Autocrine regulation of human sperm motility by tachykinins.

BACKGROUND: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. METHODS: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). RESULTS: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). CONCLUSION: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

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.

Characterization of wide dynamic range neurons in the deep dorsal horn of the spinal cord in preprotachykinin-a null mice in vivo.

We previously reported that mice with a deletion of the preprotachykinin-A (pptA) gene, from which substance P (SP) and neurokinin A (NKA) are derived, exhibit reduced behavioral responses to intense stimuli, but that behavioral hypersensitivity after injury is unaltered. To understand the contribution of SP and NKA to nociceptive transmission in the spinal cord, we recorded single-unit activity from wide dynamic range neurons in the lamina V region of the lumbar dorsal horn of urethane-anesthetized wild-type and ppt-A null mutant (-/-) mice. We found that intensity coding to thermal stimuli was largely preserved in the ppt-A -/- mice. Neither the peak stimulus-evoked firing nor the neuronal activity during the initial phase (0-4 s) of the 41-49 degrees C thermal stimuli differed between the genotypes. However, electrophysiological responses during the late phase of the stimulus (5-10 s) and poststimulus (11-25 s) were significantly reduced in ppt-A -/- mice. To activate C-fibers and to sensitize the dorsal horn neurons we applied mustard oil (MO) topically to the hindpaw. We found that neither total MO-evoked activity nor sensitization to subsequent stimuli differed between the wild-type and ppt-A -/- mice. However, the time course of the sensitization and the magnitude of the poststimulus discharges were reduced in ppt-A -/- mice. We conclude that SP and/or NKA are not required for intensity coding or sensitization of nociresponsive neurons in the spinal cord, but that these peptides prolong thermal stimulus-evoked responses. Thus whereas behavioral hypersensitivity after injury is preserved in ppt-A -/- mice, our results suggest that the magnitude and duration of these behavioral responses would be reduced in the absence of SP and/or NKA.

Peripheral tachykinin receptors as potential therapeutic targets in visceral diseases.

More than 10 years of intensive preclinical investigation of selective tachykinin (TK) receptor antagonists has provided a rationale to the speculation that peripheral neurokinin (NK)-1, -2 and -3 receptors may be involved in the pathophysiology of various human diseases at the visceral level. In the airways, despite promising effects in animal models of asthma, pilot clinical trials with selective NK-1 or -2 receptor antagonists in asthmatics have been ambiguous, whereas the potential antitussive effects of NK-1, -2 or -3 antagonists have not yet been verified in humans. In the gastrointestinal (GI) tract, irritable bowel syndrome (IBS) and pancreatitis are appealing targets for peripherally-acting NK-1 and -2 antagonists, respectively. In the genito-urinary tract, NK-1 receptor antagonists could offer some protection against nephrotoxicity and cytotoxicity induced by chemotherapeutic agents, whereas NK-2 receptor antagonists appear to be promising new agents for the treatment of neurogenic bladder hyperreflexia. Finally, there is preclinical evidence for hypothesising an effect of NK-3 receptor antagonists on the cardiovascular disturbance that characterises pre-eclampsia. Other more speculative applications are also mentioned.

Protective effect of the tachykinin NK2 receptor antagonist nepadutant in acute rectocolitis induced by diluted acetic acid in guinea-pigs.

We have evaluated the potential protective activity of nepadutant, a selective tachykinin NK2 receptor antagonist, in a model of acute rectocolitis induced by an enema with 7.5% acetic acid in guinea-pigs. The injury was quantified visually by using a macroscopic injury score, and histologically by using a necrosis score. In addition, changes in myeloperoxidase activity, a marker for neutrophil infiltration, and plasma protein extravasation were evaluated. The injury caused by 7.5% acetic acid was mild, affecting the superficial layers and producing a strong edema of the submucosa. A single administration of nepadutant (0.3-10 mg/kg s.c., 1 h before acetic acid) markedly reduced the macroscopic damage and necrosis score and the increase in plasma protein extravasation induced by 7.5% acetic acid in the early phase of the injury. Single administration of nepadutant (3 mg/kg s.c.) reduced the macroscopic score and myeloperoxidase activity at the top (24 h) of inflammation. Repeated administration (3 mg/kg s.c. three times during 24 h) or co-administration of the tachykinin NK1 receptor antagonist MEN 11467 (3 mg/kg s.c.) did not enhance the antiulcer effect obtained with the single treatment with nepadutant. These data suggest the involvement of tachykinin NK2 receptors in the first phases of inflammation induced by acetic acid.

Role of tachykinins in castor oil diarrhoea in rats.

