High Concentrations Of Substance P Research Articles

Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part II. Biomarkers of nociception, inflammation, and stress

The objective was to assess the effect of oral administration of acetylsalicylic acid after calving on the concentrations of substance P (SP), haptoglobin (HP), and cortisol in lactating dairy cows. Holstein dairy cows (n = 152) from 3 organic herds were included. At parturition, cows were blocked by parity [multiparous (MULT) and primiparous (PRIM)] and calving ease [eutocia (EUT) and dystocia (DYS)] and were randomly assigned to 1 of 2 treatment groups: aspirin (ASP; n = 76), in which within 12 h after parturition cows received 4 treatments with acetylsalicylic acid (100 mg/kg; 2 boluses) at 12-h intervals, or placebo (PLC; n = 76), in which within 12 h after parturition cows received 4 consecutive treatments with gelatin capsules (2 capsules) containing water 12 h apart. Blood samples were collected immediately before treatment and at 12, 24, 36, 48, and 168 h (7 DIM) for assessment of circulating concentration of SP, HP, and cortisol. Based on farm records, cows were classified in the following clinical disease categories: no clinical disease event (NO-EVT), a single clinical disease event (SI-EVT), and more than 1 clinical disease event (MU-EVT). The study data were analyzed as a randomized complete block design using mixed multiple linear and logistic regression models. With regard to HP, there was a tendency for an interaction between treatment and parity, where MULT cows treated with ASP had lower concentration of HP compared with MULT cows treated with PLC (ASP = 124.33 ± 6.83 µg/mL; PLC = 143.9 ± 7.24 µg/mL). Analysis by calving ease showed that cows with DYS had higher concentrations of HP (DYS = 159.17 ± 5.97 µg/mL; EUT = 138.72 ± 6.22 µg/mL) and SP (only at 168 h; DYS = 64.99 pg/mL, 95% confidence interval, CI: 2.68-2.81; EUT = 60.33 pg/mL, 95% CI: 2.91-3.06) after calving compared with EUT cows. Regardless of treatment, PRIM cows had higher concentrations of SP (MULT = 55.11 pg/mL, 95% CI: 1.27-1.30; PRIM = 57.62 pg/mL, 95% CI: 1.99-2.06), HP (MULT = 134.14 ± 4.96 µg/mL; PRIM = 163.75 ± 7.76 µg/mL), and cortisol (MULT = 18.65 µg/mL, 95% CI: 1.02-1.05; PRIM = 21.92 µg/mL, 95% CI: 1.67-1.74) compared with MULT cows. In addition, cows that experienced SI-EVT or MU-EVT had higher concentrations of HP (NO-EVT = 134.13 ± 5.95 µg/mL; SI-EVT = 142.68 ± 7.32 µg/mL; MU-EVT = 170.03 ± 9.42 µg/mL) and cortisol (NO-EVT = 17.86 µg/mL, 95% CI: 1.20-1.24; SI-EVT = 21.01 µg/mL, 95% CI: 1.61-1.67; MU-EVT = 22.01 µg/mL, 95% CI: 2.08-2.18) compared with cows with NO-EVT recorded. Results from this study suggest that a short-duration anti-inflammatory therapy after calving reduced HP in MULT cows but may not have effects on SP and cortisol concentrations. Calving ease and parity affected the concentrations of markers of inflammation, nociception, and stress regardless of treatment. Further research is warranted to assess anti-inflammatory strategies aimed at decreasing inflammation and stress in DYS and PRIM cows and therefore improve welfare and performance of these high-priority groups.

