmol. kg(-1). min(-1)), and finally during inhalation of NO (20 ppm). In preoperative patients, the lack of an additional significant change of PVR with L-Arg, Sub-P, and inhaled NO suggests little preexisting PED. Postoperative PVR was higher, with an additional pulmonary endothelial contribution that was restorable with L-Arg and Sub-P. CONCLUSIONS: Postoperatively, the rise in PVR suggested PED, which was restorable by L-Arg and Sub-P, with no additional effect of inhaled NO. These results may indicate important new treatment strategies for these patients.

Potential effect of the administration of substance P and allergen therapy on immunoglobulin E-mediated allergic reactions in human subjects.

Previously we observed and reported that immunoglobulin E-mediated (IgE-mediated) allergy in rhesus monkeys was decreased by the administration of substance P (SP) and an allergen. We extended these studies to human subjects, giving SP and 1 allergen to subjects with reactivity to more than 1 allergen, using reactivity to a second allergen as a control. SP and an allergen were initially given by aerosol delivery but subsequently were given by injection. The administration of SP and 1 allergen by aerosol delivery or injection resulted in decreased IgE-mediated reactivity to the allergen administered and also to the control allergen. This result occurred in 7 of 8 human subjects. The 2 initial subjects receiving 8 SP and allergen injections had a sharp reduction in their symptoms of ragweed hay fever lasting for 3 years to date. No significant reactions to the injection of SP occurred. Further controlled human research is necessary on the administration of SP and allergen and the mechanisms of action. Unexpected and serendipitous results first observed in rhesus monkeys and reproduced in allergic human subjects provide a new and potential mechanism for control and perhaps obliteration of common IgE-mediated allergies and even more-serious allergic problems.

Oxytocin pathways mediate the cardiovascular and behavioral responses to substance P in the rat brain.

Stimulation of brain periventricular and hypothalamic substance P receptors induces a pressor response and tachycardia associated with mesenteric and renal vasoconstriction and hindlimb vasodilation resembling thus the classical defense reaction. This cardiovascular response is brought about by the activation of the sympathoadrenal system and is accompanied by grooming behavior. To address the role of oxytocinergic pathways in the brain in the mediation of these responses, we investigated the effects of central pretreatment of rats with oxytocin antisense, mixed base, and sense oligodeoxynucleotides on mean arterial pressure, heart rate, and grooming behavior induced by intracerebroventricular injections of substance P (50 pmol). Central pretreatment of conscious rats with the oxytocin antisense oligodeoxynucleotide (intracerebroventricular injections, 8 and 4 hours before administration of substance P) attenuated the mean arterial pressure (by 55%) and heart rate responses (by 58%) as well as grooming behavior induced by the peptide. A complete recovery of all substance P-induced responses was observed 28 hours after antisense oligodeoxynucleotide pretreatment. Intracerebroventricular pretreatment of rats with mixed base and sense oligodeoxynucleotides did not affect the cardiovascular and behavioral responses to substance P. The signal for oxytocin mRNA in the paraventricular nucleus was reduced only in rats pretreated with the antisense oligodeoxynucleotide. These results demonstrate that oxytocin neurons in the paraventricular nucleus, which innervate the cardiovascular centers in the hindbrain and the spinal cord, mediate the increases in blood pressure and heart rate induced by stimulation of substance P receptors in the forebrain. These neurons may also transmit signals, which are generated by substance P in the hypothalamus and are responsible for the sympathoadrenal activation in response to stress.

Effect of the tachykinin receptor antagonists, SR 140333, FK 888, and SR 142801, on capsaicin-induced mouse ear oedema.

We examined the effect of SR 140333, a nonpeptide NK1 receptor antagonist, FK 888, a peptide NK1 antagonist, and SR 142801, a non-peptide NK3 antagonist, on ear oedema induced by topical application of capsaicin (250 micrograms/ear) in mice. SR 140333 (ED50:39 micrograms/kg, i.v.) dose-dependently inhibited the oedema response to capsaicin, whereas FK 888 (1.0 mg/kg, i.v.) and SR 142801 (3.0 mg/kg, i.v.) had no effect. Furthermore, SR 140333 significantly (p < 0.001) suppressed ear oedema in response to intradermal injection of substance P (SP) (100 pmol/site) by i.v. administration (0.1 mg/kg,) and co-injection (50 pmol/site). In contrast, FK 888 (1.0 mg/kg, i.v. and 500 pmol/site) was ineffective in the response to SP. The present results suggest that the difference in effects of the two NK1 receptor antagonists on the oedema response to capsaicin is due to species differences in affinities for the NK1 receptor in the mouse skin. Moreover, it seems unlikely that the NK3 receptor is involved primarily in capsaicin-induced mouse ear oedema.