1. We set out to ascertain the role of tachykinins, neurokinin A and substance P, in castor oil-induced diarrhoea in rats as disclosed by the inhibitory effect of the non-peptide NK1- and NK2-receptor antagonists. SR 140333 and SR 48968, respectively. 2. SR 48968 (0.02 to 20 micrograms kg-1, s.c. or p.o.), and the opioid receptor agonist loperamide (1-10 mg kg-1, p.o.), dose-dependently prevented castor oil effects: % inhibition vs castor oil, diarrhoea 0 to 100, increase in faecal mass 7 to 90 and water content 16 to 90. SR 140333 (0.02 to 20 micrograms kg-1, s.c.) and the platelet activating factor antagonist SR 27417 (5 to 500 micrograms kg-1, p.o.) did not prevent the increase in faecal water content, but reduced faecal mass (35 to 66%) and diarrhoea (0 to 57%). 3. The R-enantiomers of tachykinin NK1 and NK2 receptor antagonists, SR 140603 and SR 48605 (both at 2 or 20 micrograms kg-1, s.c.) had no effect other than reducing faecal mass at the highest dose tested. 4. SR 48968 (20 micrograms kg-1, p.o.) but not loperamide (10 mg kg-1, p.o.) given 24 h before castor oil, still slightly but significantly reduced by 30% the increase of faecal mass output; both treatments significantly reduced (30 to 70%) the effect of castor oil on faecal water content, although the incidence of diarrhoea was only slightly less than in controls. 5. In castor oil-treated rats, naloxone (2 mg kg-1, s.c.) completely blocked the antidiarrhoeal action of loperamide (10 mg kg-1, p.o.) but not of SR 48968 (20 micrograms kg-1, p.o.): a similar result was obtained on faecal mass and water content. 6. Castor oil strongly increased the occurrence of manometrically recorded propulsive giant contractions (500 to 1000% over control values) of transverse and distal colon, this effect being significantly prevented (80 to 100%) by SR 48968 and loperamide and partially by SR 140333 (35% distal colon, 70% transverse colon). 7. In castor oil free rats, loperamide but not SR 48968 or SR 140333 significantly reduced by 50% the gastrointestinal transit of a charcoal test meal, as well as 24 h faecal mass output. Consistently, loperamide, unlike the tachykinin receptor antagonists, had a dramatic effect on manometric recordings of intestinal motility, reducing all kinds of colonic contractions. 8. Our findings suggest that castor oil diarrhoea in rats entails activation of NK1 and NK2 receptors by endogenous tachykinins, whose antagonists may have a potential as antidiarrhoeal agents free from the constipating action of opioids.

Mechanisms involved in the contractile responses induced by the hydroalcoholic extract of Phyllanthus urinaria on the guinea pig isolated trachea: evidence for participation of tachykinins and influx of extracellular Ca2+ sensitive to ruthenium red.

1. The hydroalcoholic extract (HE) of stems, leaves and roots from P. urinaria (Euphorbiaceae) (1-3000 micrograms/ml), caused graded contraction in guinea pig trachea (GPT), being more effective in preparations without epithelium. 2. Response to HE was slightly affected by tetrodotoxin (0.3 microM) and nicardipine (1 microM), but was unaffected by w-conotoxin, atropine, mepyramine or staurosporine (all 1 microM). Indomethacin (3 microM) greatly inhibited HE contraction, but MK 571 (leukotriene D4 and E4 antagonist) caused partial inhibition; L-655,240 (thromboxane A2 antagonist) and WEB 2086 (PAF antagonist) (all 1 microM) were ineffective. 3. Response to HE was markedly inhibited in a Ca(2+)-free solution and was partially affected in GPT desensitized to capsaicin (10 microM). 4. Capsazepine (capsaicin antagonist, 3 microM) antagonized the contraction from capsaicin, leaving the response to HE unaffected. In contrast, ruthenium red (an ionic channel antagonist coupled to vanilloid receptors of capsaicin) (0.1-3 microM) caused graded and equipotent noncompetitive inhibition of HE- and capsaicin-induced contractions, but had no effect on carbachol- and prostaglandin E2-mediated responses. 5. FK 888 and SR 48968 (NK1 and NK2 receptor antagonists, respectively) (both 1 microM) antagonized, through a competitive mechanism, the contraction from SP and [beta-ala8]NKA (4-10) respectively, but antagonized, through a noncompetitive mechanism, HE-mediated contraction. 6. We concluded that contraction to HE in GPT is modulated by the epithelium, depends on the release of a cyclo-oxygenase metabolite, and relies largely upon an extracellular Ca2+ influx that is highly sensitive to ruthenium red, but is insensitive to L and N-type of voltage-sensitive Ca2+ channel antagonists. In addition, NK1 and NK2 tachykinins, but not vanilloid receptors, play an important role in mediating its response.

Neutral endopeptidase (NEP) and its role in pathological pulmonary change with inhalation exposure to JP-8 jet fuel.