Ubiquitin-dependent Down-regulation of the Neurokinin-1 Receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Paradoxical Exacerbation of Psoriasis in AIDS: Proposed Explanations Including the Potential Roles of Substance P and Gram-negative Bacteria

Psoriasis, a TH1-induced disorder, is not more common in human immunodeficiency virus (HIV) infection than in the general population. However, it may appear for the first time or pre-existing psoriasis may worsen and be difficult to treat in HIV disease. The paradoxical exacerbation of psoriasis in AIDS has not been fully explained. Various explanations have been proposed including (a) the reduction of Langerhans' cells (LCs) in HIV disease, (b) the direct epidermal proliferative effect of HIV, (c) the altered cytokine profile in HIV disease, (d) HIV-induced macrophage nitric oxide (NO) production, (e) the increased CD8/CD4 T-cell ratio in HIV infection and (f) the increased colonization of skin by Staphylococcus aureus. However, the observations that (a) LCs cells play an important role in the pathogenesis of psoriasis and a variety of topical and systemic psoriasis treatments cause a reversible decrease in LC function, (b) psoriasis may improve in end-stage HIV infection, (c) overproduction of some TH2 cytokines and underproduction of IL-2 in HIV infection, and (d) the presence of NO favors a TH2 response over a TH1 response make the first four explanations difficult to interpret. Since psoriasis is exacerbated in HIV infection possibly due to the increased staphylococcal colonization, and psoriatic keratinocytes could aggravate HIV infection through production of TNF-α, it could be reasoned that in HIV-positive psoriatics a strong vicious cycle is present between the degree of immune deficiency and the staphylococcal colonization, explaining the poor prognosis of both AIDS and psoriasis in these patients.With reference to the studies which indicate significant involvement of substance P (SP) in the pathogenesis of psoriasis and on the other hand increased release of this agent by HIV-infected immune cells it is proposed that SP plays an important role in creating the paradox. Since in HIV-positive psoriatics the source of SP is largely immune cells not neurons, capsaicin, which exerts its action selectively on a subpopulation of neurons, could not be of significant therapeutic value. As SP significantly enhances HIV-1 replication in latently infected immune cells, psoriatic lesions, being heavily infiltrated with immune cells and having high concentrations of SP, could serve as high HIV-replication foci, with the resultant rapid progression of the infection towards AIDS.Additionally, given that lipopolysaccharide is supposed to exacerbate psoriasis, increase of Gram-negative infections or cutaneous colonization with these organisms in AIDS may partly explain the paradox.Understanding the HIV-induced immunodysregulation that is associated with psoriasis in some HIV-seropositive patients may assist in the delineation of the immunopathogenesis of the disease in HIV-seronegative psoriatics.

Substance P modulates localized calcium transients and membrane current responses in murine colonic myocytes.

1. Neurokinins contribute to the neural regulation of gastrointestinal (GI) smooth muscles. We studied responses of murine colonic smooth muscle cells to substance P (SP) and NK(1) and NK(2) agonists using confocal microscopy and the patch clamp technique. 2. Colonic myocytes generated localized Ca(2+) transients that were coupled to spontaneous transient outward currents (STOCs). SP (10(-10) M) increased Ca(2+) transients and STOCs. Higher concentrations of SP (10(-6) M) increased basal Ca(2+) and inhibited Ca(2+) transients and STOCs. 3. Effects of SP were due to increased Ca(2+) entry via L-type Ca(2+) channels, and were mediated by protein kinase C (PKC). Nifedipine (10(-6) M) and the PKC inhibitor, GF 109203X (10(-6) M) reduced L-type Ca(2+) current and blocked the effects of SP. 4. SP responses depended upon parallel stimulation of NK(1) and NK(2) receptors. NK(1) agonist ([Sar(9),Met(O(2))(11)]-substance P; SSP) and NK(2) agonists (neurokinin A (NKA) or GR-64349) did not mimic the effects of SP alone, but NK(1) and NK(2) agonists were effective when added in combination (10(-10)-10(-6) M). Consistent with this, either an NK(1)-specific antagonist (GR-82334; 10(-7) M) or an NK(2)-specific antagonist (MEN 10,627; 10(-7) M) blocked responses to SP (10(-6) M). 5. Ryanodine (10(-5) M) blocked the increase in Ca(2+) transients and STOCs in response to SP (10(-10) M). 6. Our findings show that low concentrations of SP, via PKC-dependent enhancement of L-type Ca(2+) current and recruitment of ryanodine receptors, stimulate Ca(2+) transients. At higher concentrations of SP (10(-6) M), basal Ca(2+) increases and spontaneous Ca(2+) transients and STOCs are inhibited.

