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Biomedical subjects

L A Wheeler

Publications and source records attributed to L A Wheeler.

At least 19 recordsLinked to original sources
PubMed1991

Agonist-sensitive and -insensitive intracellular Ca2+ pools. Separate Ca(2+)-releasing mechanisms revealed by manoalide and benzohydroquinone.

The mechanism of action of a novel compound, 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ), used to modulate cell free cytosolic Ca2+ concentration ([Ca2+]i) was studied in AR42J cells and pancreatic acini by using single-cell fluorescence techniques applied to Fura-2-loaded cells. In the presence of extracellular Ca2+ (Ca(2+)out), BHQ induced a biphasic [Ca2+]i increase, an initial and rapid transient followed by a sustained increase. The initial increase was due to Ca2+ release from intracellular stores, being independent of Ca(2+)out. The sustained response was due to Ca2+ entry, being dependent on Ca(2+)out, blocked by La3+ and correlated with an increased rate of Mn2+ entry, all indicative of increased plasma-membrane permeability to Ca2+. Treatment of AR42J cells with BHQ for about 5 min reversibly blocked agonist-dependent Ca2+ release and oscillations, whereas agonist pretreatment decreased, but did not prevent, the effects of BHQ on [Ca2+]i. Accordingly, depletion of the Ins(1,4,5)P3-mobilizable pool in permeabilized AR42J cells by BHQ required 5 min of incubation, although inhibition of the internal Ca2+ pump by BHQ was rapid. These observations suggest that BHQ mobilized an additional intracellular Ca2+ pool that did not respond to changes in Ins(1,4,5)P3. Manoalide, an inhibitor of Ca2+ channels, inhibited agonist-evoked [Ca2+]i oscillation and [Ca2+]i increase in a dose- and time-dependent manner without significant effect on internal Ca2+ pumps and Ca2+ content of the internal stores. Manoalide also inhibited the BHQ-evoked [Ca2+]i increase in the absence and presence of Ca(2+)out. Neither BHQ nor manoalide affected Ins(1,4,5)P3 levels in resting or stimulated cells. Therefore, the effect of BHQ appears to involve unmasking of passive Ca(2+)-permeation pathways in the plasma and intracellular membranes that do not respond to cholecystokinin octapeptide, following its described inhibition of the internal-store Ca2+ pumps responsible for accumulating Ca2+ in these pools.

Animals↗
PubMed1991

AGN 190383, a novel phospholipase inhibitor with topical anti-inflammatory activity.

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

Administration, Topical↗
PubMed1991

Synthesis of chiral and achiral pyranenamine derivatives. Potent agents with topical ocular antiallergic activity.

The SS, RR and meso stereoisomers of pyranenamine SK&F 84210 were synthesized stereospecifically starting from commercially available (R)-(-)- or (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol. In addition, two achiral pyranenamines 19 and 26 were also synthesized. When evaluated by intravenous and topical routes in the rat passive ocular anaphylaxis (POA) assay, (SS)- and meso-2 as well as achiral compounds 19 and 26 were found to be more potent antiallergic agents than (RR)-2.

Anaphylaxis↗
PubMed1991

Transforming growth factor alpha: in vivo release by normal human skin following UV irradiation and abrasion.

Transforming growth factor alpha (TGF alpha) is a keratinocyte-growth-stimulating factor which may have a role in epidermal hyperproliferation, psoriasis, and wound healing. Since increased epidermal proliferation occurs in response to UV radiation, we have measured the amount of TGF alpha in exudates from normal and UVB-irradiated human skin. Cutaneous exudates were obtained using the skin chamber abrasion technique from one side of the back of volunteers (n = 10) with normal skin (collected following skin contact times of 2 and 30 min). Exudates were similarly obtained from the contralateral side of the back at sites irradiated 2 h previously with 3 x the minimum erythemal dose UVB. Levels of TGF alpha were measured by radioimmunoassay. Normal human skin released TGF alpha immediately after abrasion: unirradiated, 63 +/- 18 ng/ml; irradiated, 89 +/- 15 ng/ml. Levels of TGF alpha increased within 30 min to 110 +/- 14 ng/ml in unirradiated skin and to 190 +/- 17 ng/ml in irradiated skin. Irradiated sites at 30-min time points were significantly higher (p less than 0.05) than all other samples. The presence of releasable TGF alpha in normal skin suggests a role for TGF alpha in wound repair mechanisms.

