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M M Whalen

Publications and source records attributed to M M Whalen.

16 recordsLinked to original sources
PubMed2001

Butyltin exposure causes a rapid decrease in cyclic AMP levels in human lymphocytes.

Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells, and antibody-coated cells. Butyltins (BTs) are used in a variety of consumer products and industrial applications. Tributyltin (TBT) is found in dairy products, meat, and fish. Dibutyltin (DBT) is found in plastic products, beverages stored in PVC pipes during manufacturing, and poultry products. BTs appear to increase the risk of cancer and viral infections in exposed individuals. This increased risk may be due in part to the inhibitory effect of these compounds on the cytotoxic function of NK cells. A 24-h exposure of NK cells to 200 nM TBT or 1.5 microM DBT decreased the cytotoxic function of NK cells by greater than 90%. Higher concentrations of TBT and DBT decreased the cytotoxic function of NK cells (by greater than 90%) after only a 1-h exposure. A 24-h exposure to either TBT or DBT decreased intracellular ATP levels by about 30%. However, as much as a 1-h exposure to either 300 nM TBT or 10 microM DBT caused no significant decrease in ATP levels. Thus, a decrease in ATP levels is a longer-term consequence of BT exposure. Intracellular levels of cAMP are decreased by as much as 80% within 5 min of exposure to either TBT or DBT. This rapid decline in cAMP levels in NK cells may be a consequence of BT exposure that is related to the rapid decrease in the cytotoxic function of NK cells.

Adenosine Triphosphate↗
PubMed2000

Inhibition of a phosphodiesterase III in the lysis-sensitive target-induced elevation of cyclic AMP (cAMP) in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Phenyltin inhibition of the cytotoxic function of human natural killer cells.

Phenyltin (PT) contamination has been reported in water, sediment, and fish. However, the role of PT in weakening human immune function mediated through natural killer (NK) lymphocytes has not been elucidated. In this study, we report the effects of in vitro exposure to triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT) on the function of human NK cells. Exposure to TPT (1 microM, for 1 h) inhibited the tumor killing capacity of NK cells by 85%. Exposure of NK cells to DPT for 1 h (5 microM) and 24h (1.5 microM) reduced tumor lysis by greater than 90%. A 24-h exposure of NK cells to 5 microM MPT reduced tumor lysis by greater than 80%. Assays assessing the ability of NK cells to bind to tumor cells showed that a 24-h pretreatment with TPT, DPT, or MPT reduced NK cell binding to tumor cells by greater than 50%. The toxic potential of the PTs followed the order TPT > DPT > MPT. In comparison with butyltins (BTs), in vitro effects of PTs revealed that these compounds are relatively less toxic to NK cells than BTs. The results of this study provide evidence that phenyltin compounds are immunotoxic to human NK cells under in vitro experimental conditions.

Cell Adhesion↗
PubMed1999

Immunotoxicity of environmentally relevant concentrations of butyltins on human natural killer cells in vitro.

The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans.

Adult↗
PubMed1999

Lysophosphatidylcholine and arachidonic acid are required in the cytotoxic response of human natural killer cells to tumor target cells.

Treatment of human natural killer (NK) cells with phospholipase A(2) (PLA(2)) inhibitors, mepacrine and 4-bromophenacyl bromide (BPB), diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was significantly affected by BPB above 2 microM, but not by mepacrine at any concentration tested. This indicates that BPB is having effects on NK cells unrelated to its inhibition of PLA(2) activity at concentrations above 2 microM. The activation of phospholipase C in response to K562 cell binding (as measured by inositol phosphate turnover) was unaffected by inhibition of the PLA(2) activity. The products of PLA(2) catabolism are a fatty acid (often arachidonic acid) and a lysophospholipid. Inhibition of NK cytotoxicity by mepacrine or BPB is reversed significantly when lysophosphatidylcholine, but no other lysolipid, is added back to the NK cells before assaying for cytotoxicity. Arachidonic acid, but not linoleic acid, also significantly reverses inhibition of NK cytotoxicity. Finally, the 15-lipoxygenase product, 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE), is also able to reverse mepacrine-induced inhibition of NK cytotoxicity. The 5-lipoxygenase product 5S-HPETE was not effective. These data indicate that PLA(2) activation is a necessary signal in human NK cytotoxicity and that it is not involved in protein tyrosine kinase and subsequent phospholipase C activation; these latter two enzymes are also required in the cytotoxic response. Thus PLA(2) activation is either a more distal signal, dependent on activation of some earlier signal, or an independent cosignal stimulated by tumor-target binding which generates lysophosphatidylcholine, arachidonic acid, and/or a lipoxygenase product(s).

