Biomedical subjects

K E Akerman

Publications and source records attributed to K E Akerman.

At least 55 records · Page 3Linked to original sources

PubMed1995

Phenotypic modification of human osteosarcoma cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

Treatment of the U-2 OS human osteosarcoma cell line with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically decreased the rate of DNA synthesis. This decrease in proliferation as well as the change in morphology of the TPA-treated cells can be blocked by the protein kinase C inhibitor GF 109203X. The U-2 OS cells are known to express the c-sis oncogene [platelet-derived growth factor (PDGF) B-chain], PDGF-A, and receptors for PDGF, thus providing a potential autocrine loop of growth stimulation. TPA was found to induce the expression of both the PDGF-A and the PDGF-B chains. However, the levels of the PDGF receptor beta subunits and of the PDGF-BB inducable tyrosine phosphorylation of the PDGF receptor were markedly reduced. The TPA treatment of the U-2 OS cells also induced changes typical for maturing bone cells, such as increased expression levels of alkaline phosphatase and osteopontin. The expression levels of type I collagen and bone sialoprotein were reduced. The results show a TPA-dependent down-regulation of the PDGF receptor beta subunits that correlates with an increased expression of osteoblast phenotypic markers.

Cell Division↗

PubMed1994

Does protein kinase C regulate receptor agonists-mediated elevation in the cytosolic Ca2+ in human neutrophils?

The feedback regulation of elevated cytosolic free Ca2+ concentrations ([Ca2+]i) by protein kinase C (PKC) in neutrophils was studied by two approaches. First, the PKC-activity was induced by phorbol 12-myristate 13-acetate (PMA) before the stimulation of Ca2+-signal with N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles. Pretreatment of cells with PMA inhibited agonists-dependent Ca(2+)-transients. This inhibition was reversed by staurosporine. Second, the PKC-activity was induced simultaneously with the Ca(2+)-signal by the receptor agonists. In these conditions staurosporine had no effect on the Ca(2+)-response. In addition, the enhancement of PKC-activity, obtained by accumulating the endogenous PKC-activator diacylglycerol (DAG) with a DAG-kinase inhibitor, failed to change the agonists-stimulated elevations of [Ca2+]i. It is concluded that activation of PKC by PMA inhibits neutrophil Ca(2+)-responses, whereas receptor-mediated activation of PKC does not yield a negative feedback in elevated [Ca2+]i.

Alkaloids↗

PubMed1994

Different sensitivity of alpha 2A-C10 and alpha 2C-C4 receptor subtypes in coupling to inhibition of cAMP accumulation.

Two human alpha 2-adrenergic receptor subtypes, alpha 2A-C10 and alpha 2C-C4, were compared with respect to their ability to inhibit stimulated cAMP-production. The inhibition was transduced with about one order of magnitude higher sensitivity in the alpha 2C-C4 subtype than in the alpha 2A-C10 subtype. The phorbol ester, TPA, known to desensitize alpha 2-adrenergic receptor function, possible through phosphorylation of Gi, almost completely abolished the inhibition of cAMP-production in the alpha 2C-C4 subtype, while only a partial effect was seen in the alpha 2A-C10 subtype. These results suggest that the receptor subtypes differ with respect to their coupling efficiency to adenylyl cyclase.

Adenine↗

PubMed1994

Coupling of human alpha 2-adrenoceptor subtypes to regulation of cAMP production in transfected S115 cells.

Stable S115 mouse mammary tumour cell lines, expressing separately alpha 2A-C10, alpha 2B-C2 and alpha 2C-C4 adrenoceptors were used to compare the receptor binding properties of alpha 2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 microM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the alpha 2A-C10 subtype, smaller in alpha 2C-C4, and minimal in alpha 2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the alpha 2B-C2 subtype (35%) than in the alpha 2A-C10 and alpha 2C-C4 subtypes (50-70%). After treatment of cells expressing alpha 2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by alpha 2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) alpha 2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected S115 cells, (2) all three alpha 2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in S115 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in S115 cells is different among the subtypes so that the alpha 2A-C10 subtype is coupled with high efficacy but with low sensitivity, the alpha 2B-C2 subtype with low efficacy but high sensitivity, and the alpha 2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least alpha 2B-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through Gs.

Adrenergic alpha-Agonists↗

PubMed1994

Production and release of matrix vesicles in the cell processes of TPA-treated human osteoblast-like cells.