Substance P modulates nicotinic responses of intracardiac neurons to acetylcholine in the guinea pig.

Application of substance P (SP) to intracardiac neurons of the guinea pig causes slow depolarization and increases neuronal excitability. The present study was done to determine the influence of SP on fast excitatory responses of intracardiac neurons to ACh. Intracellular recording methods were used to measure responses of intracardiac neurons in whole mount preparations of atrial ganglionated nerve plexus from guinea pig hearts. Local pressure ejection of 100 microM SP (1 s) from a glass micropipette caused slow depolarization of all neurons (n = 38) and triggered action potential generation in 47% of the cells tested. Bath application of SP (0.5-100 microM) caused a dose-dependent depolarization of intracardiac neurons but rarely evoked action potentials, even at the highest concentration. However, such treatment with SP enhanced nicotinic responses evoked by local pressure ejections of ACh (10 mM, 10- to 100-ms duration) in 77% of intracardiac neurons studied (n = 52). A significant increase in amplitude of ACh-evoked fast depolarization occurred during treatment with 0.5 microM SP (13.0 +/- 1.8 mV for control vs. 17.7 +/- 1.9 mV with SP present, n = 7, P = 0.019). At higher concentrations of SP, enhancement of the response to ACh resulted mainly in action potential generation. However, responses to ACh were attenuated by SP in 15% of the intracardiac neurons studied. This attenuation occurred primarily during exposure to 10 and 100 microM SP and was manifest as a reduction in amplitude of nicotinic fast depolarization or inhibition of ACh-evoked action potentials. These findings support the conclusion that SP could function as a neuromodulator and neurotransmitter in intracardiac ganglia of the guinea pig.

Effects of substance P on [Ca2+]i and force in intact guinea pig ileal smooth muscle

In intact sheets of the guinea pig ileal longitudinal muscle, loaded with fura-2, both substance P (SP)- and K(+)-induced contractile responses are preceded by cytoplasmic free Ca2+ ([Ca2+]i) variations. In response to SP (10(-10)-10(-7) M), [Ca2+]i and force increased concentration dependently. From 10(-9) M on, the response was biphasic: an initial Ca2+ spike and force transient were followed by a tonic component. The [Ca2+]i and force vs. log [SP] curves were sigmoidal for the initial phasic component, while a homologous receptor desensitization caused a reduced tonic component of the [Ca2+]i and contractile response at higher concentrations of SP. Both intracellular Ca2+ release and Ca2+ influx play a role in the effect of the peptide. In depolarizing solutions (140 mM K+) and in Ca2(+)-free medium (2 mM EGTA), SP induced a transient increase in [Ca2+]i and force. The Ca2+ stores used by SP and acetylcholine (ACh) overlap. [Ca2+]i and force fell to base-line levels when the extracellular Ca2+ was reduced from 1.2 to 0.2 mM during stimulation with SP. Verapamil reduced the tonic response. We also studied the relation between [Ca2+]i and force for the peak and steady-state values after stimulation with increasing concentrations of SP and K+. The phasic force response was linearly related to log [Ca2+]i. During the sustained response to K+, the Ca2+ sensitivity of the contractile/regulatory proteins was decreased, whereas no changes were observed during prolonged stimulation with the peptide.

Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody-coated microelectrodes

The localization of the sites of release of immunoreactive substance P (SP) in the spinal cord after peripheral nerve stimulation has been examined in the anaesthetized cat. A new technique using antibodies bound to the outside of glass microelectrodes has allowed the identification of these sites with a spatial precision previously unobtainable. A basal release of SP was detected and this was not increased by electrical stimulation of large myelinated primary afferent fibres. Excitation of unmyelinated primary afferents resulted in the release of high concentrations of SP confined to the region of the substantia gelatinosa and lamina V–VI.

