Magnetic resonance imaging assessment of articular disc position in temporomandibular disorder subjects with various bite registrations.

Objective: This study aimed to evaluate the validity and reliability of three bite registrations on articular disc position in temporomandibular disorder patients using magnetic resonance imaging (MRI). Materials and Methods: Fifteen clinically symptomatic and orthodontically untreated temporomandibular disorder patients within the age range of 17-40 years (mean age: 28.5 years) were examined. Each patient was subjected to three bite registrations, namely maximum intercuspation, initial contact bite and Roth power centric bite, and evaluated with MRI. Results: On the right side, the mean vertical and horizontal measurement values of the point in the most posterior aspect of the posterior band of the articular disc in relation to horizontal reference line (HRL) and vertical reference line (VRL) in the sagittal view in the Roth power centric bite were lesser (2.720 ± 1.239 mm and 2.380 ± 1.185 mm, respectively), in comparison with the other two bites, and on the left side too, it was lesser in the Roth power centric bite (2.293 ± 0.979 mm and 2.360 ± 1.078 mm, respectively), when compared to the other two bites. Statistical analysis also showed the significance of Roth power centric bite over the other two bites. Conclusions: Favourable articular disc positional changes were observed in the Roth power centric bite followed by the initial contact bite and that maximum disc recapture was observed in most patients with the Roth power centric bite rather than in initial contact bite and maximum intercuspation positions. The Roth power centric bite could be assumed to be the ideal method for articulation and fabrication of gnathological splints for treating patients with temporomandibular disorders.

MicroRNA Regulation of Airway Inflammation and Airway Smooth Muscle Function: Relevance to Asthma.

Genetic and environmental factors contribute to the onset and severity of asthma. Molecular pathogenesis of asthma involves changes in gene expression by a variety of inflammatory mediators acting in autocrine and paracrine fashion on effector cells of the airways. Transcriptional regulation of gene expression in resident airway cells has been studied extensively. However, protein function in a target cell can be regulated at multiple levels starting from transcription followed by post-transcription, translation, and post-translation steps. In this context, small noncoding RNAs known as microRNAs (miRNAs) have evolved as one of the key regulators of gene expression post-transcriptionally. Most importantly, miRNA expression is dynamic in nature and can be regulated by a variety of external stimuli. Altered expression of individual or a group of miRNAs is thought to contribute to human diseases. Recent studies have implicated differential expression of miRNAs in the lungs during inflammation. Most importantly, advanced biochemical and molecular tools could be used to manipulate miRNA expression thereby effecting functional changes in target cells and organ systems. This review summarizes the current understanding of miRNA in the regulation of airway function in health and disease, and highlights the potential clinical utility of mRNAs as biomarkers of airway diseases and as potential therapeutic targets.

Comparison of frictional resistance of esthetic and semi-esthetic self-ligating brackets.

AIM: The frictional resistance encountered during sliding mechanics has been well established in the orthodontic literature, and it consists of complex interactions between the bracket, archwire, and method of ligation the claim of reduced friction with self-ligating brackets is often cited as a primary advantage over conventional brackets. This study was done to compare and evaluate the frictional forces generated between fully esthetic brackets and semi-aesthetic self-ligating brackets, which are of passive form and SEM (scanning electron microscope) study of the Brackets after Frictional evaluation. MATERIALS AND METHODS: Two types of self-ligating esthetic brackets, Damon clear (Ormco) made of fully ceramic and Opal (Ultradent Products, USA) and, Two types of self-ligating semi-esthetic brackets, Clarity SL (3M Unitek) and Damon 3 (Ormco) both of which are made of ceramic with metal slot. Arch wires with different dimensions and quality 17 × 25, 19 × 25 Titanium Molybdenum Alloy (TMA) and 17 × 25, 19 × 25 stainless steel that came from plain strands of wire were used for frictional comparison test. The brackets used in this study had 0.022 × 0.028 inch slot. RESULTS: The statistical tests showed significantly smaller amount of kinetic frictional forces is generated by Damon 3 (semi-esthetic self-ligating brackets). For each wire used, Damon 3 displayed significantly lower frictional forces (P ≤ 0.05) than any of the self-ligating system, followed by Opal (fully esthetic self-ligating brackets) which generated smaller amount of frictional forces but relatively on the higher side when compared with Damon 3. Damon clear (fully esthetic self-ligating brackets) generated the maximum amount of kinetic forces with all types of wire dimensions and properties when compared to the other three types of self-ligating system. Clarity SL (semi-esthetic self-ligating brackets) generated smaller amount of frictional forces when compared with Damon clear and relatively higher amount of frictional forces when compared to Opal and Damon 3.