Adolescent↗
PubMed1990

Inhibition by manoalide of fMLP-stimulated elastase release from human neutrophils.

Incubation of human polymorphonuclear leukocytes (PMNLs) with the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) resulted in a concentration-dependent release of the neutral protease elastase. This response was inhibited by pretreatment of the PMNLs with manoalide (IC50 approximately 0.08 microM). To understand the mechanism of this inhibition, we examined the effect of manoalide on the signal-transduction pathway believed to mediate fMLP stimulation. We observed in fura-2 loaded cells that pretreatment with manoalide blocked fMLP-induced increases in cytosolic free-calcium (IC50 approximately 0.15 microM). However, manoalide had no effect on inositol 1,4,5-trisphosphate (IP3) production at concentrations which completely inhibited the Ca2+ signal. Furthermore, manoalide was approximately 50-fold less potent as an inhibitor of phospholipase C activity in membrane preparations of PMNLs than as an inhibitor of calcium mobilization in whole cells. These data indicate that manoalide can block stimulation of human PMNLs through inhibition of Ca2+ mobilization, but that this occurs at a site beyond phospholipase C activation and inositol phosphate turnover.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1990

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: separation of growth factor and bradykinin pathways.

Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration [( Ca2+]i). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]i response to these agonists was examined by four methods. Low concentrations of TPA (2 x 10(-10) M) had no effect on Ca2+ release due to EGF, TGR-alpha or bradykinin but resulted in a rapid return of [Ca2+]i to baseline levels for EGF or TGF-alpha. Addition of the PKC inhibitor staurosporine (1 and 10 nM) completely inhibited the action of TPA on EGF-induced [Ca2+]i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 microM TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-alpha. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF-alpha resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-alpha in this cell type. A431 cells treated with higher concentrations of TPA (5 x 10(-8) M) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-alpha but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF-alpha gave a single transient of [Ca2+]i, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-alpha) and bradykinin resulted in two [Ca2+]i transients equal in size to those obtained with a single agonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗
PubMed1990

Novel dihydrofolate reductases isolated from epidemic strains of trimethoprim/sulfamethoxazole-resistant Shigella sonnei.

Two strains of trimethoprim-resistant Shigella sonnei bearing R plasmids pBH600 and pBH700 each elaborated a dihydrofolate reductase (DHFR) and were moderately resistant to trimethoprim (minimum inhibitory concentrations, 128 and 256 micrograms/ml, respectively). Neither plasmid hybridized to probes for DHFR types I, II, or III. The trimethoprim resistance genes from the R plasmids resided on a 1600-base pair (bp) PstI fragment of pBH600 and an 1800-bp PstI fragment of pBH700. Isoelectric focusing showed distinct isoelectric points for the enzymes coded for on pBH600 (5.3) and pBH700 (5.6-5.7). Trimethoprim-resistant S. sonnei from 10 locations in nine states were examined. Isolates from 8 locations hybridized only to a pBH700-derived probe and one isolate hybridized to a pBH600-derived probe. These two trimethoprim resistance genes appear novel. The gene on plasmid pBH700 codes for an enzyme that seems widespread among S. sonnei isolates in the USA.

Ampicillin Resistance↗
PubMed1990

Trans retinoic acid enhances the growth response of epidermal keratinocytes to epidermal growth factor and transforming growth factor beta.

Retinoids have been shown to either stimulate or inhibit epidermal keratinocyte proliferation. We have observed that in serum and growth factor free medium (basal medium), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) stimulated DNA synthesis in mouse epidermal keratinocyte cultures (mKC) in a time- and dose-dependent manner. Incubation with all-trans retinoic acid (RA) greatly enhanced the stimulatory effect of EGF. Transforming growth factor beta (TGF beta) inhibited the EGF-induced DNA synthesis in a dose-dependent manner, and the inhibition was greatly enhanced by a low dose of RA. Treatment of growth-factor deprived human keratinocyte cultures (hKC) with RA before incubation in basal medium containing EGF or a mixture of EGF, bovine pituitary extract (BPE), and insulin caused a dose-related increase in DNA synthesis and cell growth (cell number), respectively. A low concentration of RA also enhanced the inhibitory effect of TGF beta on growth-factor-induced DNA synthesis and cell growth in hKC. These findings suggest that the differential effects of retinoids on epidermal keratinocyte proliferation are in part due to an enhancement of the response of keratinocytes to positive and negative peptide growth factors.

