Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.     

Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes

We have investigated the effects of hyperosmolarity induced by sucrose on the fluid phase endocytosis of the fluorescent dye lucifer yellow CH (LY) and the endocytosis of 125I-asialo-orosomucoid (ASOR) by the galactosyl receptor system in isolated rat hepatocytes. Continuous uptake of LY by cells at 37 degrees C is biphasic, occurs for 3-4 h, and then plateaus. Permeabilized cells or crude membranes do not bind LY at 4 or 37 degrees C. Intact cells also do not accumulate LY at 4 degrees C. The rate and extent of LY accumulation are concentration- and energy-dependent, and internalized LY is released from permeabilized cells. Efflux of internalized LY from washed cells is also biphasic and occurs with halftimes of approximately 38 and 82 min. LY is taken up into vesicles throughout the cytoplasm and the perinuclear region with a distribution pattern typical of the endocytic pathway. LY, therefore, behaves as a fluid phase marker in hepatocytes. LY has no effect on the uptake of 125I-ASOR at 37 degrees C. The rate of LY uptake by cells in suspension is not affected for at least 30 min by up to 0.2 M sucrose. The rate of endocytosis of 125I-ASOR, however, is progressively inhibited by increasing the osmolality of the medium with sucrose (greater than 98% with 0.2 M sucrose; Oka and Weigel (1988) J. Cell. Biochem. 36, 169-183). Hyperosmolarity completely inhibits endocytosis of 125I-ASOR by the galactosyl receptor, whereas fluid phase endocytosis of LY is unaffected. Cultured hepatocytes contained about 100 coated pits/mm of apical membrane length as assessed by transmission electron microscopy. In the presence of 0.4 M sucrose, only 17 coated pits/mm of membrane were observed, an 83% decrease. Only a few percent of the total cellular fluid phase uptake in hepatocytes is due to the coated pit endocytic pathway. We conclude that the fluid phase and receptor-mediated endocytic processes must operate via two separate pathways.     

Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin.

Propranolol inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyl-1-methylxanthine (IBMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10(-7) M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 min). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized cells than in intact cells. Other beta-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (beta 2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the cells prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized cells to exogenous cAMP was markedly increased, indicating that propranolol neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.     

Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells

The capacity of plant heterotrophic organs to transport and accumulate incoming nutrients (mostly in the form of sucrose) directly impacts their final size, crop productivity and nutritional value. Endocytosis as a mechanism for nutrient uptake in heterotrophic cells was investigated using suspension culture cells of sycamore (Acer pseudoplatanus L.) and the endocytic inhibitors wortmannin and LY294002. Time course analysis of sucrose uptake in intact walled cells revealed a two-phase process involving an initial 90 min wortmannin- and LY294002-insensitive sucrose uptake period, followed by a prolonged phase of rapid sucrose accumulation which was greatly inhibited by the two endocytic inhibitors. Walled cells were assessed for their capacity to incorporate the fluorescent endocytosis marker lucifer yellow-CH (LY) in the presence or absence of sucrose. Rates of sucrose and LY accumulation were virtually identical, as was their response to wortmannin. In addition, LY incorporation increased as a function of external sucrose concentration. When sucrose was substituted by other sugars or amino acids, uptake of LY greatly diminished, indicating that sucrose itself is the primary signal of endocytosis. Microscopic observations revealed the formation of vesicles containing LY and its eventual accumulation on the vacuole when sucrose was present in the incubation medium. These results demonstrate the existence of a sucrose-inducible endocytic process as a viable mechanism for solute transport into the vacuole of storage cells.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Modulation of ion uptake across posterior gills of the crab Chasmagnathus granulatus by dopamine and cAMP

Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10 per thousand salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na(+) influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose-response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 micromol l(-1). When the effects of forskolin and DA on the Na(+)/K(+)-ATPase activity were tested, they correlated well with the Vte and Na(+) influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 °C (18.7±1.8 kcal/mol) than below that critical temperature (7.5±1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1±1.6 kcal/mol above 15 °C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 °C, and two smaller transitions at 26 and 37 °C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 °C and smaller transitions at 26 and 35 °C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-β-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 °C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.     

Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.     

Internalization of lucifer yellow in Candida albicans by fluid phase endocytosis

Lucifer yellow (LY), an impermeable fluorescent dye used as a marker for fluid phase endocytosis, was internalized by Candida albicans. As observed by fluorescence microscopy, incubation of C. albicans with LY in potassium phosphate buffer (pH 6.0) and glucose (2%, w/v) resulted in localization of the dye inside vacuoles. Sodium azide and carbonyl cyanide m-chlorophenylhydrazone, which are inhibitors of energy metabolism, decreased the uptake of the dye. The optimum temperature for uptake was 30 degrees C; no internalization was observed at 0 degrees C. Quantification of cell-associated LY by fluorescence spectrometry showed an uptake linear with time and not saturable over a 400-fold range of concentration. Thus, C. albicans internalized LY into vacuoles by a nonsaturable and time-, temperature- and energy-dependent process consistent with fluid phase endocytosis. Both the yeast and mould phase of this dimorphic fungus endocytosed LY. Growth in complex medium appeared to be required to enable the cells to internalize LY. However, addition of peptone or yeast extract to the phosphate buffer/glucose assay medium interfered with LY uptake by causing an apparent increase of exocytosis. These studies provide the first evidence of fluid phase endocytosis in C. albicans and may explain how some large molecules, such as toxins and cationic proteins, enter C. albicans.     

Stimulation of bile acid active transport related to increased mucosal cyclic AMP content in rat ileum in vitro

The regulation of bile acid transport in rat ileum was studied in vitro using the adenylate cyclase stimulator forskolin, or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Forskolin 20 microM as well as 100 microM IBMX enhanced mucosal cyclic AMP to 3-fold the control levels. As a physiological response, net fluid absorption in everted ileal sacs was reduced. Taurocholate (10-500 microM) transfer in everted perfused segments of rat ileum was measured using a three compartment dual label method suitable for measuring active transport. Transport asymmetry with absorption exceeding its counterflux by 26-fold, was measured at 500 microM taurocholate. Forskolin increased absorption of taurocholate still further, by 68%, and reduced the serosal to mucosal flux. Enhanced intracellular accumulation of taurocholate indicated a stimulatory action of forskolin on active transport at the mucosal brush-border membrane. In uptake studies, accumulation of taurocholate was enhanced by 100 microM IBMX also. Forskolin-induced uptake stimulation could also be shown for chenodeoxycholate and cholate. In the presence of the neuronal blocker tetrodotoxin, uptake stimulation was still effective. Results indicate that the ileal bile acid transporter is included within the group of sodium-dependent cotransporters of the rat small intestine which are subject to a cyclic AMP-related stimulation at the mucosal cellular level.     

The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent

In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent.     

The cyclic AMP-protein kinase A pathway restrains islet phospholipase A(2) activation

Although phospholipase A(2) (PLA(2)) is of importance for insulin secretion, it is not established how it relates to other signalling mechanisms. This study examined the crosstalk between PLA(2) and the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in isolated rat islets. Forskolin, IBMX, and dbcAMP reduced [(3)H]arachidonic acid ([(3)H]AA) efflux from prelabelled islets during PLA(2) activation by mellitin or cholecystokinin (CCK-8), while efflux induced by carbachol was unaffected. The PKA inhibitor myrPKI(14-22) prevented this reduction of CCK-8-induced efflux. Glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and vasoactive intestinal polypeptide (VIP) diminished CCK-8-induced efflux. Also in the absence of Ca(2+), forskolin/IBMX and dbcAMP reduced CCK-8-induced efflux. In parallel with effects on [(3)H]AA, the expected additive insulin secretion induced by mellitin or CCK-8 in combination with forskolin or GLP-1, respectively, was reduced. In conclusion, the cAMP-PKA pathway restrains both Ca(2+)-dependent and Ca(2+)-independent PLA(2) activation, indicating a regulating crosstalk between these two pathways.     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Forskolin inhibits 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca+2 Mobilisation in canine cultured aorta smooth muscle cells.