A new approach for evaluation of canine dento alveolar distraction using cone-beam computed tomography.

OBJECTIVE: The aim was to evaluate and plan the canine dento alveolar distractions (DADs) with the use of cone-beam computed tomography (CBCT). MATERIALS AND METHODS: 5 patients are requiring 10 canine DADs were selected for the study. A custom-made DAD distractor was fabricated for the study. CBCT scans were taken prior to and post thedistraction. DAD parameters such as Canine retraction, canine and molar rotation, molar anchor loss and level of the osteotomy cut above the canine was evaluated. RESULTS: Average canine retraction was 7.5 mm in 17 days, molar anchor loss was 0.5 mm, canine and molar rotations were 8° and 0.40° and thedistance of the osteotomy cut to the canine was1.93 mm. CONCLUSION: The CBCT can be used to accurately evaluate the canine DADtechnique.

Comparison of essential drug list in a rural secondary care hospital in south India with Indian & World Health Organization list 2011.

OBJECTIVE: Fixed drug combinations are a major marketing strategy in India but it can compromise the rational use of medicines. In this study we compared the fixed drug combinations and dosage forms in the hospital pharmacy before and after introducing the essential drug list. We also compared the Hospital Essential Drug List (HEDL) 2011 with the World Health Organization (WHO) Essential Drug List (EDL) 2011 and the National Essential Drug List of India (NEDL) 2011. METHODS: The study was done in a secondary level care charity hospital at Anantapur, AP with a bed size of 315 and an average OP per day of 1200-1700 visits. We compared the three essential drug lists (HEDL, WHOEDL and NEDL) and the hospital drug list before introducing EDL. Drugs which were present in NEDL and not present in the HEDL were also screened. Microsoft excel was used to tabulate the results and for graphs. RESULTS: The number of medicines used in the hospital before and after the introduction of the HEDL was 1627 and 424 respectively. On comparison, WHOEDL 2011 have 350 and NEDL of India have 348 medicines. While preparing the HEDL, 46 double drug combinations decreased to 15 and 9 triple drug combinations decreased to 1. In the case of injections, 20 double drug combinations decreased to 6 and 1 triple drug combination increased to 2. The number of tablets, capsules, injections, syrups, powders and inhalers was reduced to almost half. The great reductions were in 51 ointments to 9, 69 drops to 5, 11 paste to 0, 21 solutions to 3 and 14 creams to 1. The dosage forms removed included elixirs, insulin pens, gums, paste, paints, gargles and mouthwashes. CONCLUSIONS: There was drastic reduction in the number of medicines and dosage forms when the HEDL was implemented. Many of the fixed drug combinations were also removed for improving the rational use of medicines. The WHO essential drug list 2011, national essential drug list of India 2011 and the hospital essential drug list 2011 were comparable with few exceptions.

TNF-alpha induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-kappaB and AP-1.

In human airway smooth muscle (HASM) cells, the expression of CD38, which synthesizes the calcium-mobilizing molecule cyclic ADP-ribose, is augmented by TNF-alpha, a cytokine implicated in asthma. We determined the role of mitogen-activated protein kinase (MAPK) in the activation of NF-kappaB and AP-1 in the regulation of CD38 expression in HASM cells. In HASM cells exposed to TNF-alpha (40 ng/ml), the inhibitors of extracellular signal-regulated kinase (ERK), p38, or c-Jun NH(2)-terminal kinase (JNK) decreased CD38 expression and ADP-ribosyl cyclase activity. Transfection of HASM cells with a dominant negative MEK decreased while a wild-type ERK increased TNF-alpha-induced CD38 expression. Electrophoretic mobility shift assays (EMSAs) were performed using nuclear proteins and consensus sequences to detect the effect of the MAPKs on NF-kappaB and AP-1 activation. EMSAs confirmed the role of p38 and JNK in mediating NF-kappaB and AP-1 activation. Transfection of a dominant negative c-Jun decreased TNF-alpha-induced CD38 expression indicating involvement of AP-1. Stability of TNF-alpha-induced CD38 transcripts were determined in the presence of MAPK inhibitors after arresting the transcription with actinomycin D. Transcript stability decreased in the presence of ERK and p38 MAPK, but not the JNK, inhibitors. These results indicate that regulation of CD38 expression through p38 and JNK MAPKs involves NF-kappaB and AP-1 activation, and ERK and p38 MAPKs also regulate expression posttranscriptionally through message stability.

Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18.

To map the site involved in Mannheimia haemolytica leukotoxin (LktA) binding and biological activity within bovine CD18, bovine x human CD18 chimeric constructs were generated and coexpressed with bovine CD11a in K562 cells. Studies with the chimeric leukocyte function-associated antigen 1 transductants demonstrate that the site required for LktA binding and biological effects resides within amino acid residues 500 and 600 of the extracellular region of bovine CD18.

Biological effects of two genetically defined leukotoxin mutants of Mannheimia haemolytica.

Mannheimia(Pasteurella)haemolytica serotype 1 is the primary causative agent responsible for bovine pneumonic mannheimiosis, also known as shipping fever in cattle. The bacterium produces a variety of virulence factors, foremost of which is the exotoxic leukotoxin. The leukotoxin is a calcium-dependent cytolysin that is a member of the RTX (repeats in toxin) family and exhibits a narrow cell-type and species specificity and has biological effects only on ruminant leukocytes and platelets. The genetic organization of the leukotoxin is comprised of four genes: lktC, lktA, lktB and lktD. The lktA structural gene encodes the protoxin (pro-LktA) and lktC encodes a transacylase that post-translationally modifies the inactive pro-LktA to a biologically active wild-type leukotoxin (LktA). The LktA has been implicated as the key factor that contributes to the pathogenesis of lung injury associated with the disease and considerable efforts have been employed in abrogating toxin function while retaining immunogenicity, with an eye towards design of attenuated vaccines. We hypothesized that the pro-LktA retains the ability to cause biological effects on target cells as has been reported in the case of the closely related RTX toxin alpha-hemolysin (HlyA). We also examined the biological effects of an amino-terminal truncation mutant leukotoxin DeltaLktA on target cells. Thus the objectives of our study were to investigate whether two different mutant leukotoxins, one a nonacylated pro-LktA, and the other lacking 344 amino acids at the N-terminal end of the LktA protein; DeltaLktA, are capable of (i). binding to the beta2-integrin leukotoxin receptor, (ii). inducing the elevation of second messenger intracellular calcium ([Ca(2+)](i)), and (iii). inducing inflammatory gene expression, reactive oxygen metabolites (ROMs) and cytolysis in target cells. Our results demonstrate that neither acylation nor the amino terminal 344 amino acids are required for LktA binding but are essential for LktA-induced [Ca(2+)](i) elevation, generation of ROM, generation of the inflammatory cytokine IL-8 and cytolysis in target cells.

Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects.

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.

Effects of halothane on sarcoplasmic reticulum calcium release channels in porcine airway smooth muscle cells.

BACKGROUND: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels. METHODS: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy. RESULTS: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 microM Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45+/-13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65+/-13% and 68+/-22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 microM ryanodine blunted the [Ca2+]i response to halothane by 32+/-13% and 39+/-21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane. CONCLUSIONS: The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.

Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity.

Effects of intestinal ischemia on in vitro activity of adjacent jejunum in samples obtained from ponies.

OBJECTIVE: To determine whether intestinal ischemia would alter activity of the jejunum in vitro or alter staining characteristics for certain types of enteric neurotransmitters. SAMPLE POPULATION: Jejunal samples obtained from 10 ponies. PROCEDURE: Jejunal samples were obtained from locations proximal and distal to an area of small intestine made ischemic for 60 minutes. A portion of each sample was stained to detect substance P-like immunoreactivity, cholinergic and adrenergic neurons, and nitric oxide synthase. Portions of the remaining samples were suspended in muscle baths. General activity patterns (frequency and amplitude of contraction), responses to neuronal depolarization induced by electrical field stimulation (EFS), and responses to 1 microM norepinephrine (NE) were compared with responses of a normal section of small intestine obtained prior to ischemic insult. RESULTS: Staining patterns were not altered. Proximal and distal sections had evidence of decreased contractility, compared with the normal section. Contraction frequency also was decreased, and distal sections had lower contraction frequency than proximal sections. Relaxation responses were decreased in distal sections. Responses to NE differed significantly for distal and proximal sections, compared with normal sections. CONCLUSIONS AND CLINICAL RELEVANCE: Short-term ischemia can significantly affect adjacent bowel. Contractile and relaxation responses are impaired. Discrepancies in intestinal motility patterns and alterations in response to NE for sections proximal and distal to ischemic intestine could lead to clinical ileus or slowed transit of ingesta.

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

Evaluation of substance P as a neurotransmitter in equine jejunum.

