Functional and molecular characterization of tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK1 receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RT-PCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin NK1 receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK2 receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK3 receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.     

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory,motor and vascular functions under normal and neuropathic conditions

Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK₁ tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP^−/−, Tac1^−/−, and Tacr1^−/−) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30–40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1^−/− and Tacr1^−/− mice, but was absent in PACAP^−/− animals. Motor coordination of the PACAP^−/− and Tac1^−/− groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1^−/− mice. Basal postoperative microcirculation on the plantar skin of PACAP^−/− mice did not differ from the wildtypes, but was significantly lower in Tac1^−/− and Tacr1^−/− ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP^−/− mice, but not in Tacr1^−/− and Tac1^−/− animals. Both PACAP and SP/NKA, but not NK₁ receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications.     

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in β-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.     

Antimicrobial activity of PLUNC protects against Pseudomonas aeruginosa infection

Epithelial antimicrobial activity may protect the lung against inhaled pathogens. The bactericidal/permeability-increasing protein family has demonstrated antimicrobial activity in vitro. PLUNC (palate, lung, and nasal epithelium associated) is a 25-kDa secreted protein that shares homology with bactericidal/permeability-increasing proteins and is expressed in nasopharyngeal and respiratory epithelium. The objective of this study was to determine whether PLUNC can limit Pseudomonas aeruginosa infection in mice. Transgenic mice (Scgb1a1-hPLUNC) were generated in which human PLUNC (hPLUNC) was directed to the airway epithelium with the Scgb1a1 promoter. The hPLUNC protein (hPLUNC) was detected in the epithelium throughout the trachea and bronchial airways and in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid from transgenic mice exhibited higher antibacterial activity than that from wild type littermates in vitro. After in vivo P. aeruginosa challenge, Scgb1a1-hPLUNC transgenic mice displayed enhanced bacterial clearance. This was accompanied by a decrease in neutrophil infiltration and cytokine levels. More importantly, the overexpressed hPLUNC in Scgb1a1-hPLUNC transgenic mouse airway significantly enhanced mouse survival against P. aeruginosa-induced respiratory infection. These data indicate that PLUNC is a novel antibacterial protein that likely plays a critical role in airway epithelium-mediated innate immune response.     

Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites

To investigate the sites of the free fatty acid (FFA) effects to increase basal hepatic glucose production and to impair hepatic insulin action, we performed 2-h and 7-h Intralipid + heparin (IH) and saline infusions in the basal fasting state and during hyperinsulinemic clamps in overnight-fasted rats. We measured endogenous glucose production (EGP), total glucose output (TGO, the flux through glucose-6-phosphatase), glucose cycling (GC, index of flux through glucokinase = TGO - EGP), hepatic glucose 6-phosphate (G-6-P) content, and hepatic glucose-6-phosphatase and glucokinase activities. Plasma FFA levels were elevated about threefold by IH. In the basal state, IH increased TGO, in vivo glucose-6-phosphatase activity (TGO/G-6-P), and EGP (P < 0.001). During the clamp compared with the basal experiments, 2-h insulin infusion increased GC and in vivo glucokinase activity (GC/TGO; P < 0.05) and suppressed EGP (P < 0.05) but failed to significantly affect TGO and in vivo glucose-6-phosphatase activity. IH decreased the ability of insulin to increase GC and in vivo glucokinase activity (P < 0.01), and at 7 h, it also decreased the ability of insulin to suppress EGP (P < 0.001). G-6-P content was comparable in all groups. In vivo glucose-6-phosphatase and glucokinase activities did not correspond to their in vitro activities as determined in liver tissue, suggesting that stable changes in enzyme activity were not responsible for the FFA effects. The data suggest that, in overnight-fasted rats, FFA increased basal EGP and induced hepatic insulin resistance at different sites. 1) FFA increased basal EGP through an increase in TGO and in vivo glucose-6-phosphatase activity, presumably due to a stimulatory allosteric effect of fatty acyl-CoA on glucose-6-phosphatase. 2) FFA induced hepatic insulin resistance (decreased the ability of insulin to suppress EGP) through an impairment of insulin's ability to increase GC and in vivo glucokinase activity, presumably due to an inhibitory allosteric effect of fatty acyl-CoA on glucokinase and/or an impairment in glucokinase translocation.     