The effect of forskolin on 5-hydroxytryptamine (5-HT)-induced inositol phosphate (IP) and Ca2+ mobilisation was investigated in canine cultured aorta smooth muscle cells (ASMCs). Pretreatment of ASMCs with forskolin attenuated 5-HT-induced IP accumulation and Ca2+ mobilisation in a time- and concentration-dependent manner. The half-maximal effects (pEC50) of forskolin to attenuate IP and Ca2+ responses to 5-HT occurred at concentrations of 6.28 and 6.64, respectively. Pretreatment of ASMCs with cholera toxin caused a similar inhibition on 5-HT-induced responses. Even after treatment with forskolin for 24 h, the 5-HT-induced responses were still inhibited. The inhibitory effect of forskolin resulted from both a depression of the maximal response and a shift to the right of the concentration-effect curves of 5-HT in these responses. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated IP accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on IP response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-iosquinolinesulphonamide] and HA-1004 [N-(2-guanidinoethyl)-5-iosquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IP in ASMCs. These results indicate that activation of cAMP/PKA might inhibit the 5-HT-stimulated IP accumulation and consequently reduce Ca2+ mobilisation, or inhibit both responses independently.     

The biphasic induction of p21 and p27 in breast cancer cells by modulators of cAMP is posttranscriptionally regulated and independent of the PKA pathway.

Cyclic AMP (cAMP) elevation affects growth arrest and differentiation in a wide variety of breast cell lines; however, the mechanisms associated with this process are poorly understood. Previous studies linked cAMP-mediated growth arrest in breast tumor cells to increased levels of cyclin kinase inhibitor (CKI), p21. In the present study we examined the role of cAMP-dependent protein kinase (PKA) on p21 and p27 induction in the breast cancer cell line, MDA-MB-157. The induction of the CKIs by modulators of cAMP such as cholera toxin (CT) + 1-isobutyl-3-methylxanthine (IBMX) and lovastatin fluctuates with biphasic kinetics (although the kinetics of CKI induction with CT + IBMX treatment are different from that of lovastatin) and is depicted by the periodic accumulation of lower molecular weight forms of p21 and p27 which also correlate with fluctuations in CDK2 activity. Using three different approaches we show that the cAMP-mediated induction of CKIs is independent of PKA activity. In the first approach we treated MDA-MB-157 cells with a variety of cAMP modulators such as CT + IBMX, and forskolin in the presence or absence of H-89, a potent PKA inhibitor. This analysis revealed that the cAMP activators were capable of inducing p21 even though PKA activity was completely eliminated. In the second approach PKA dominant negative stable clones of MDA-MB-157 treated with CT + IBMX or forskolin also resulted in p21 induction, in the absence of any PKA activity. Last, treatment of MDA-MB-157 cells with lovastatin, another known cAMP modulator which also causes growth arrest, resulted in the induction of p21 and p27 without any increase in PKA activity. Collectively, the above results suggest that the induction of p21 by cAMP is through a novel pathway, independent of PKA activity.     

Effects of Forskolin, Dibutyryl Cyclic AMP, and 5''N-Ethylcarboxamide Adenosine on 22Na Uptake by Rat Brain Synaptosomes Stimulated by Veratridine

The effects of forskolin, dibutyryl cyclic AMP, and 5'-N-ethylcarboxamide adenosine on specific 22Na uptake by synaptosomes stimulated by veratridine were investigated. All substances inhibited 22Na uptake, with forskolin more potent than 5'-N-ethylcarboxamide and this latter one more potent than dibutyryl cyclic AMP. In the absence of preincubation with forskolin, this substance caused little or no effect on 22Na uptake by synaptosomes. In the presence of the adenosine antagonist dipropylsulfophenylxanthine, the inhibitory effect of 5'-N-ethylcarboxamide adenosine on 22Na uptake was consistently antagonized. The results were interpreted as forskolin and 5'-N-ethylcarboxamide adenosine increasing cyclic AMP accumulation, and dibutyryl cyclic AMP mimicking it, and by these mechanisms decreasing sodium uptake through the sodium channels.     

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in a dose-response manner (IBMX ED₅₀ = 41 μM for COCs; forskolin ED₅₀ = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro. © 1996 Wiley-Liss, Inc.