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Biomedical subjects

D L Ochs

Publications and source records attributed to D L Ochs.

17 recordsLinked to original sources
PubMed1990

Lack of formyl-methionyl-leucyl-phenylalanine receptors on porcine neutrophils.

The response of blood neutrophils to the chemotactic peptide formyl-methyl-leucyl-phenylalanine varies among species. Our results indicate that this peptide does not activate the respiratory burst of porcine neutrophils. Specifically, concentrations less than or equal to 10(-6) M did not cause production of either superoxide or hydrogen peroxide. Studies designed to delineate the biochemical deficit responsible for these results indicated that these cells do not express specific chemotactic peptide receptors on the external surface of the plasma membrane. Although these data do not rule out the possibility that internal stores of chemotactic peptide receptor exist, attempts to induce expression of the receptor by priming the cells with either lipopolysaccharide or calcium ionophore were unsuccessful.

Animals↗
PubMed1989

Immunodeficiency in young pigs.

Immunodeficiency in neonatal and young pigs was studied in terms of T-cell function. Generalized T-cell deficiency did not exist in young pigs on the basis of the in vitro response of blood mononuclear cells to a polyclonal T-cell mitogen, phytohemagglutinin. However, immunodeficiency that extended from birth up to 4 weeks, was observed in serum antibody concentration and in vitro proliferative responses of blood mononuclear cells from young pigs exposed to a low antigen dose of a T-cell dependent antigen, egg white lysozyme. The low in vitro proliferative response to lysozyme was not attributable simply to a lack of interleukin-2 production, because supplementation with human interleukin-2 did not enhance the in vitro cellular response. Also, pokeweed mitogen-stimulated B cells from young pigs up to the age of 5 to 6 weeks produced immunoglobulin concentration, which also was not affected by the addition of human interleukin-2 to the in vitro cultures. The blood mononuclear cells obtained from pigs within the first 5 to 6 weeks after birth and incubated with monoclonal antibodies reactive to all T cells (MSA4), helper T cells (74-12-4) or suppressor/cytotoxic T cells (76-2-11) did not yield consistent excess of suppressor/cytotoxic T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Calcium-stimulated ATPase activity in plasma membrane vesicles from pancreatic acinar cells.

Plasma membrane vesicles were prepared from rat pancreatic acinar cells using nitrogen cavitation and magnesium precipitation. The vesicles exhibited ATPase activity that was stimulated by submicromolar concentrations of free calcium and was dependent upon the presence of magnesium. This enzyme activity was localized to the cytoplasmic surface of the plasma membrane by two criteria. First, no activity was observed when intact cells replaced the membrane vesicles in the assay. Second, right side-out and inside-out vesicles were separated using concanavalin A sepharose-B. The calcium-stimulated, magnesium-dependent ATPase activity per mg protein in the inside-out fraction was 60% greater than that occurring in the mixed vesicle preparation. These results indicate that the plasma membrane of pancreatic acinar cells has a calcium-stimulated, magnesium-dependent ATPase located on its cytoplasmic surface and that this enzyme is stimulated by submicromolar concentrations of free calcium.

Adenosine Triphosphate↗
PubMed1988

Cellular defense of the avian respiratory system: effects of Pasteurella multocida on respiratory burst activity of avian respiratory tract phagocytes.

The respiratory tract of healthy chickens contain few free-residing phagocytic cells. Intratracheal inoculation with Pasteurella multocida stimulated a significant (P less than 0.05) migration of cells to the lungs and air sacs of White Rock chickens within 2 hours after inoculation. We found the maximal number of avian respiratory tract phagocytes (22.9 +/- 14.0 x 10(6] at 8 hours after inoculation. Flow cytometric analysis of these cells revealed 2 populations on the basis of cell-size and cellular granularity. One of these was similar in size and granularity to those of blood heterophils. Only this population was capable of generating oxidative metabolites in response to phorbol myristate acetate. The ability of the heterophils to produce hydrogen peroxide, measured as the oxidation of intracellularly loaded 2',7'-dichlorofluorescein, decreased with time after inoculation. These results suggest that the migration of heterophils, which are capable of high levels of oxidative metabolism, to the lungs and air sacs may be an important defense mechanism of poultry against bacterial infections of the respiratory tract.

Animals↗
PubMed1986

Cell calcium of ejaculated rabbit spermatozoa before and following in vitro capacitation.

Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2. Measurements of trypan blue exclusion indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of quin-2 loaded sperm was calculated to be 144 +/- 14 nM. Spermatozoa capacitated in vitro either before or following quin-2 loading had intracellular free calcium levels similar to that of non-capacitated sperm. These studies indicate that the concentration of intracellular free calcium of ejaculated rabbit spermatozoa does not change as a result of in vitro capacitation.

Acrosome↗
PubMed1985

Relation between free cytosolic calcium and amylase release by pancreatic acini.