POSSIBLE INVOLVEMENT OF SUBSTANCE P IN THE CONTRACTION PRODUCED BY PERIARTERIAL NERVE STIMULATION IN THE RAT ILEUM

The effect of substance P (SP) on the contractile responses produced by periarterial (mesenteric) nerve stimulation was studied in the rat isolated ileum. Periarterial nerve stimulation at 1-50 Hz, with 10 V (maximum) and 0.2 msec pulse duration, for 15-20 sec, produced frequency-dependent contractions in the rat ileum. In the presence of guanethidine (10 microM) and 6-hydroxydopamine (1 microM), to block noradrenergic responses, periarterial nerve stimulation at 1-20 Hz still produced small contractions which were reduced by atropine (1 microM) and morphine (1 microM). In the presence of atropine, morphine, guanethidine and 6-hydroxydopamine, the contraction produced by periarterial nerve stimulation was readily abolished by tetrodotoxin (1 microM), capsaicin (3.3 microM) and an SP-antagonist (SPA1, 10 microM). SP in low concentrations (0.01-1.0 microM) potentiated the contractions produced by periarterial nerve stimulation at 1-2 Hz by 20-30%. High concentrations of SP (1.0-10.0 microM) reduced the contractile response by 40-50%. Indomethacin (2.8 microM) amd mepyramine (1 microM) had no effect on these responses. When the mesenteric nerve supply to the gut was cut, periarterial nerve stimulation produced no contraction in the rat ileum. However, SP in low concentrations, still produced small contractions which were abolished by an SP-antagonist but not by tetrodotoxin. SP in low concentrations, slightly increased the contractions produced by ACh (0.5-50 microM) or TEA (2.4-12 mM). High concentrations of SP significantly reduced the ACh and TEA-induced contractions in the rat ileum.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of substance P antagonists on the atropine-sensitive and atropine-resistant responses of guinea-pig ileum to substance P

The effects of substance P (SP) antagonists on the atropine-sensitive and atropine-resistant responses to SP of guinea-pig isolated ileum were investigated. The atropine-resistant response to SP was the contractile response on untreated preparations, and the atropine-sensitive response was the response to high concentrations of SP (5 × 10 −7 M) on preparations desensitized to SP with a concentration of SP of 3 × 10 −7 M. The SP antagonists tested, ( d-Pro 2, d-Phe 7, d-Trp 9)-SP, ( d-Pro 2, d-Trp 7,9)-SP, ( d-Arg 1, d-Pro 2, d-Trp 7,9,Leu 11)-SP and ( d-Arg 1, d-Trp 7,9,Leu 11)-SP (spantide), did not have similar orders of potency on the atropine-sensitive and atropine-resistant responses to SP, spantide being more potent than the other antagonists on the atropine-resistant response but no more potent on the atropine-sensitive response than ( d-Pro 2, d-Phe 7, d-Trp 9)-SP or ( d-Arg 1, d-Pro 2, d-Trp 7,9,Leu 11)-SP. These findings are consistent with the hypothesis that neuronal receptors for SP differ from those on smooth muscle in guinea-pig ileum.

Substance P can contract the longitudinal muscle of the guinea-pig small intestine by releasing intracellular calcium.

The contraction of the longitudinal muscle of the guinea-pig isolated ileum in response to substance P (SP) in high [K+] medium and in Ca2+-free solution which contained EGTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca2+ from an intracellular store. In tissues contracted by K+, high concentrations of SP (greater than or equal to 0.1 microM) were still capable of causing a slight, transient contraction. Bathing the ileum in a Ca2+-free medium for 2.5 min greatly diminished the potency and efficacy of SP in contracting the longitudinal muscle but concentrations of 2.2-22 microM SP were still able to produce a response 30-40% of maximal. The responsiveness to SP was completely lost within 10 min of bathing in Ca2+-free medium but was partially restored by a brief exposure (0.5-2 min) to high concentrations of Ca2+ (9-72 mM). The restorative effect of Ca2+ depended on the concentration of Ca2+ and on the time for which the tissue was exposed to this Ca2+ concentration. The fact that high concentrations of SP were able to elicit a contraction in media containing high [K+] or no Ca2+, suggested that they may do so by releasing Ca2+ from an intracellular store which is not as sensitive to removal of extracellular Ca2+ or as easily accessible to EGTA as the extracellular space of the muscle. The location of this store is not known; it may be associated with the internal side of the cell membrane. There is apparently an overlap between the Ca2+ pool associated with the action of SP and the Ca2+ pools utilized by acetylcholine, histamine or tetraethylammonium, which accounts for the cross-desensitization observed between these agonists. It was not possible to determine whether autodesensitization to SP also results from depletion of an intracellular Ca2+ store.

The dog common carotid artery: a sensitive bioassay for studying vasodilator effects of substance P and of kinins.

In order to develop a sensitive pharmacological preparation which would allow the measurement of the inhibitory effects of kinins and substance P (SP) in vascular smooth muscles, several large arteries of the dog were studied in vitro. The common carotid artery was found to be one of the most sensitive preparations to SP and kinins. When contracted with low concentrations of noradrenaline (between 3.0 x 10(-8) and 3.0 x 10(-7) M), this artery responds to SP (6.5 x 10(-11)-6.5 x 10(-9) M) and bradykinin (BK) (8.1 x 10(-11)-9.1 x 10(-8) M) with relaxations that are proportional to the concentrations of the two peptides. SP and BK appear to exert their relaxant effects through the activation of specific receptors as the exposure of the common carotid artery to concentrations of [Leu8]-angiotensin II, propranolol, methysergide, cimetidine, or atropine sufficient to inhibit the effects of the corresponding agonists do not affect the relaxing effect of SP and BK. [Leu8]-des-Arg9-BK (1.0 x 10(-6) M), indomethacin (2.8 x 10(-5) M), and lioresal (4.7 x 10(-5) M) are also inactive. When the dog common carotid artery is desensitized with high concentrations of SP, BK, eledoisin, and physalaemin a cross-desensitization is observed only between SP and physalaemin. These results support the conclusion that SP and kinins act on different receptors. The order of potency of kinins is the following: BK = [Tyr(Me)8]-BK greater than des-Arg9-BK, suggesting that the receptor for kinins is of the B2 type. The order of potency of peptides related to SP is SP greater than C-terminal 4-11 greater than C-terminal hexapeptide 6-11, similar to that observed in other vascular preparations. The results summarized in this paper indicate that the dog common carotid artery is a preparation sensitive to SP and BK and useful for studying the relaxant effect of these two peptides on vascular smooth muscles.

POTENTIATION BY SUBSTANCE P OF CONTRACTIONS OF THE ISOLATED VAS DEFERENS OF THE MOUSE ELICITED BY ELECTRIC FIELD STIMULATION AND BY DRUGS

1 Isolated vasa deferentia from the mouse were opened longitudinally and suspended in Krebs solution at 37 degrees C in an organ bath. Contractions of the muscle were elicited by electric field stimulation, noradrenaline (10(-6) M) and acetylcholine (10(-6) M). Continued transmural stimulation evoked a biphasic response comprising a rapid twitch followed about 10 s later by a smaller, sustained rise in muscle tone.2 The amplitudes of nerve-mediated and drug-induced responses were considerably potentiated by substance P (SP) in the dose range 10(-12) to 10(-7) M. Higher concentrations of SP were directly spasmogenic. The sensitizing property of SP was dose-dependent and was usually well maintained, but always disappeared quickly on washing the preparation. In some experiments SP facilitated the twitch, but not the subsequent phase of the electrically-induced contraction or the response to externally applied noradrenaline.3 Phentolamine (10(-6) M) failed to block this effect of SP, but itself potentiated the nerve-mediated twitch, and completely abolished the sustained secondary contraction.4 Desmethylimipramine (10(-6) M) enhanced the delayed contraction but not the immediate contraction.5 The uptake of tritiated noradrenaline (3 x 10(-7) M) by vasa was inhibited by desmethylimipramine (10(-6) M) and increased by nialamide (3 x 10(-5) M), but was not modified by SP (10(-6) M).6 Nerve-mediated release of accumulated radioactivity was accelerated by phentolamine, but not by SP or desmethylimipramine.7 These findings suggest that SP sensitizes the muscle cells to depolarizing stimuli but that it has no facilitatory effect on sympathetic neural elements.