Inhibition of brown fat activity during hypothalamic stimulation in the rat.

Previous work has shown that low-level electrical stimulation of the ventromedial hypothalamus (VMH) in anesthetized rats produces a sustained decrease (phase 1) in interscapular brown adipose tissue (IBAT) temperature followed by a rise (phase 2) after the stimulus has stopped [Woods, A. J., and M. J. Stock. Am. J. Physiol. 266 (Regulatory, Integrative Comp. Physiol. 35): R328-R337, 1994]. In this study, rat oxygen consumption was found to decrease (24%) and then increase (74%) during phase 1 and 2, respectively. The effect of norepinephrine, alpha-adrenoceptor antagonists, substance P, and neuropeptide Y, with and/or without VMH stimulation, suggested that vasoconstriction was unlikely to account for the phase 1 decreases in thermogenesis and temperature. However, measurement with radio-labeled microspheres showed that IBAT capillary blood flow was reduced by 70% during phase 1, and this, plus a 50% decrease in blood oxygen extraction, indicated that phase 1 could be due to vasodilatation of arteriovenous anastomoses. It was postulated that phase 1 resulted from release of neuropeptides, such as substance P, causing diversion of arterial blood away from IBAT capillaries, thereby increasing convective heat loss and inhibiting heat production during phase 1.

Demonstration of a 'septide-sensitive' inflammatory response in rat skin.

1. Measurement of plasma protein extravasation induced by the natural tachykinins following intradermal administration in rat skin indicated equipotency between substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). The selective NK1 receptor agonist, [Sar9]SP sulphone was 10-100 times more potent than SP. The synthetic hexapeptide, septide, [pGlu6, Pro9]SP-(6-11), which has been proposed to act on a distinct NK1 receptor subtype/binding site was equipotent with [Sar9]SP sulphone. 2. The selective NK2 receptor agonist [beta Ala8]NKA(4-10) (0.1-1 nmol) and the selective NK3 receptor agonist, senktide (0.1-1 nmol) were both ineffective in producing oedema. The selective NK2 receptor antagonist, SR 48, 968 (0.3 mumol kg-1) had no significant inhibitory effects upon oedema induced by approximately equiactive doses of SP (0.2 nmol), septide (0.002 nmol), [Sar9]SP sulphone (0.002 nmol), or NKB (0.3 nmol). These results together suggest that neither NK2 nor NK3 receptors are involved in oedema formation in rat skin. 3. The non-peptide tachykinin NK1 receptor antagonist, RP 67,580 (1-3 mumol kg-1), inhibited plasma protein extravasation induced by septide (0.002 nmol) to a greater extent than that to SP (0.2 nmol). RP 67,580 (1 mumol kg-1) produced a significant inhibition of approximately 66% of the response to septide (0.002 nmol) only. Increasing the dose of RP 67,580 3 fold resulted in inhibition of the response to SP (0.2 nmol) and [Sar9]SP sulphone (0.002 nmol) by approximately 66% and 64% respectively with the response to septide being inhibited by approximately 70%. 4. Co-administration of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME)(0.1 micromol) with the relevant tachykinin, resulted in a significant attenuation of the oedemaresponse to septide (0.1 nmol) producing only an approximate 56% inhibition of the response. The response to 0.2 nmol SP was unaffected whereas the response to a higher dose of 1 nmol was lowered byL-NAME but this did not reach significance.5. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg-1) for 3 consecutive days, significantly inhibited the oedema responses to only high dose SP (1 nmol) and[Sar9SP sulphone (0.002 nmol). SP (0.2 nmol), septide (0.002 nmol), NKA (0.2 nmol) and NKB(0.3 nmol) were unaffected by this treatment.6. RP 67,580 (0.3-3 microM kg-1) inhibited oedema induced by both 0.002 nmol and 0.1 nmol of septide.When using equiactive doses of SP only the response to the lower dose of 0.2 nmol SP was significantly inhibited, while RP 67,580 (3 micromol kg1) did not affect the response to 1 nmol SP.7 These results suggest distinct mechanisms of action for SP and septide in producing plasma protein extravasation in rat skin. The response induced by septide is blocked by RP 67,580 and is both NO dependent and mast-cell independent. In contrast the response to SP is only partially blocked by RP67,580 and is NO-independent. These data support the existence of a distinct 'septide-sensitive' receptor/binding site and suggest that this site is involved in tachykinin-induced oedema formation in rat skin.