OBJECTIVE: To determine whether substance P (SP) functions as a neurotransmitter in equine jejunum. SAMPLE POPULATION: Samples of jejunum obtained from horses that did not have lesions in the gastrointestinal tract. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscle, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 2 intensities (30 and 70 V) and various frequencies on muscle strips that were maintained at low tension or were under contraction. A neurokinin-1 receptor blocker (CP-96,345) was added to baths prior to EFS to interrupt SP neurotransmission. Additionally, direct effects of SP on muscle strips were evaluated, and SP-like immunoreactivity was localized in intestinal tissues, using indirect immunofluorescence testing. RESULTS: Substance P contracted circularly and longitudinally oriented muscle strips. Prior treatment with CP-96,345 altered muscle responses to SP and EFS, suggesting that SP was released from depolarized myenteric neurons. Depending on orientation of muscle strips and stimulation variables used, CP-96,345 increased or decreased the contractile response to EFS. Substance P-like immunoreactivity was detected in the myenteric plexus and circular muscle layers. CONCLUSIONS AND CLINICAL RELEVANCE: Substance P appears to function as a neurotransmitter in equine jejunum. It apparently modulates smooth muscle contractility, depending on preexisting conditions. Effects of SP may be altered in some forms of intestinal dysfunction. Altering SP neurotransmission in the jejunum may provide a therapeutic option for motility disorders of horses that are unresponsive to adrenergic and cholinergic drugs.

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes.

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.

Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle.

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

Spatial and temporal aspects of calcium sparks in porcine tracheal smooth muscle cells.

Spontaneous, localized intracellular Ca(2+) concentration ([Ca(2+)](i)) transients (Ca(2+) sparks) in skeletal, cardiac, and smooth muscle cells are thought to represent Ca(2+) release through ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle (TSM) cells, ACh induces propagating [Ca(2+)](i) oscillations that also represent Ca(2+) release through RyR channels. We used real-time confocal imaging to examine the spatial and temporal relationships of Ca(2+) sparks to propagating [Ca(2+)](i) oscillations in TSM cells. Ca(2+) sparks within an intracellular region displayed different spatial Ca(2+) distributions with every occurrence. The amplitudes of Ca(2+) sparks within a region were approximately integer multiples of the smallest response. However, across different regions, the attributes of Ca(2+) sparks varied considerably. Individual sparks were often grouped together and coupled across adjacent regions. Fusion of individual sparks produced large local elevations in [Ca(2+)](i) that occasionally triggered a propagating [Ca(2+)](i) wave. The incidence of sparks was increased by ryanodine and caffeine but was unaffected by removal of extracellular Ca(2+). Exposure to ACh triggered repetitive, propagating [Ca(2+)](i) oscillations that always originated from foci with a high spark incidence. The [Ca(2+)](i) oscillations disappeared with the removal of ACh, and Ca(2+) sparks reappeared. We conclude that agonist-induced [Ca(2+)](i) oscillations represent a spatial and temporal integration of local Ca(2+)-release events through RyR channels in TSM cells.

Adrenergic, cholinergic, and nonadrenergic-noncholinergic intrinsic innervation of the jejunum in horses.

OBJECTIVE: To determine the major neurotransmitters that regulate contractile activity in the jejunum of horses. SAMPLE POPULATION: Jejunal specimens from 65 horses without gastrointestinal tract lesions. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscular layer, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 30 and 70 V intensities and at various frequencies on muscle strips maintained at low or high muscle tone. To detect residual nonadrenergic-noncholinergic neurotransmission, the response of muscle to EFS in the presence of adrenergic and cholinergic blockade was compared with the response in the presence of tetrodotoxin. RESULTS: Atropine (ATR) decreased the contractile response of muscle strips to EFS under most conditions. However, ATR increased the contractile response of high-tone circular muscle. Adrenergic blockade generally increased the muscle responses to 30 V EFS and in high-tone longitudinal muscle but decreased contractile responses in high-tone circular muscle. Tetrodotoxin significantly altered the responses to EFS, compared with adrenergic and cholinergic receptor blockade. CONCLUSIONS: Acetylcholine and norepinephrine appear to be important neurotransmitters regulating smooth muscle contractility in the equine jejunum. They induce contraction and relaxation, respectively, in most muscle preparations, although they may cause opposite effects under certain conditions. In addition, nonadrenergic-noncholinergic excitatory and inhibitory influences were detected. CLINICAL RELEVANCE: Acetylcholine or norepinephrine release within the myenteric plexus of horses may alter gastrointestinal motility.

Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kappaB activation and calcium elevation.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity.