Enhanced airway responses to substance P after repeated challenge in guinea pigs

We performed three consecutive dose-response curves to rapid intravenous infusions of substance P (SP) in anesthetized, mechanically ventilated guinea pigs. The dose of SP required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) decreased 2.8-fold (P less than 0.002) and 3.3-fold (P less than 0.001) on the second and third dose-response curves, respectively, compared with the first. SP did not alter airway responses to intravenous histamine but did cause a significant (3.7-fold) decrease in ED50GL for dose-response curves to intravenous capsaicin, an agent that causes bronchoconstriction by release of endogenous tachykinins. The neutral metalloendopeptidase inhibitor thiorphan (0.5 mg) and the angiotensin-converting enzyme inhibitor captopril (1.7 mg) both caused a marked enhancement of airway responses to SP observed on the first dose-response curve but did not alter the enhancement of SP-induced airway responses produced by repeated SP challenge. The anticholinergic atropine (5 mg/kg iv), the antihistamine mepyramine (8 mg/kg iv), and the cyclooxygenase inhibitor indomethacin (30 mg/kg ip) had no effect on the first SP dose-response curve. Atropine and mepyramine did not prevent the enhancement of SP responses observed with repeated challenge, but after pretreatment with either indomethacin or acetylsalicylic acid, dose-response curves to SP were reproducible. Our results indicate that airway responses to intravenous SP are enhanced with repeated SP challenge and suggest that cyclooxygenase products of arachidonic acid metabolism are involved in the mediation of this phenomenon.     

Comparison of airway and blood eosinophil function after in vivo antigen challenge.

Eosinophils (EOS) are important effector cells in allergic diseases and asthma. However, functional characteristics of the EOS have been derived primarily from studies of blood cells, and it is unlikely that such assessments reflect events occurring in tissues or airways. To establish more precisely the function of airway EOS, segmental Ag challenge was used to elicit and isolate large numbers of these cells. Airway, as well as blood, EOS were isolated from allergic patients 48 h after segmental Ag challenge. Both blood and bronchoalveolar lavage (BAL) EOS were fractionated over Percoll density gradients; by using this protocol, three density-distinct populations of pure (>90%) EOS were obtained from BAL fluid (1.100, 1.095, and 1.090 g/ml) and one from blood (1.100 g/ml). The functions of these various populations were compared by measuring superoxide generation, adherence to collagen and endothelial cell monolayers, cell surface receptors, and in vitro survival. BAL EOS of all three densities had greater superoxide generation and adherence with FMLP activation than did corresponding blood EOS. In contrast, blood and airway EOS responded similarly to PMA. BAL EOS also had increased expression of CD11b/CD18 and HLA-DR. The intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent marker indo-1/acetoxymethyl ester. FMLP caused a greater and more sustained increase in [Ca2+]i with BAL than blood EOS. EGTA blocked the sustained component of the [Ca2+]i response to FMLP. Our findings indicate that BAL EOS have an enhanced [Ca2+]i response to activation that may contribute to their functional up-regulation.     

Cell-mediated and humoral immune responses to aspermatogenic antigen in experimental allergic orchitis in the guinea-pig

Guinea-pigs were immunized with a defined and highly potent aspermatogenic antigen, G75m, and the occurrence of orchitis was correlated with (1) cell-mediated immune response to G75m, determined by lymph node cell proliferation and by secretion of macrophage migration inhibitory factor (MIF) by peritoneal exudate cells, and (2) humoral antibodies to G75m and to cell surface antigens of guinea-pig testicular cells, by radioimmunometric assays. A consistent temporal relationship between cell-mediated immune responses and disease was found: lymph node cell proliferation was positive by Day 4, followed 3 days later by maximum secretion of MIF, and orchitis lesions were manifest on Day 10. In contrast, maximal IgG antibodies to G75m or to the surface antigens of spermatozoa/testicular cells were detected at a time when cell-mediated immune responses and active testicular lesions had subsided. In individual animals, lymph node cell proliferation increased with severity of orchitis, while MIF secretion by peritoneal cells increased with orchitis only late in the disease. Early in disease, MIF response showed a negative correlation with orchitis. Moreover, peritoneal injection of oil reduced the incidence of early lymph node cell proliferative responses, and delayed the onset of testicular disease. These findings are consistent with competition between different inflammatory sites for recently antigen-activated T lymphocytes. We conclude that (1) the development of orchitis correlates with cell-mediated immune responses to purified aspermatogenic antigens but not with IgG antibody responses, and (2) when the same animal is used to assess different aspects of cellular immunity and autoimmune disease, one study may significantly influence the other.     