Through a simulated flightline exposure protocol, Fischer 344 rats (F344) were subjected to an aerosol/vapor mix of the military jet fuel, JP-8. Previous studies with this model of lung injury have revealed significant increases in pulmonary resistance, increased alveolar clearance of 99mTcDTPA, and a decrease in bronchoalveolar lavage fluid (BALF) concentration of the neuropeptide substance P (SP). Exposures to JP-8 were nose-only and for one hour daily. Six groups of Fischer 344 rats were exposed for 7, 28, or 56 days at two JP-8 concentrations (low dose = 469-520 mg/m3/hr, high dose = 814-1263 mg/m3/hr). Exposed groups were matched with longitudinal controls. In response to JP-8 inhalation, exposure animals demonstrated a dose-dependent as well as duration-determined reduction in BALF SP concentration. Both JP-8 concentrations caused significant pathological changes in lower pulmonary structures.

Temporal regulation by estrogen of beta-preprotachykinin mRNA expression in the rat ventromedial nucleus of the hypothalamus.

In the rat, reproduction and sexual behavior are controlled by the gonadal steroid regulation of synaptic interactions within the sexually dimorphic limbic-hypothalamic system. The effects of estrogen on the ventromedial nucleus of the hypothalamus, one nucleus within the circuit, are central to the modulation of this behavior. Involvement of the neuropeptide substance P, a member of the tachykinin family of neuropeptides, has been implicated in the regulation of both lordosis behavior and gonadotropin release. However, previous studies have provided conflicting evidence as to whether levels of substance P in the ventromedial nucleus of the hypothalamus are modulated by circulating estrogens. To study this question further, in situ hybridization histochemistry was used to examine levels of beta-preprotachykinin mRNA, which encodes substance P and other tachykinins, in the ventrolateral subdivision of the ventromedial hypothalamus at 10 consecutive timepoints over a 4 day period subsequent to an acute administration of estrogen. Following estrogen treatment, beta-preprotachykinin mRNA expression was increased in cells of the ventrolateral portion of the ventromedial nucleus of the hypothalamus which constitutively express beta-preprotachykinin mRNA; however, there were no statistically significant changes in the number of cells that express detectable levels of beta-preprotachykinin mRNA in the ventrolateral portion of the ventromedial nucleus. Estrogen treatment produced two peaks of beta-preprotachykinin mRNA expression, the first at 2 h and the second at 48 h after the injection of estrogen. These data indicate that estrogen has both rapid and prolonged effects on beta-preprotachykinin mRNA levels, suggesting that estrogen may affect different cellular mechanisms relevant to the induction of beta-preprotachykinin mRNA expression.

Tachykinin systems in the spinal cord and basal ganglia: influence of neonatal capsaicin treatment or dopaminergic intervention on levels of peptides, substance P-encoding mRNAs, and substance P receptor mRNA.

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro.

Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.

Nerve-induced tachykinin-mediated vasodilation in skeletal muscle is dependent on nitric oxide formation.

Nerve-induced vasodilatation was studied by intravital microscopy of the rabbit tenuissimus muscle, pretreated with pancuronium, phentolamine, and guanethidine. Nerve stimulation of the tenuissimus nerve induced a vasodilatation which was frequency and pulse duration-dependent and insensitive to atropine and propanolol but abolished by tetrodotoxin. The nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), but not its enantiomer, D-NAME, markedly inhibited the vasodilation induced by nerve stimulation or by exogenous substance P or neurokinin A. Vasodilatation due to calcitonin gene-related peptide, prostaglandin E2 or nitroprusside was unaffected. The substance P antagonist, spantide (30 microM), significantly attenuated nerve-induced vasodilatation, in parallel with L-NAME. Our results indicate that nerve-induced vasodilatation in skeletal muscle can be attributed to the release of substance P and/or other tachykinins and that nitric oxide subsequently mediates the response to endogenous tachykinins released from nerves.

Capsaicin inhibits airway hyperresponsiveness but not lipoxygenase activity or eosinophilia after repeated aerosolized antigen in guinea pigs.

To evaluate the role of tachykinins in airway hyperresponsiveness following repeated aerosolized antigen challenge in guinea pigs, we treated 12 guinea pigs with capsaicin (105.6 mg cumulative dose given subcutaneously over 5 days) after sensitization to ovalbumin (OA) and before three repeated OA aerosol challenges per wk for 4 to 5 wk. Ten guinea pigs received identical OA sensitization and challenges without capsaicin treatment, and four of eight nonsensitized controls received capsaicin followed by saline challenges. Capsaicin treatment did not alter antibody responses to OA as assessed by passive cutaneous anaphylaxis, nor did it alter lipoxygenase products from OA-stimulated bronchial tissue in vitro. Capsaicin completely inhibited the increased pulmonary resistance (RL) to acetylcholine produced by repeated aerosolized OA, whereas it did not alter baseline RL or acetylcholine responses of controls. Capsaicin did not alter airway eosinophilia induced by repeated aerosolized OA. We conclude that neuropeptides play an important role in antigen-induced airway hyperresponsiveness without altering antibody levels, lipoxygenase mediator production, or airway eosinophilia.