Acetophenones↗
PubMed1998

Lysis-sensitive targets stimulate an elevation of cAMP in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumour cells and virally infected cells (cytolysis) without prior sensitization. When cAMP is artificially elevated in NK cells, it is a potent inhibitor of their cytolytic function. We investigated whether NK-cell cAMP levels are modulated in response to tumour target cells to determine the potential of cAMP as a physiological regulator of NK cytotoxic function. When NK cells are exposed to a range of lysis-sensitive (LS) tumour-target cells there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. The peak increase in cAMP (200-400% above control) occurs at 30 min for all LS targets tested. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumour-target cells. The cAMP elevation may be dependent on both LS-target-stimulated adenylyl cyclase (AC) activation and LS-target-stimulated phosphodiesterase (PDE) inhibition. When the NK cells are pretreated with the protein tyrosine kinase (PTK) inhibitor, genistein (30 micrograms/ml), the AC-activation component of the cAMP elevation is abolished. Thus, the AC-activation component appears to require PTK activation. When NK cells are pretreated with the protein kinase C (PKC) inhibitor, chelerythrine chloride (10 microM) the cAMP elevation in response to LS targets was not diminished. This indicates that neither the AC-activation component nor any PDE-inhibition component require PKC activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Phospholipase C activation in the cytotoxic response of human natural killer cells requires protein-tyrosine kinase activity.

Treatment of highly purified natural killer (NK) cells with the protein-tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was not affected by PTK inhibition. However, activation of phospholipase C (PLC) in response to K562 cell binding (as measured by inositol phosphate turnover) was decreased by as much as 75% when PTK activity was inhibited. Furthermore, there was an increase in tyrosine phosphorylation of NK cell PLC gamma 2 after exposure to K562 target cells. These data indicate that a PTK is involved in the activation of NK PLC by tumour target cells in the cytotoxic response.

Benzoquinones↗
PubMed1992

Effects of pertussis toxin treatment on human natural killer cell function.

Membranes from highly purified natural killer (NK) cells were ADP ribosylated by treatment with pertussis toxin (PTX). PTX treatment resulted in a single band of 32P incorporation at M(r) 41,600. PTX treatment of NK cells diminished their ability to lyse K562 tumour cells by about 50%. However PTX treatment had no measurable effect on cAMP levels in NK cells. PTX pretreatment also had no effect on the ability of target cells to induce phosphoinositide turnover or on the ability of the NK cells to conjugate with the K562 tumour cells. Movement toward the chemoattractants interleukin-2 (IL-2) and formylmethionylleucylphenylalanine (FMLP) was significantly inhibited indicating that a PTX substrate in NK cells may be involved in the transduction of signals which are involved in cell motility.

Cell Adhesion↗
PubMed1990

Effects of beta-adrenergic receptor activation, cholera toxin and forskolin on human natural killer cell function.

Membranes from highly purified natural killer (NK) cells were ADP-ribosylated by treatment with cholera toxin (CTX). CTX resulted in a single band of specific 32P incorporation at Mr 43,600. CTX treatment of intact NK cells caused a 9-fold increase in cyclic AMP (cAMP) concentrations. Pretreatment of NK cells with CTX diminished their ability to lyse K562 tumour cells by up to 79%. Forskolin treatment elevated NK cell cAMP levels 8-fold and decreased lysis of K562 cells by up to 45%. Adrenaline and isoprenaline (isoproterenol) both inhibited lysis of K562 cells by approx. 35% and elevated cAMP by at least 2.5-fold, and their inhibition of lysis was reversed by propranolol. These data suggest that the stimulatory guanine-nucleotide-binding protein GS coupled to beta-adrenergic receptors is involved in transducing signals which inhibit NK cell lysis of tumour cells. CTX and forskolin also diminish the ability of NK cells to bind K562 cells (binding is necessary for lysis). This suggests that the NK-cell receptor(s) for the tumour cell may be altered as a consequence of cAMP-mediated events or by activation of GS.