Pressor and tachycardic responses to intravenous substance P in anesthetized rats

Intravenous injection of 3–33 nmol/kg of substance P (SP) caused pressor and tachycardic responses in anesthetized rats. The responses were not blocked by a ganglion nicotinic receptor antagonist or by pithing. Pretreatment with reserpine blocked both responses. β-Adrenoceptor blockade attenuated only the tachycardic response, and -adrenoceptor blockade attenuated only the pressor response. These findings indicated that the effects of SP to increase blood pressure and heart rate are due to sympathetic ganglion stimulation. Studies with adrenalectomized rats showed that stimulation of the adrenals by SP contributes to both responses but makes a greater contribution to the tachycardic response. These observations raise the possibility that the tachykinin innervation of sympathetic ganglia and the adrenal medulla may be involved in the local regulation of blood pressure and heart rate.     

Enhanced ganglionic responses to substance P in spontaneously hypertensive rats

Intravenous injection of substance P (SP) increases blood pressure in normotensive rats by stimulating sympathetic ganglia. This study compared the effects of SP to increase renal nerve firing and blood pressure in normotensive and hypertensive rats treated with chlorisondamine. The increase in renal nerve firing was greatest in spontaneously hypertensive rats (SHR), intermediate in Wistar rats, and least in Wistar-Kyoto (WKY) rats. Blood pressure was increased more in SHR than in Wistar rats. Blood pressure was not increased in WKY rats. Responses to the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium were the same in the three strains. These results suggest that there is a selective increase in the action of SP on sympathetic ganglia of SHR and that ganglion responsiveness to SP is correlated with its effect on blood pressure.     

Adenoviral transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation

Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.     

Distribution and origin of substance P and neuropeptide Y-immunoreactive nerves in the guinea-pig heart

Summary The localization and origin of substance P (SP)-, neuropeptide Y (NPY)-, and noradrenaline/tyrosine hydroxylase (NA/TH)-immunoreactive (IR) nerves in the guinea-pig heart were investigated by means of immunohistochemistry; quantitative analysis was performed by radioimmunoassay (NPY) and high performance liquid chromatography (NA). Both untreated animals and animals subjected to stellatectomy, combined stellatectomy and local capsaicin pretreatment of the vagal nerves or systemic application of capsaicin were studied. A dense network of SP-IR nerves was observed in the right atrium in different locations: (1) around local cardiac ganglion cells, (2) close to blood vessels, (3) within the myocardium, and (4) close to and within peri and endocardium.A moderately dense SP-innervation, mainly related to blood vessels, was found in the ventricles. Very dense networks of NPY and TH-IR nerve fibers with an overlapping distributional pattern around blood vessels and in the myocardium were seen in both the atria and the ventricles. In addition, some cell bodies in local cardiac ganglia were NPY-IR. Bilateral stellatectomy resulted in a reduction of SP-IR in the right atrium (55% of control), which was more pronounced after additional capsaicin pretreatment of the vagal nerves (44% of control).In the left ventricle no significant depletion of SP-IR was seen by either stellatectomy or combined stellatectomy and capsaicin treatment of the vagal nerves. It was not possible to establish any defined target areas within the heart for vagal or spinal SP-IR afferents by use of immunohistochemical methods. Systemic capsaicin treatment caused a total loss of SP-IR nerves in the heart. After bilateral stellatectomy the levels of NPY-IR and NA were reduced to about 10% of control in both the right atrium and left ventricle. In accordance, NPY and TH-IR nerves were also almost totally absent in the heart after bilateral stellatectomy.     

Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the third ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of substance P (SP) or neurotensin (NT) and plasma GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of physiologic saline iv or into the third ventricle did not modify plasma hormone levels. Intraventricular injection of SP or NT at doses of either 0.5 or 2 μg elevated plasma GH concentrations within 5 min and they remained elevated for 60 min. Third ventricular injection of similar doses of SP or NT had no effect on plasma TSH. An intermediate dose of 1 μg of SP or NT given iv had no effect on plasma GH but NT elevated plasma TSH. Incubation of hemipituitaries from OVX rats with varying doses of SP or NT did not alter GH release into the medium but TSH release was enhanced with NT at doses of 100 or more ng/ml of medium. It is suggested that SP acts centrally to stimulate growth hormone-releasing factor (GRF) or to inhibit somatostatin release and thereby enhance GH release and that NT acts directly on the pituitary to stimulate TSH release.     

Tracheal smooth muscle responses to substance P and neurokinin A in the piglet.

The tachykinins substance P (SP) and neurokinin A (NKA) have been shown to induce airway smooth muscle contraction in mature animals, and the enzyme neutral endopeptidase (NEP) modulates this effect. We evaluated maturation of SP- and NKA-induced tracheal smooth muscle contraction and modulation of their effects by NEP in anesthetized, paralyzed, and artificially ventilated piglets less than 4 days, 2-3 wk, and 10 wk of age. Tracheal smooth muscle tension was measured in vivo from an open tracheal segment by use of a force transducer. Intravenous SP caused a dose-dependent increase in tracheal tension in all three age groups; however, the response in less than 4-day-old piglets was significantly weaker than in 2- to 3- and 10-wk-old piglets. NKA caused a dose-dependent increase in tracheal tension only in 2- to 3- and 10-wk-old piglets. The response of tracheal tension to NKA was weaker than the response to SP in all age groups. Atropine (2 mg/kg) significantly diminished the responses of tracheal tension to SP and NKA, indicating a cholinergic contribution to these responses at all ages. Intravenous thiorphan, a known NEP inhibitor, potentiated the effects of SP only in 2- to 3- and 10-wk-old piglets and did not affect the response of tracheal tension to NKA at any age. Biochemical analyses demonstrated a significant increase in tracheal NEP activity in comparably aged piglets over the first 10 wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular responses to substance P in the nucleus tractus solitarii: microinjection study in conscious rats

The cardiovascular effects of substance P (SP) microinjections in the nucleus tractus solitarii (NTS) were evaluated in conscious rats. We chose this model because it is an effective way to access some of the cardiovascular effects of neurotransmitters in the NTS without the inconvenience of blunting pathways with anesthetic agents or removing forebrain projections by decerebration. The cardiovascular responses to SP injections were also evaluated after chronic nodose ganglionectomy. We found that, in conscious rats, SP microinjections into the NTS induced hypertension and tachycardia. Unilateral and bilateral SP injections into the NTS caused a slow increase in blood pressure and heart rate that peaked 1.5-5 min after injection and lasted for 20-30 min. Nodose ganglionectomy increased the duration of the pressor and tachycardic effects of SP and enhanced the pressor response. These data show that SP in the NTS is involved in pressor pathways. The supersensitivity to SP seen after nodose ganglionectomy suggests that vagal afferent projections are involved in those pressor pathways activated by SP in the NTS.     

Biological activity and enzymic degradation of substance P analogs: implications for studies of the substance P receptor.

Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.     

