Jeffrey Bingham Smith

Bryostatins: potent, new mitogens that mimic phorbol ester tumor promoters.

Bryostatins (2 ng/ml), when combined with insulin in serum-free culture medium, are strongly mitogenic for Swiss 3T3 cells that have been arrested in the G1/G0 phase of the cell cycle. The mitogenic effect of the bryostatins is similar to that of 12-O-decanoylphorbol-13-acetate (TPA). A prior treatment of the cultures with TPA eliminated the mitogenic response to bryostatin and to a second addition of TPA. Conversely, a prior treatment of the cultures with bryostatin eliminated the mitogenic response to TPA. Bryostatin potently inhibited the binding of [3H]phorbol dibutyrate to a high affinity receptor in the cells. The findings suggest that the bryostatins and TPA act via the same receptor, possibly protein kinase C.

Na+/K+/Cl- cotransport in cultured vascular smooth muscle cells: stimulation by angiotensin II and calcium ionophores, inhibition by cyclic AMP and calmodulin antagonists.

SummaryThe specific activity of the Na+/K+/Cl− cotransporter was assayed by measuring the initial rates of furosemide-inhibitable86Rb+ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 μm, respectively. The dependence of furosemide-inhibitable86Rb+ influx on external Na+ and K+ was hyperbolic with apparentKm values of 46 and 4mm, respectively. The dependence on Cl− was sigmoidal. Assuming a stoichiometry of 1∶1∶2 for Na+/K+/Cl−, aKm of 78mm was obtained for Cl−. In quiescent smooth muscle cells cotransport activity was approximately equal to Na+ pump activity with each pathway accounting for 30% of total86Rb+ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na+ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca2+ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited86Rb+ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.

Caspase processing activates atypical protein kinase C ζ by relieving autoinhibition and destabilizes the protein

Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system.

Activation of Protein Kinase C Induces Nuclear Translocation of RFX1 and Down-regulates c-myc via an Intron 1 X Box in Undifferentiated Leukemia HL-60 Cells

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA onmyc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.

Initiation of DNA synthesis in quiescent Swiss 3T3 and 3T6 cells by lanthanum

La3+ stimulated quiescent Swiss 3T3 and 3T6 ceils to enter the DNA-synthesizing S phase of the cell cycle. La3+ and insulin interacted synergistically to increase DNA synthesis. A brief exposure of the cells to soluble LaCl3 optimally stimulated entry into S. La3+ was similar to Al3+ in its mitogenic properties (J. Cell. Physiol.118 , 298, 1984), but La3+ was 10 times more potent than Al3+.

A putative lectin-binding receptor mediates cadmium-evoked calcium release

Nanomolar concentrations of cadmium (Cd2+) produce an immediate rise in free Ca2+ in human dermal fibroblasts, which is mostly caused by the release of stored Ca2+ via inositol trisphosphate. Here we have used lectins to evaluate the hypothesis that a cell surface glycoprotein mediates the response to Cd2+. A prior incubation with wheat germ agglutinin (WGA) or certain other lectins inhibited calcium release evoked by Cd2+. WGA reversibly inhibited Cd(2+)-evoked calcium release as indicated by measurements of cytosolic free Ca2+ and 45Ca2+ efflux. WGA half-maximally inhibited Ca2+ release at 1.2 x 10(-7) M. The Kd for the binding of fluoresceinylated WGA was 2.8 x 10(-7) M. Chitotriose dissociated fluoresceinylated WGA from the cells and restored cadmium responsiveness. WGA inhibited Cd(2+)-evoked 45Ca2+ efflux similarly at 18 and 37 degrees C. A brief incubation with chitotriose at 18 or 10 degrees C reversed the inhibition by WGA. WGA neither bound 109Cd2+ nor affected 109Cd2+ uptake by the cells. Succinylated WGA, which binds N-acetylglucosamine but not N-acetylneuraminic acid, failed to inhibit Ca2+ release evoked by Cd2+. WGA probably inhibits Ca2+ release produced by Cd2+ by binding to N-acetylneuraminic acid in the external domain of a plasma membrane receptor.