Inhibition by actinomycin D of neurogenic mouse ear oedema.

We have investigated the effects of actinomycin D on mouse ear oedema induced by capsaicin, neuropeptides, and established inflammatory mediators. Actinomycin D (0.5 mg/kg, i.v.) significantly (P < 0.01) inhibited ear oedema induced by topical application of capsaicin, while adriamycin (6.0 mg/kg, i.v.) and cycloheximide (6.0 mg/kg, i.v.) had no effect on oedema. The ear oedema induced by intradermal injection of neuropeptides such as mammalian tachykinins, calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), was markedly (P < 0.05, P < 0.01 or P < 0.001) suppressed by actinomycin D. The drug was also effective (P < 0.01 or P < 0.001) in inhibiting bradykinin (BK)- and compound 48/80-induced ear oedema, but did not inhibit oedema induced by histamine, 5-HT, leukotriene C4 (LTC4), and platelet activating factor (PAF) at a dose of 1 mg/kg. In mast cell-deficient W/WV mice, actinomycin D (1.0 mg/kg, i.v.) failed to inhibit substance P (SP)-induced ear oedema whereas spantide (0.5 mg/kg, i.v.) was an effective (P < 0.01) inhibitor of oedema formation. Furthermore, actinomycin D (10-100 microM) dose-dependently prevented histamine release from rat peritoneal mast cells evoked by SP, compound 48/80, and the ionophore A23182, respectively. These results strongly suggest that an inhibitory effect of actinomycin D on neurogenic inflammation is due primarily to the prevention of mast cell activation mediated by neuropeptides, rather than an interaction with DNA or receptors of neuropeptides.

Inhibition of hind-paw edema and cutaneous vascular plasma extravasation in mice by acetylshikonin.

Acetylshikonin, a naphthoquinone isolated from the Chinese herb medicine, tzu ts'ao, was demonstrated to inhibit the polymyxin B-induced hind-paw edema in normal as well as in adrenalectomized mice. Liver glycogen content was increased in adrenalectomized mice pretreated with dexamethasone, but not with acetylshikonin. Like diphenhydramine, methysergide and isoproterenol, acetylshikonin reduced the plasma exudation evoked in dorsal hind-paw skin by antidromic stimulation of the saphenous nerve, and in passive cutaneous anaphylactic reaction, bradykinin-, substance P-, compound 48/80-, histamine- and serotonin-induced ear edema. Indomethacin was ineffective in these respects. Bradykinin- and substance P-induced plasma exudation were also significantly reduced when [Thi5,8,D-Phe7]bradykinin and [D-Pro2,D-Trp7,9]substance P were coinjected with bradykinin and substance P, respectively. In isolated rat peritoneal mast cell preparation, acetylshikonin produced a concentration-dependent inhibition of histamine and beta-glucuronidase release from mast cells challenged by compound 48/80. In compound 48/80-pretreated mice, acetylshikonin and isoproterenol produced significantly more inhibitory effect on bradykinin- and substance P-induced plasma exudation than did diphenhydramine in combination with methysergide. Pretreatment with diphenhydramine/methysergide in compound 48/80-pretreated mice significantly further reduced the bradykinin- and substance P-induced plasma exudation if [Thi5,8,D-Phe7]bradykinin and [D-Pro2,D-Trp7,9]substance P were coinjected with bradykinin or substance P, respectively. The results suggest that the inhibitory effect of acetylshikonin on the edematous response is due neither to the release of steroid hormones from the adrenal gland nor to the glucocorticoid activity, but probably partly to the suppression of mast cell degranulation and partly to protection of the vasculature from mediator challenge.