Adenosine Diphosphate Ribose↗
PubMed1990

The effect of the gamma-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme.

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Life, evolution, and the pursuit of single photon sensitivity.

Evolutionary forces have designed a large family of rod and cone photoreceptors, each member of which suits the lifestyle requirements and circadian patterns of a particular species. The three-segment architecture of signal transduction is conspicuous in the biochemistry of photoreceptors and supports their demonstrated properties of extreme sensitivity, low noise levels, extended dynamic range, and light adaptation. The designs elaborated by evolution reflect a gradual process of modification, with the sequential elaboration of layers of control and refinements in control. The end results of this long evolutionary labor are the functional efficiency and dynamic range that give the rod its utility. Our conceptual problems in deriving observed rod properties from the collective features of known rod gene products may well give way when we have learned more about the true composition and topology of the outer segment gene set and both bound and free nucleotide concentrations. The invertebrates have developed alternative solutions to the problems of photoreceptor sensitivity and wide dynamic range. The vertebrate rod represents a truly optimized way to capture and interpret low-intensity photon signals. One may anticipate, with some enthusiasm, those molecular and kinetic data that will permit an understanding of how cones differ from rods and how release from the requirement for single photon detection has shaped the design of this wavelength-specific companion photoreceptor. The utilization by evolution of the three-segment architecture of GTP-dependent signal transduction for other modalities of sensory perception, such as olfaction (Lancet et al., this volume) and gustation (Jones et al., this volume), is certainly a reasonable and successful choice.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

The major gangliosides of the bovine pineal body.

Acetone powders of fresh-frozen pineals were extracted with chloroform/methanol mixtures. By column chromatography on silicic acid, mild alkaline methanolysis, ion-exchange high performance liquid chromatography and a final thin layer chromatography on silicic acid, the major glycosphingolipids were purified from the extracts of a total of 300 bovine pineal bodies. Chromatographically purified fractions were characterized by gas chromatographic analysis. The most prominent glycosphingolipid appeared to be cerebroside. In addition, five different gangliosides were found in detectable levels. The two major gangliosides have the chromatographic and component characteristics of GD3 and GM3, with disialoganglioside predominating. Gangliosides indistinguishable from purchased standards of GM1 and GD1a were third and fourth, respectively, in amount. The fatty acid profiles of the two lactosyl gangliosides are similar and significantly different from those of the two gangliotetraose gangliosides. The fifth most prominent ganglioside, present at a level of 1.09% of total recovered ganglioside sialic acid, appears to be a novel trisialoganglioside, called GTx. This new molecule has a component ratio of gal:glc:sialic acid:amino sugar of approximately 1:2:3:1. Similarities between bovine pineal and rod outer segments are discussed.

Animals↗
PubMed1986

Separation of underivatized gangliosides by ion exchange high performance liquid chromatography.

A procedure is described which separates neutral glycolipids from gangliosides, and which separates the gangliosides into classes, based on their number of sialic acid residues. In addition to separation into mono-, di- and trisialoganglioside classes, there is purification of individual disialoganglioside species. The procedure uses a commercially available--NH2 high performance liquid chromatography (HPLC) column, which normally is used as an adsorption column. In this method the column is modified by protonation, to pH 5.4, so that it exhibits ion exchange as well as adsorption properties. Sensitive, nondestructive detection of eluent peaks is accomplished by monitoring continuously at 210 nm, a wavelength near which underivatized glycosphingolipids have absorbance maxima and which also will clearly detect most possible contaminants. An entire run, including re-equilibration of the column, takes two hours.

Animals↗