Occurrence and effects of multiple tachykinins; substance P, neurokinin A and neuropeptide K in human lower airways

In the present work we have studied the occurrence of different tachykinins (substance P (SP), neurokinin A (NKA) and neuropeptide K (NPK)) in human distal bronchi and pulmonary arteries by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). We have also compared the biological effects of different tachykinins on isolated human bronchi and pulmonary arteries in vitro. The concentration of immunoreactive SP using antiserum SP2 in the pulmonary arteries was higher (1.34 +/- 0.15 pmol/g) than in the bronchi (0.56 +/- 0.05 pmol/g). The contents of other tachykinins than SP measured using antiserum K12 was on the other hand considerably higher in the bronchi (0.33 +/- 0.14 pmol/g) than in pulmonary arteries (0.13 +/- 0.02 pmol/g). Immunoreactive materials corresponding to SP, NKA and NPK were identified in bronchial extracts by RIA combined with HPLC, which also indicated the presence of an eledoisin (ELE)-like component. In vitro studies showed that NKA was the most potent of the tachykinins as a bronchoconstrictor agent, being several hundred-fold more active than SP, acetylcholine and histamine. NPK had an intermediate potency. The bronchoconstrictor effect of NKA was unaffected by atropine, mepyramine and cimetidine. The tachykinins SP and NKA had on the other hand, a rather equal potency in inducing relaxation of serotonin precontracted pulmonary arteries. In conclusion, multiple tachykinins are present in lower airways of man. These peptides exert different biological activities whereby NKA is a very active bronchoconstrictor agent compared to SP while both NKA and SP have rather similar relaxatory activities of vascular smooth muscle.     

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.     

Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 +/- 5 vs. 24 +/- 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR-/-) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2--> LDLR-/-; n = 19) or wild-type C57BL/6 littermates (C57 BL/6-->LDLR-/-; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2-->LDLR-/- and C57BL/6-->LDLR-/- mice. Aortic root atherosclerosis was increased by 57% in sPLA2-->LDLR-/- mice compared with C57BL/6-->LDLR-/- controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2alpha-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2-->LDLR-/- compared with C57BL/6-->LDLR-/- mice, indicating significantly increased in vivo oxidative stress in sPLA2--> LDLR-/-. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism.     

Effects of some neurokinin A analogues on tachykinin-induced contraction of guinea pig trachea.

A series of analogues of neurokinin A (NKA) has been synthesized and characterized by testing for their abilities, in vitro, to contract guinea pig tracheal smooth muscle or to antagonize NKA-, NKB- and SP-induced contraction of this tissue. Substitution of NKA residues Gly8 or Leu9 by conformationally restricting amino acids produced peptides that were antagonists of NKA action, but the type and specificity of the antagonism depended on the size of the peptide. Thus, while [Ala5, Aib8, Leu10]NKA(2-10) showed no agonism and was a specific, competitive antagonist of NKA, [Ala5, Aib8, Leu10]NKA(4-10) was a noncompetitive antagonist of NKA and substance P (SP) and was itself a weak agonist at concentrations above 10(-7) M.     

Ventilatory behavior after hypoxia in C57BL/6J and A/J mice.

Given the environmental forcing by extremes in hypoxia-reoxygenation, there might be no genetic effect on posthypoxic short-term potentiation of ventilation. Minute ventilation (VE), respiratory frequency (f), tidal volume (VT), and the airway resistance during chemical loading were assessed in unanesthetized unrestrained C57BL/6J (B6) and A/J mice using whole body plethysmography. Static pressure-volume curves were also performed. In 12 males for each strain, after 5 min of 8% O2 exposure, B6 mice had a prominent decrease in VE on reoxygenation with either air (-11%) or 100% O2 (-20%), due to the decline of f. In contrast, A/J animals had no ventilatory undershoot or f decline. After 5 min of 3% CO2-10% O2 exposure, B6 exhibited significant decrease in VE (-28.4 vs. -38.7%, air vs. 100% O2) and f (-13.8 vs. -22.3%, air vs. 100% O2) during reoxygenation with both air and 100% O2; however, A/J mice showed significant increase in VE (+116%) and f (+62.2%) during air reoxygenation and significant increase in VE (+68.2%) during 100% O2 reoxygenation. There were no strain differences in dynamic airway resistance during gas challenges or in steady-state total respiratory compliance measured postmortem. Strain differences in ventilatory responses to reoxygenation indicate that genetic mechanisms strongly influence posthypoxic ventilatory behavior.     

Degradative enzymes modulate airway responses to intravenous neurokinins A and B

We studied the effects of the neutral endopeptidase (NEP) inhibitor thiorphan (1.7 mg/kg iv) and the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg iv) on airway responses to rapid intravenous infusions of neurokinin A (NKA) and neurokinin B (NKB) in anesthetized, mechanically ventilated guinea pigs. The dose of NKA required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) was fivefold less (P less than 0.0001) in animals treated with thiorphan compared with controls. NKA1-8, a product resulting from cleavage of NKA by NEP, had no bronchoconstrictor activity. Similar results were obtained by using NKB as the bronchoconstricting agent. Captopril had no significant effect on airway responses to NKA or NKB. In contrast, both thiorphan and captopril decrease the ED50GL for substance P (SP). We also compared the relative bronchoconstrictor potency of NKA, NKB, and SP. In control animals, the rank order of ED50GL values was NKA much less than NKB = SP. NKA also caused a more prolonged bronchoconstriction than SP or NKB. Thiorphan had no effect on the rank order of bronchoconstrictor potency, but in animals treated with captopril, the rank order of ED50GL values was altered to NKA less than SP less than NKB. These results suggest that degradation of NKA and NKB by NEP but not by ACE is an important determinant of the bronchoconstriction induced by these peptides. The degradation by ACE of SP but not NKA or NKB influences the observed relative potency of the three tachykinins as bronchoactive agents.     