Animals↗
PubMed1989

Prostaglandin F2 alpha effects on intraocular pressure negatively correlate with FP-receptor stimulation.

According to the current working classification for prostanoid receptors, the prostaglandin F2 alpha-sensitive receptor (FP-receptor) may be identified by comparing the rank order of activity of prostaglandin F2 alpha (PGF2 alpha) and its analogues. In order to further understand the pharmacology of PGF2 alpha-induced ocular hypotension, the intraocular pressure response to PGF2 alpha and selected analogues was compared with their rank order of activity in typical FP-receptor preparations such as contraction of the cat iris sphincter and affinity for corporal luteal membrane binding sites. The rank order of potency for decreasing intraocular pressure was as follows: PGF2 alpha greater than PGF1 alpha greater than 16-phenoxytetranor PGF2 alpha greater than 17-phenyltrinor PGF2 alpha = fluprostenol (inactive). For cat iris sphincter contraction, the rank order of potency appears to be fluprostenol = 17-phenyltrinor PGF2 alpha greater than 16-phenoxytetranor PGF2 alpha = PGF2 alpha greater than PGF1 alpha. The rank order of potency for PGF2 alpha analogues in decreasing intraocular pressure appears to negatively correlate with the rank order for cat iris sphincter contraction and literature values for corporal luteal membrane binding. It is concluded that the ocular hypotensive effect of PGF2 alpha is not mediated by the FP-receptor.

Administration, Topical↗
PubMed1988

Preferential inhibition of 5-lipoxygenase activity by manoalide.

Treatment of human polymorphonuclear leukocytes (PMNLs) with micromolar concentrations of the anti-inflammatory drug manoalide inhibited production of leukotriene B4 (LTB4) and LTC4/LTD4 in response to the calcium ionophore A23187. In an attempt to further define the mechanism(s) of action of this agent, we have examined its interaction with several lipoxygenase enzymes. In RBL-1 cells, manoalide inhibited 5-lipoxygenase (5-LO) activity with an approximate IC50 of 0.3 microM. This was equipotent in our system with the known lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Manoalide was virtually inactive, however, against 12-lipoxygenase activity in both human platelets and mouse epidermis, with little inhibition seen at concentrations up to 100 microM. Manoalide showed some activity against soybean lipoxygenase, although it was 30- to 50-fold less potent than as an inhibitor of the 5-lipoxygenase enzyme. These data indicate that manoalide is a selective 5-LO inhibitor and suggest the possibility that its anti-inflammatory actions may be due, at least in part, to inhibition of leukotriene synthesis.

Anti-Inflammatory Agents↗
PubMed1988

Differential effects of retinoids on DNA synthesis in calcium-regulated murine epidermal keratinocyte cultures.

To study the possibility that the state of proliferation of epidermal keratinocytes can influence the action of retinoids, the rate of proliferation of murine epidermal keratinocytes was manipulated by growing the cells in media containing high or low concentrations of Ca++. In contrast to what other investigators have reported, keratinocytes cultured in medium containing 1.4 mM Ca++ proliferate faster, instead of slower, than cells cultured in medium with 0.09 mM Ca++. Other experiments showed that Ca++ was stimulatory to keratinocytes in medium containing a low level of growth factors, and inhibitory in medium containing a high level of growth factors, suggesting that the discrepancy could be due to a difference in the sera used. The high Ca++ cells prominently expressed the 48kD/56kD pair of keratin, showing that they were in a hyperproliferative state. Exposure of the faster growing high Ca++ cells to all-trans retinoic acid, 13-cis retinoic acid, etretinate, etretin, and arotinoid ethyl ester caused dose-dependent inhibition of DNA synthesis. In contrast, exposure of the slower growing low Ca++ cells to these retinoids resulted in dose-dependent stimulation of DNA synthesis. In addition, all-trans retinoic acid caused dose-related increases in cell number in the low Ca++ cultures. These findings correlate with the reported differential effects of retinoids on normal and hyperproliferative epidermis, and suggest that Ca++ and low growth factor-regulated keratinocyte cultures are useful for studying the mechanism of hyperproliferation and retinoid actions.