At the onset of the mineralization of bone, small membranous matrix vesicles are often observed. The information available on the production and release of these vesicles is limited. When treated with 10-20 nM of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the human osteosarcoma cell line U-2 OS developed long cytoplasmic processes connecting adjacent cells. SEM and TEM show that TPA triggers a production and release of matrix vesicle-like membrane vesicles, mainly from the cellular processes. Tetracycline HCl was used to label intracellular bound calcium. The tetracycline HCl label was primarily localized to the end-feet of the cytoplasmic processes, indicating that these contain high concentrations of Ca2+, and to endoplasmic reticulum-like structures in the cell bodies. Together with our previous demonstration of the release of alkaline phosphatase-containing vesicles into the culture medium (Ringbom-Anderson T, Akerman KEO 1992 Calcif Tissue Int 50:533-540), the results presented here indicate that TPA induces a rapid induction of the primary steps of mineralization in U-2 OS osteosarcoma.

Actins↗

PubMed1994

Alaproclate acts as a potent, reversible and noncompetitive antagonist of the NMDA receptor coupled ion flow.

We studied the effect of alaproclate [2-(4-chlorophenyl)-1,1-dimethyl 2-aminopropanoate] on the N-methyl-D-aspartate (NMDA)-induced changes in membrane potential and intracellular free Ca++ in cerebellar granule cells by using the fluorescent indicators DiBaC4(3) and fura-2, respectively. The NMDA-induced responses were blocked by the 5-hydroxytryptamine reuptake blocker alaproclate in a noncompetitive manner with an IC50 value of 0.3 microM. The effect of alaproclate was stereoselective because the S-(-)-enantiomer was more potent than the R-(+)-enantiomer. The inhibitory response was rapidly reversed if alaproclate was removed by perfusion. The same was the case with the reversible noncompetitive NMDA receptor antagonists dextromethorphan, dextrorphan, amitriptyline and desipramine. The inhibition caused by the noncompetitive antagonist dizolcipine could not be reversed by perfusion. The glycine-sensitivity of the NMDA response was unaffected by alaproclate, and high concentrations of glycine were unable to reverse the inhibition of alaproclate. Alaproclate also did not affect the sensitivity of the responses to Mg++. The results suggest that alaproclate, which has previously been in clinical trials for depressive illness, acts as a reversible noncompetitive antagonist of the NMDA receptor.

Alanine↗

PubMed1993

Determination of the intracellular free chloride concentration in rat brain synaptoneurosomes using a chloride-sensitive fluorescent indicator.

The chloride-sensitive fluorescent indicator MQAE (N-(6-methoxyquinolyl) acetoacetyl ester) has been used for determination of the intracellular free chloride concentration in rat brain synaptoneurosomes. Loading of the synaptoneurosomes with MQAE occurs by transmembrane diffusion. Calibration of the intracellular MQAE was done by determining the correlation between fluorescence intensity and intrasynaptoneurosomal Cl- concentration in the presence of the Cl-/OH- exchanger tributyltin and the K+/H+ exchanger nigericin, starting from zero Cl- concentration. The total quenchable signal of MQAE was determined by adding KSCN in the presence of the K+ ionophore valinomycin. The correlation between the reciprocal of the fluorescence intensity and the chloride concentration was linear at least up to 50 mM Cl-. The fluorescence of freshly prepared synaptoneurosomes was then measured and the obtained value was plotted into the calibration curve and the corresponding Cl- was read. The mean intrasynaptoneurosomal chloride concentration was 14 +/- 4 mM. We also quantitatively estimated the Cl- flux after addition of the barbiturate, pentobarbitone that opens GABAA receptor-Cl(-)-channels, to the synaptoneurosomes. An addition of 1 mM pentobarbitone corresponded to an approx. 0.59 mM change in the intrasynaptoneurosomal free chloride concentration. The results show that the chloride-sensitive fluorescent indicator MQAE is a useful tool when determining intracellular chloride activity, and in quantitative determination of chloride fluxes in living cells and subcellular preparations.

Animals↗

PubMed1993

Inhibition of inositol 1,4,5-trisphosphate 5-phosphatase by micromolar concentrations of disulfiram and its analogues.