Pancreatic acini were loaded with the Ca-selective fluorescent indicator quin-2 by incubation with its acetyoxymethyl ester. Loading acini with 844 +/- 133 microM quin-2 altered neither their ultrastructure nor their viability. The rate of amylase release from quin-2-loaded acini in response to the secretagogue carbachol, however, was significantly smaller than that of control acini. Studies in which acini were loaded with both quin-2 and a similar Ca-chelating compound, BAPTA, indicated that this reduced amylase release was related to the Ca buffering properties of quin-2. The concentration of free intracellular Ca calculated from the fluorescence of quin-2 was 90 +/- 18 nM. Stimulation by carbachol of acini suspended in media containing 1.25 mM Ca caused a rapid, transient enhancement of this value. After stimulation amylase release, the onset of the rise in free cytosolic Ca levels was observed in 1.1 +/- 0.1 s following the addition of agonist, and peak Ca levels (545 +/- 112 nM) were obtained within 5.3 +/- 0.3 s. For concentrations of carbachol less than or equal to 10(-6) M, a stoichiometric relation was found between stimulated amylase release and the peak concentration of free cytosolic Ca achieved. At higher concentrations of carbachol, however, the peak free cytosolic Ca remained constant while amylase release declined. The latency of the rise in intracellular Ca following stimulation of acini suspended in Ca-free media was not different from that observed for acini suspended in normal media, but the rise time was significantly prolonged. In the presence of extracellular Ca, the intracellular level of Ca remained elevated 2.8-fold above basal levels for at least 15 min following stimulation with 10(-6) M carbachol, whereas it had returned to near resting levels by 15 min when either 3 X 10(-7) or 3 X 10(-5) M carbachol was the stimulus. The Ca ionophore ionomycin (10-6 M) induced changes in the level of free cytosolic Ca similar to those caused by 10(-6) M carbachol. Ionomycin, however, stimulated only approximately one-third as much amylase release. These data suggest that factors in addition to changes in free cytosolic Ca may be important in regulating enzyme secretion by pancreatic acinar cells.

Aminoquinolines↗
PubMed1985

Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells.

The involvement of Ca2+ and cyclic nucleotides in neurohormonal regulation of Na+-K+-ATPase (Na+-K+ pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86Rb+ uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [3H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca2+-free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca2+, while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca2+ chelator. Basal intracellular Ca2+ concentration ([Ca2+]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively. Forskolin, secretin, and cAMP analogues had no effect on [Ca2+]i, nor did they either reduce or potentiate the rise in [Ca2+]i evoked by CCh.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1984

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport.

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.

Animals↗
PubMed1983

Intracellular free calcium concentrations in isolated pancreatic acini; effects of secretagogues.

Isolated pancreatic acini were loaded with the calcium selective fluorescent indicator, quin-2. Measurements of cellular K+ content and lactic dehydrogenase release indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of unstimulated acinar cells was calculated to be 180 +/- 4 nM. When cells suspended in media containing millimolar calcium were exposed to the secretagogues carbachol and cholecystokinin a rapid increase in [Ca2+]i occurred. Both the amplitude and rate of rise of the concentration increase were dose dependent with [Ca2+]i reaching a maximum of 860 +/- 41 nM. The dose-response relationship coincides with the known concentration dependence of the stimulation of amylase release by these agents. In the absence of extracellular calcium, carbachol was still able to elicit a rise in [Ca2+]i. These studies indicate that pancreatic secretagogues induce an increase in [Ca2+]i of acinar cells, both in the presence or absence of extracellular calcium.

Aminoquinolines↗
PubMed1983

ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes.

Plasma membrane vesicles were prepared from guinea pig peritoneal exudate neutrophils, using nitrogen cavitation to rupture the plasma membrane and differential centrifugation to separate the vesicles. The vesicles were enriched 13.2-fold in (Na+, K+)-ATPase activity and had a cholesterol:protein ratio of 0.15, characteristic of plasma membranes. Contamination of the vesicle preparation with DNA or marker enzyme activities for intracellular organelles was very low. Studies designed to determine vesicle sidedness and integrity indicated that 33% were sealed, inside-out; 41% were sealed, right side-out, and 26% were leaky. The vesicles accumulated 45Ca2+ in a linear fashion for 45 min. The uptake was dependent on the presence of oxalate and MgATP in the incubating medium. Uptake showed a Ka for free Ca2+ of 164 nM and a Vmax of 17.2 nmol/mg . min (based on total protein). GTP, ITP, CTP, UTP, ADP, or AMP supported uptake at rates less than or equal to 11% of ATP. Ca2+ uptake was maximal at pH 7-7.5. Calcium stimulated the hydrolysis of ATP by the vesicles with a Ka for free Ca2+ of 440 nM and Vmax of 17.5 nmol/mg . min (based on total protein). When the Ca2+ uptake rate was based upon those vesicles expected to transport Ca2+ (33% sealed, inside-out vesicles) and Ca2+-stimulated ATPase activity was based upon those vesicles expected to express that activity (26% leaky + 33% sealed, inside-out vesicles), the molar stoichiometry of Ca2+ transported:ATP hydrolyzed was 2.12 +/- 0.12. Calmodulin did not increase either Vmax or Ka for free Ca2+ of the uptake system in the vesicles, even when they were treated previously with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The high affinity of this system for Ca2+, specificity for ATP, physiological pH optimum, and stoichiometry of Ca2+ transported:ATP hydrolyzed suggest that it represents an important mechanism by which neutrophils maintain low levels of cytoplasmic free Ca2+.

Adenosine Triphosphate↗