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

SummaryKinetic properties of Na+−Ca2+ exchange in a renal epithelial cell line (LLC-MK2) were assessed by measuring cytosolic free Ca2+ with fura-2 and45Ca2+ influx. Replacing external Na+ with K+ produced relatively small increases in free Ca2+ and45Ca2+ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na+ and strongly potentiated the effect of replacing external Na+ with K+ on free Ca2+ and45Ca2+ uptake.45Ca2+ influx in 140mm K+ or N-methyl-d-glucamine minus influx in 140mm Na+ was used to quantify Na+−Ca2+ exchange activity of Na+-loaded cells. The dependence of exchange on cell Na+ was sigmoidal; theK0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na+ partly account for the small increase in Ca2+ influx when all external Na+ is replaced by K+. Besides raising cell Na+ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg2+ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na+. Potassium also increased theVmax of exchange by 86% and had no effect on theKm for Ca2+. The exchanger does not cause detectable22Na+−Mg2+ exchange and does not appear to require K+ or transport86Rb+. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na+−Ca2+ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.

Inhibition of sodium-calcium and sodium-proton exchangers by amiloride congeners in arterial muscle cells.

The inhibitory potencies of several amiloride congeners towards Na(+)-Ca2+ and Na(+)-H+ exchange were compared in rat aortic myocytes. N-(2,4-Dimethylbenzyl)amiloride (DMB) was 10 times more potent towards Na(+)-Ca2+ than Na(+)-H+ exchange. Amiloride and ethylisopropylamiloride were about 5,000 and 10,000 times more potent toward Na(+)-H+ than Na(+)-Ca2+ exchange respectively. N-(3,4-Dichlorobenzyl)amiloride was almost equipotent towards both exchangers. About 40 nM ethylisopropylamiloride inhibited Na(+)-H+ exchange by 50%. Ethylisopropylamiloride (10 microM) had no effect on basal or angiotensin-evoked 45Ca2+ efflux or net Ca2+ efflux. In contrast to ethylisopropylamiloride, 25-50 microM DMB, which strongly inhibits Na(+)-Ca2+ exchange, markedly decreased both 45Ca2+ efflux and net Ca2+ efflux produced by angiotensin. Replacing extracellular Na+ with N-methyl-D-glucamine inhibited angiotensin-evoked 45Ca2+ efflux similarly to DMB. Neither DMB nor Na+ placement had any effect on basal or angiotensin-evoked production of [3H]inositol phosphates. These findings suggest that Na(+)-H+ exchange has no major influence on short-term Ca2+ regulation and provide evidence that Na(+)-Ca2+ exchange is a major pathway of rapid Ca2+ efflux in stimulated arterial muscle cells.

Bryostatins mimic the effects of phorbol esters in intact human platelets

Bryostatin-7 induces aggregation of human platelets and the phosphorylation of specific platelet proteins. Both the rate and extent of aggregation are similar to that induced by the tumor promoter phorbol ester 12-myristate 13-acetate (PMA); however, the rate of response is markedly reduced compared to that induced by thrombin. The addition of bryostatin-7 to 32P-labeled platelets results in a time-dependent incorporation of 32P into proteins of 20, 47 and 250 kDa; proteins of similar molecular mass are phosphorylated in response to the addition of thrombin or PMA. The time courses and dose responses of the phosphorylations induced by bryostatin-7 are similar to those found with PMA. In addition, bryostatin-7 increases the level of 32P incorporation into platelet polyphosphoinositides, which also occurs in response to PMA. These results suggest that, in intact human platelets, bryostatin-7 mimics the phorbol ester tumor promoter by directly activating protein kinase C.

Rapid calcium mobilization by vasopressin and prostaglandin F2α is independent of sodium influx in quiescent 3T3 cells

Vasopressin (VP) rapidly increased 45Ca2+ efflux. A VP antagonist prevented VP from mobilizing Ca2+ and stimulating DNA synthesis. Prostaglandin F2 alpha (PGF2 alpha) also stimulated rapid 45Ca2+ release. The effectiveness of different prostaglandins corresponded to their effectiveness as mitogens. The removal of external Na+ or Ca2+ had no effect on VP-or PGF2 alpha-induced 45Ca2+ release. The present results indicate that the mobilization of intracellular Ca2+ by these hormones is independent of Na+ or Ca2+ influx and that Ca2+ mobilization is important for growth stimulation.