Following a preincubation period of 10 min, disulfiram and its analogues FLA 46, FLA 63, FLA 99, EWP 815 and EWP 840 inhibited the breakdown of 10 microM [3H]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH3 cells, with IC50 values (in microM), for soluble/particulate enzymes respectively, of: disulfiram, 24/24; FLA 46, 23/30; FLA 63, 24/6; FLA 99, 50/48; EWP 815, 8/6; EWP 840, 11/8. The inhibition produced by FLA 99 was time-dependent in nature, although inhibition was found in the absence of a preincubation period. EWP 815 and EWP 840 were more potent inhibitors of Ins(1,4)P2 phosphatase than of Ins(1,4,5)P3 5-phosphatase. Thyrotropin-releasing hormone (TRH; 3/100 microM)-stimulated inositol phospholipid breakdown in prelabelled GH3 cells was inhibited by disulfiram (IC50 values 63/52 microM respectively), FLA 46 (89/110 microM), EWP 815 (83/71 microM) and EWP 840 (220/200 microM), without affecting basal breakdown rates. FLA 99 did not inhibit either basal or TRH-stimulated activity at any of the concentrations tested (30, 100 and 300 microM). [3H]Ins(1,4,5)P3 binding to its cerebellar receptor was not inhibited by any of the compounds over a concentration range of 3-300 microM, although an increased level of binding was seen at high concentrations. FLA 99 and EWP 840 increased the basal intracellular Ca2+ concentration in GH3 cells, but with no corresponding effect on the Ca2+ response to TRH stimulation. These compounds did not increase the cellular permeability to Trypan Blue, but did affect cell proliferation. It is concluded that disulfiram and related compounds produce dramatic effects on Ins(1,4,5)P3 metabolism in GH3 cells.

Animals↗

PubMed1993

Enhanced IL-4-mediated D10.G4.1 proliferation with suboptimal concentrations of anti-IL-4 receptor monoclonal antibodies.

Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, an enhanced proliferative response to the IL-4-responsive cell line D10.G4.1, compared to IL-4 alone. Increasing amounts of the M1 antibody inhibited the IL-4-dependent proliferation in a normal fashion. The enhanced IL-4-induced proliferation was only inhibited with the anti-IL-4 mAb 11B11, whereas the anti-IL-2 receptor antibodies had no effect. Addition of M1 or M2 antibodies to the IL-2-dependent cell line CTLL, known to express small amounts of IL-4 receptors and thereby also slightly responsive to IL-4, gave no enhanced IL-4-induced proliferation. Instead these antibodies were found to inhibit both the IL-4 as well as the IL-2-mediated CTLL proliferation.

Animals↗

PubMed1993

Histamine modulation of Ca2+ homeostasis in human neutrophils.

The influence of histamine on the basal intracellular free Ca2+ concentration ([Ca2+]i) and agonist-induced increases of [Ca2+]i was studied in Fura-2-loaded neutrophils. Histamine was unable to change the basal [Ca2+]i at concentrations (10(-6)-10(-4) M) that have been shown to cause a rapid increase in [Ca2+]i in a variety of cell types. Histamine, in contrast, was found to inhibit dose-dependently the rise in [Ca2+]i induced by two neutrophil receptor agonists, N-formylmethionyl-leucylphenylalanine (fMLP) and serum-opsonized zymosan particles. The histamine inhibition was shown to be specific for H2 receptor activation by blocking experiments with selective H1 and H2 receptor antagonists. In the absence of extracellular Ca2+, histamine failed to inhibit the agonist-induced rise in [Ca2+]i, indicating that histamine does not affect the release of Ca2+ from internal pools. Forskolin, which mimics the biochemical effects of H2 receptor activation by directly stimulating adenylate cyclase, also decreased the Ca2+ transients induced by receptor agonists. Similarly, 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, reduced the Ca2+ response of activated neutrophils. These data suggest that in human neutrophils (1) no functional H1 receptors are present or alternatively H1 receptors are not coupled to cellular Ca2+ metabolism, and (2) H2 receptors modulate the receptor-triggered Ca2+ flux via the cAMP second messenger system.

Calcium↗

PubMed1993

Ca2+ and Zn2+ dependence of DNA synthesis in untransformed and in Ha-ras(val-12)-expressing NIH 3T3 cells.

The role of Ca2+ and Zn2+ in the initiation of DNA synthesis in NIH 3T3 fibroblasts and c-Ha-ras(val-12) oncoprotein-expressing (NIH 3T3) cells has been studied. Entrapment of the Ca2+ chelator, BAPTA (30 microM), into the cells totally blocked a serum-induced rise in cytosolic free Ca2+ ([Ca2+]i) as determined with fura-2. Serum starvation for 24 h considerably reduced DNA synthesis in control NIH 3T3 fibroblasts. BAPTA treatment reduced serum-induced DNA synthesis and totally inhibited platelet-derived growth factor-induced DNA synthesis in these cells. DNA synthesis of the c-Ha-ras(val-12)-expressing fibroblasts was little affected by serum starvation and unaffected by entrapment of BAPTA into the cells. Intracellular Zn2+ was measured using the fluorescent probe TSQ in intact cells. As determined using image analysis the TSQ fluorescence was distributed throughout the cytoplasm and concentrated around the nucleus. The permeable Zn2+ chelator, TPEN, at a concentration of 10 microM, caused a maximal reduction in TSQ-available Zn2+. This concentration of TPEN totally blocked DNA synthesis both in control and c-Ha-ras(val-12)-expressing fibroblasts. Upon addition of 11 microM Zn2+ DNA synthesis was restored even after TPEN addition. [3H]Thymidine incorporation itself was also sensitive to TPEN treatment. The results suggest that c-Ha-ras(val-12)-induced proliferation is independent of changes in [Ca2+]i. A specific role of Zn2+ in c-Ha-ras-induced proliferation is unlikely since ras-expressing and control cells reacted similarly to Zn2+ deprivation. There seems to be a constant requirement for the presence of Zn2+ in cell proliferation.

3T3 Cells↗

PubMed1992

Apparent noncompetitive antagonism of muscarinic receptor mediated Ca2+ mobilization by some muscarinic antagonists.

Ca2+ mobilizations in SH-SY5Y and IMR-32 human neuroblastoma cell lines were measured using the fluorescent Ca2+ indicator fura-2. A variety of antagonists (atropine, pirenzepine, 4-DAMP and N-methyl-scopolamine) inhibited carbamyl choline-induced transient Ca2+ mobilization both in a competitive and a noncompetitive manner. The apparent noncompetitive inhibition constants were lower in IMR-32 than in SH-SY5Y cells even when the competitive inhibition constants were similar. This may relate to the previously reported differential expression of muscarinic receptor subtypes in these cell lines.

Atropine↗

PubMed1992

Intracellular free [Ca2+] and [Na+] in response to capsaicin in cultured dorsal root ganglion cells.

Intracellular free Ca2+ and Na+ concentrations in cultured rat dorsal root ganglion cells were studied using the intracellular fluorescent probes fura-2 and SBFI, respectively. Capsaicin increased both intracellular [Ca2+] and [Na+]. A rise in cytosolic free Ca2+ could still be seen if Na+ was removed from the outer medium. The results suggest that capsaicin opens up ion channels which are permeable both to Ca2+ and Na+.

Animals↗

PubMed1992

A phorbol ester induces secretion of alkaline phosphatase activity in human osteosarcoma cells.

The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS. The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium. The secretion of APase appeared after a lag period of 4-6 hours of TPA treatment, and it could also be visualized with histological staining. Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles. The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes. It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.

Alkaline Phosphatase↗

PubMed1992

Differential expression of two alpha 2-adrenergic receptor subtype mRNAs in human tissues.

Genetic subtypes of alpha 2-adrenergic receptors (AR) may mediate distinct physiological functions, and undergo differential cell type-specific regulation. Thus, these distinct receptor subtypes are possible targets for the development of subtype-selective drugs. We have analyzed the tissue distribution of two human alpha 2-adrenoceptor subtype gene mRNAs, alpha 2-C4 and alpha 2-C10, in normal human fetal and adult tissues. Both receptor subtype mRNAs were abundantly expressed in fetal brain and choroid plexus. In non-neural fetal tissues, alpha 2-C10 mRNA was detected in spleen, kidney, adrenal gland, and skin, while alpha 2-C4 transcripts were observed only in kidney and skin. Most regions of the adult brain also expressed both subtypes, but with marked quantitative differences. For example, cerebral cortex contained predominantly alpha 2-C10 mRNA, whereas the caudate nucleus expressed mostly alpha 2-C4 mRNA. In adult peripheral tissues, alpha 2-C10 mRNA expression was most abundant in spleen and renal cortex, and expression of alpha 2-C4 mRNA was strongest in renal cortex and medulla. These different expression patterns provide evidence for the differential regulation of the two alpha 2-adrenergic receptor genes and warrant further investigation with techniques capable of improved anatomical resolution. Regional differences in receptor subtype expression may be valuable for the development of new, subtype-selective pharmacological agents with more targeted actions compared to currently used alpha 2-adrenoceptor agonists and antagonists.

Animals↗

PubMed1992

Muscarinic receptor subtypes in human neuroblastoma cell lines SH-SY5Y and IMR-32 as determined by receptor binding, Ca++ mobilization and northern blotting.

Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH-SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.

Blotting, Northern↗

PubMed1991

Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe.

Cl- fluxes through the GABAA receptor gated ion channels in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl- ions. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor-coupled Cl- fluxes through the GABAA receptor-gated channels in living cells.

Animals↗

PubMed1991

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.

Adenylate Cyclase Toxin↗