Animals↗
PubMed1988

Effect of forskolin on beta-adrenergic hyporesponsiveness in skin.

beta-Adrenergic receptor hyporesponsiveness has been observed in psoriasis and after exposure of epidermis to phorbol esters. It was the purpose of our studies to determine if forskolin, which is known to act synergistically with receptor agonists in elevating endogenous levels of cyclic AMP, could return these responses to those seen under control conditions. It was observed that topical application of phorbol ester to mouse ears in vivo led to a significant reduction in isoproterenol stimulation of cyclic AMP in vitro. Low doses of forskolin (10(-7) M) were able to enhance isoproterenol's effect under these conditions. Similarly, human keratinocyte cell cultures treated with phorbol esters and human psoriatic epidermis in vitro were both hyporesponsive to isoproterenol. Again, pretreatment of these samples with forskolin restored the beta-agonist stimulation to control values. These data indicate that forskolin is still able to act synergistically with beta-agonists in hyporesponsive systems and suggest that forskolin may be a useful probe in defining the mechanism of this decreased responsiveness both in phorbol-ester-treated skin and in psoriasis.

Animals↗
PubMed1987

Manoalide, a natural sesterterpenoid that inhibits calcium channels.

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.

Animals↗
PubMed1986

Depletion of cutaneous glutathione and the induction of inflammation by 8-methoxypsoralen plus UVA radiation.

The purpose of this study was to examine the dose response and time course relationships between PUVA (psoralen + UVA) depletion of skin glutathione (GSH) and the induction of inflammation. Dorsal skin fold thickness (DSFT), an index of cutaneous edema, was used as a noninvasive measure of inflammation. Ornithine decarboxylase (ODC) was used as a measure of epidermal damage. Female hairless mice were given 8-methoxypsoralen (8-MOP) (dissolved in corn oil) by gavage at different doses, and 2 h later the mice were irradiated with 5 J/cm2 UVA. At 24 h, DSFT measurements were taken, the mice were killed, and reduced GSH, glutathione disulfide (GSSG), and glutathione-S-transferase were measured in the epidermis and dermis. Epidermal GSH was depleted 0, 11, 45, 87, and 98% from vehicle and/or UVA-treated levels (0.7 mM) after 0.1, 0.5, 5, 25, and 50 mg/kg, respectively. In the dermis GSH decreased from 0.3 mM by 47, 87, and 91% after 5, 25, and 50 mg/kg 8-MOP, respectively. Increases in DSFT of 20, 141, and 242% were observed after 5, 25, and 50 mg/kg doses, respectively. GSSG accounted for a small portion of total GSH in the skin after PUVA treatment. The maximal decreases in GSH were not observed until 24-48 h after PUVA treatment. PUVA treatment leads to dose-related increases in dermal edema, epidermal ODC, and depletion of GSH levels from both compartments in the skin. The time course of glutathione loss suggests that PUVA may interfere with its resynthesis or utilization from the circulation.

Animals↗
PubMed1986

Stimulation of endogenous cyclic AMP levels in ciliary body by SK&F 82526, a novel dopamine receptor agonist.

The effect of dopamine and SK&F 82526 on cyclic AMP metabolism in ocular tissues has been examined. The in vitro incubation of human and bovine ciliary body with these agonists produced a dose-dependent increase in endogenous levels of cyclic AMP. This stimulation was blocked by the selective DA1 receptor antagonist SK&F 83566, but not by the beta-receptor antagonist propranolol. The response to SK&F 82526 was stereoselective, with SK&F R-82526 being approximately 100 times more potent than SK&F S-82526 in this preparation. This stimulation of ciliary body cyclic AMP content was not observed in the rabbit or cat, nor was it seen in human, bovine or rabbit iris tissue. These data suggest that adenylate cyclase linked dopamine receptors are present in both human and bovine ciliary body.

Animals↗
PubMed1985

Hematologic malignancies associated with primary mediastinal germ-cell tumors.

Three men with primary mediastinal germ-cell tumors subsequently developed a malignant hematologic disorder characterized by pancytopenia and marrow infiltration with hematopoietic blast cells. Two of these patients were classified as having acute megakaryocytic leukemia and the third was believed to have a myelodysplastic syndrome with a prominent megakaryocytic component. Analysis of clinical characteristics of these patients and review of the literature suggest that the proximate association of mediastinal germ-cell tumors with malignant hematologic disorders is neither a coincidence nor a consequence of chemotherapy given for the germ-cell tumor. We believe this association represents the evolution of a neoplastic disorder that initially involves a totipotent germ cell. These germ cells, when located in the mediastinum, apparently acquire hematologic phenotypes and are manifested clinically as a hematologic malignancy.

Acute Disease↗