Macrophage immunity to influenza virus: in vitro and in vivo studies.

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.     

The cardiac effects of amitraz in the guinea-pig in vivo and in vitro

Intravenous amitraz caused significant hypotension and bradycardia in pentobarbitone anaesthetized guinea-pigs. Depression of blood pressure reached a plateau with a dose of 10 mg/kg but heart rate continued to fall in a dose-dependent manner, up to a fall of 90 beats per minute after a total of 160 mg/kg/min. Amitraz was then tested on spontaneously beating guinea-pig isolated atria. The maximum bath concentration approximated a blood concentration produced by 5 mg/kg amitraz in the guinea-pig (2.3 X 10(-4) M). Amitraz did not significantly shift the dose-response curve to isoprenaline or acetylcholine but antagonized histamine rate responses competitively in the presence of propranolol (2 X 10(-6) M). Propranolol unmasked a dose-dependent depressant effect of amitraz on atrial rate, an effect abolished with atropine (1 X 10(-5) M). Amitraz increased atrial force of contraction, an effect which was not seen when propranolol was present in the bath solution. Amitraz also depressed atrial rate directly, but this effect was minor in comparison to bradycardia seen in the guinea-pig. It is likely that the cardiovascular depression seen in the guinea-pig following amitraz i.v. is caused by an alteration in autonomic drive rather than a significant direct cardiac effect.     

Vascular response in a non-uterine site to implantation-stage embryos in the rat and guinea-pig: in vivo and ultrastructural studies

Summary Preimplantation-stage embryos were transferred to the anterior eye chamber of recipient rats and guinea-pigs. After implantation had occurred the influence of the embryo on the iris vasculature was examined ultrastructurally. In both species, the earliest effect of embryonic implantation was an increased stromal oedema. Under increasing embryonic influence the vascular endothelial cells showed an increased number of projections into the vascular lumen, while in the rat, endothelial projections were also found pushing back into the basement membrane. In the rat, the endothelium became very irregular in thickness prior to complete disintegration and loss during more advanced stages of implantation. Rat embryonic trophoblast was found invading iris vasculature, particularly in areas where the iridial endothelium was partially or completely missing. Other cells in the iris, including the stroma, appeared to be less affected. In the guinea-pig, however, trophoblast cells appeared to be capable of invading the vasculature by displacing endothelial cells that still appeared morphologically normal.     

The role of substance P in stress and anxiety responses

Summary. Substance P (SP) is one of the most abundant peptides in the central nervous system and has been implicated in a variety of physiological and pathophysiological processes including stress regulation, as well as affective and anxiety-related behaviour. Consistent with these functions, SP and its preferred neurokinin 1 (NK1) receptor has been found within brain areas known to be involved in the regulation of stress and anxiety responses. Aversive and stressful stimuli have been shown repeatedly to change SP brain tissue content, as well as NK1 receptor binding. More recently it has been demonstrated that emotional stressors increase SP efflux in specific limbic structures such as amygdala and septum and that the magnitude of this effect depends on the severity of the stressor. Depending on the brain area, an increase in intracerebral SP concentration (mimicked by SP microinjection) produces mainly anxiogenic-like responses in various behavioural tasks. Based on findings that SP transmission is stimulated under stressful or anxiety-provoking situations it was hypothesised that blockade of NK1 receptors may attenuate stress responses and exert anxiolytic-like effects. Preclinical and clinical studies have found evidence in favour of such an assumption. The status of this research is reviewed here.     

Electrical responses of frog olfactory mucosa to the administration of acetylcholine and substance P

The effects of acetylcholine and substance P were studied on the Frog's olfactory mucosa. Stimulation with these chemicals elicited low-threshold slow electrical potentials. Moreover, prior application of substance P strongly depressed the electrical response of the mucosa to acetylcholine. These results are discussed in relation to the possibility that acetylcholine and substance P could act on the functioning of the olfactory neuroreceptors.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.