Tachykinin release from rat spinal cord in vitro and in vivo in response to various stimuli

The aim of the present study was to establish an experimental model, previously used in cat, for studying tachykinin release from the rat spinal cord in vivo and to compare the results with those obtained in vitro. Stimulation with pulses of 40 mM potassium or 10 microM capsaicin in the spinal cord superfusion fluid increased the release of substance P (SP)- and neurokinin A (NKA)-like immunoreactivity (LI) both in vivo and in vitro. The amounts of SP-LI and NKA-LI released by potassium in vitro were 1.02 +/- 0.12 and 1.17 +/- 0.22 fmol/mg tissue, respectively. Also the ratio between the amounts released by two consecutive potassium stimulations were similar for SP-LI and NKA-LI. Reversed-phase high performance liquid chromatography of the NKA-LI released in vitro by potassium or capsaicin revealed a major immunoreactive component coeluting with synthetic NKA. Despite the use of highly sensitive radioimmunoassays, basal release of SP-LI and NKA-LI was found only in 9 of 31 in vivo experiments. In these, peripheral electrical stimulation of the sciatic nerves (50 Hz, 50 V and 0.05 ms or 10 Hz, 10 V and 5 ms) induced an increase of the SP-LI and NKA-LI levels in the superfusates. This increase persisted for more than 40 min after a 2 min stimulation. In most experiments, however, no SP-LI or NKA-LI could be detected in the superfusates, neither at basal conditions nor following electrical nerve stimulation. Similarly, no release of SP-LI could be detected in response to various noxious mechanical, thermal or chemical stimuli applied to the skin. The present results demonstrate that the superfused rat spinal cord may be used to study in vivo release of tachykinins in response to intense chemical stimulation of the entire spinal cord. However, the method seems to be less suitable for studies of tachykinin release in response to electrical activation engaging only a few spinal segments or in response to natural noxious stimuli. The results obtained in vitro suggest that SP and NKA are released in equimolar amounts from the spinal cord upon stimulation with potassium.     

Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-dependent pathway

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~ 3-fold increase in proliferation and ~ 3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O₂^??) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.     

Effects of trigeminal neurotransmitters on piglet pial arterioles.

We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.     

Coexisting peptides in capsaicin-sensitive sensory neurons: release and actions in the respiratory tract of the guinea-pig

Release of substance P (SP) and neurokinin A (NKA), was demonstrated in the isolated perfused guinea-pig lung. Significant release was obtained by perfusion with capsaicin, high potassium, histamine, bradykinin dimethylphenylpiperazinium, and by electrical vagal nerve stimulation. Capsaicin-induced peptide release was not blocked by 1 microM clonidine. SP and NKA contracted respiratory smooth muscle, NKA being 42 times more potent. Both tachykinins were equipotent in relaxing pulmonary artery. It is concluded that multiple tachykinin can be released from capsaicin-sensitive sensory nerves in the respiratory tract, exerting multiple effects on the target tissues.     

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)     

Cerebellar LTD and pattern recognition by Purkinje cells

Many theories of cerebellar function assume that long-term depression (LTD) of parallel fiber (PF) synapses enables Purkinje cells to learn to recognize PF activity patterns. We have studied the LTD-based recognition of PF patterns in a biophysically realistic Purkinje-cell model. With simple-spike firing as observed in vivo, the presentation of a pattern resulted in a burst of spikes followed by a pause. Surprisingly, the best criterion to distinguish learned patterns was the duration of this pause. Moreover, our simulations predicted that learned patterns elicited shorter pauses, thus increasing Purkinje-cell output. We tested this prediction in Purkinje-cell recordings both in vitro and in vivo. In vitro, we found a shortening of pauses when decreasing the number of active PFs or after inducing LTD. In vivo, we observed longer pauses in LTD-deficient mice. Our results suggest a novel form of neural coding in the cerebellar cortex.     

Intermittent hypoxia activates peptidylglycine alpha-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.     

Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice

To assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar (BAL) lavage fluid was increased 2- or 4-fold relative to that in WT mice. Lung structure and cellular composition of BAL were not altered by the expression of lysozyme. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. To determine whether killing of bacteria was enhanced by expression of rat lysozyme, 5-wk-old transgenic mice and WT littermates were infected with 10(6) CFU of group B streptococci or 10(7) CFU of a mucoid strain of Pseudomonas aeruginosa by intratracheal injection. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h postinfection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. Increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection.     

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia.

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20-36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress (P < 0.005), twitch half-relaxation time (P < 0.02), and tetanic stress at 20 or 30 Hz (P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals (P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.