Biomedical subjects

H C Palfrey

Publications and source records attributed to H C Palfrey.

At least 37 records · Page 2Linked to original sources

PubMed1994

Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells.

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.

Animals↗

PubMed1994

Prolactin regulation of the calmodulin-dependent protein kinase III elongation factor-2 system in the rat corpus luteum.

A M(r) 100,000 phosphoprotein in the corpus luteum was identified as elongation factor 2 (EF-2). Since prolactin (PRL) is necessary for optimal luteal development and protein synthesis, we determined whether this hormone affects the content and/or phosphorylation of EF-2 in the corpus luteum. PRL treatment enhanced the Ca2+/calmodulin (CaM)-dependent phosphorylation of endogenous EF-2 in luteal cytoplasmic extracts. Immunoblot analysis revealed that PRL had no effect on EF-2 levels, but examination of luteal EF-2 by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis showed that PRL increased the relative amount of the most basic dephosphorylated forms of EF-2. This suggests that PRL induces net dephosphorylation of the protein in vivo. Since EF-2 phosphorylation is regulated by both Ca2+/CaM-dependent kinase III (CaM kinase III) and protein phosphatase 2A, we examined the effect of PRL on both enzymes. Paradoxically, PRL enhanced the in vitro activity of CaM kinase III, possibly reflecting increased kinase levels, but had no effect on phosphatase activity. These results suggest that PRL maintains luteal EF-2 in a relatively dephosphorylated state in vivo by limiting the availability of Ca2+ and/or CaM to CaM kinase III. These data provide strong evidence for a role of the EF-2/CaM kinase III system in PRL action in the corpus luteum.

Animals↗

PubMed1994

Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.

Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na(+)-K(+)-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was approximately 20 pmol.min-1.oocyte-1 in the presence of blockers of Na(+)-K(+)-ATPase and conductive K+ transport. Bumetanide (10 microM) inhibited > 90% of this uptake, suggesting involvement of Na(+)-K(+)-2Cl- cotransport. This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide [50% inhibitory concentration (IC50), 2 x 10(-11) M] > bumetanide (IC50, 7 x 10(-8) M) > furosemide (IC50, 2.5 x 10(-6) M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by approximately 75%, whereas a doubling of medium osmolarity increased it by approximately 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an approximately 140-kDa oocyte membrane protein. These results demonstrated that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Rapid and sustained phosphorylation of a calmodulin-binding protein (CaM-BP100) in NGF-treated PC12 cells.

Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.

Animals↗

PubMed1993

Early nerve growth factor-induced events in developing rat septal neurons.

A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this "bilaminar" system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2-5 min) and transiently induced expression of c-fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.

Animals↗

PubMed1993

Purification and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas.

Calmodulin-dependent protein kinase III (CaM kinase III) phosphorylates and thereby inactivates eukaryotic elongation factor-2 (EF-2). This enzyme, purified to homogeneity from either rabbit reticulocytes or rat pancreas, had similar properties: it migrated with an apparent M(r) of 140,000 by gel filtration, was comprised of a major polypeptide of M(r) 95,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a high affinity for CaM (half-maximal activation < 1 nM). The M(r) 95,000 polypeptide was autophosphorylated by an intramolecular mechanism on seryl residues in the presence of Ca2+, CaM, and ATP, and phosphopeptide mapping indicated that several sites were phosphorylated. Autophosphorylation of CaM kinase III resulted in the generation of a partially Ca2+/calmodulin-independent activity. The enzyme could also be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Amino acid sequencing of CaM kinase III indicated that it is distinct from other known proteins including the heat-shock protein hsp90, which was recently suggested to be identical to CaM kinase III (Nygärd, O., Nilsson, A., Carlberg, U., Nilsson, L., and Amons, R. (1991) J. Biol. Chem. 266, 16425-16430). Furthermore, hsp90 did not copurify with CaM kinase III, and the M(r) 95,000 protein did not cross-react with antibodies to hsp90. CaM kinase III exhibited Michaelis-Menten kinetics toward its substrates ATP and EF-2, with Km values of 15 and 2 microM, respectively. CaM kinase III was able to phosphorylate yeast EF-2 with an Km of 2 microM, but the enzyme did not significantly phosphorylate a variety of other substrates, confirming its identification as a novel protein kinase.

Amino Acid Sequence↗

PubMed1993

High-affinity nerve growth factor receptor (Trk) immunoreactivity is localized in cholinergic neurons of the basal forebrain and striatum in the adult rat brain.

Trk-immunoreactivity was observed in basal forebrain and striatal cholinergic neurons, whereas low-affinity NGF receptor immunoreactivity was observed in basal forebrain but not striatal cholinergic neurons. Since NGF exerts trophic actions on both basal forebrain and striatal cholinergic populations, the presence of Trk in these neurons lends strong support for an essential role of Trk in NGF-responsive neurons, but suggests that the low affinity receptor is not necessary for NGF actions in the striatum.

Animals↗

PubMed1993

Calmodulin binding to and cAMP-dependent phosphorylation of kinesin light chains modulate kinesin ATPase activity.

Kinesin is an ubiquitous heterotetrameric microtubule-based motor which translocates membrane-bound organelles. Since organelle motility and motor protein function can be regulated by components of signaling pathways, the ability of purified bovine brain kinesin (kinesin) to be phosphorylated and to recognize calmodulin (CaM) was tested. Extensively purified "kinesin" was found to consist of several forms of both heavy (KHC) and light (KLC) chains. Phosphorylation of kinesin by a variety of protein kinases was examined; cAMP-dependent protein kinase (cAMP-PK) was the most active enzyme leading to the incorporation of up to 8 mol P/mol kinesin. Phosphorylation occurred predominantly on the KLCs and led to substantial acidic pI shifts. Peptide maps indicated that multiple phosphorylation sites exist on each KLC. Incubation of kinesin in vitro with protein kinase C (PKC) led to the phosphorylation of both KHCs and KLCs. In vivo phosphorylation of KHC and KLCs was demonstrated by immunoprecipitation of [32P]-labeled kinesin from cultured rat hippocampal pyramidal neurons; kinesin phosphorylation was stimulated by 8-chlorophenyl-thio-cAMP or 12-O-tetradecanoylphorbol-13-acetate. Native bovine brain kinesin was shown to bind 125I-CaM by nucleotide-dependent pelleting with stable microtubules. Specific calcium-dependent binding of 125I-CaM to KLCs but not KHC was found using a ligand blotting assay. cAMP-PK phosphorylated kinesin bound 125I-CaM less well than untreated protein in both ligand blotting and microtubule-pelleting paradigms. Calcium-dependent binding of CaM to kinesin inhibited the ATPase activity of native kinesin but not of cAMP-PK phosphorylated kinesin. These results suggest that the KLCs have a regulatory function and integrate information coming from diverse signaling pathways to modulate the activity and function of kinesin.

Adenosine Triphosphatases↗

PubMed1993

The ATP and Mg2+ dependence of Na(+)-K(+)-2Cl- cotransport reflects a requirement for protein phosphorylation: studies using calyculin A.

Na(+)-K(+)-2Cl- cotransport activity has previously been shown to depend on both intracellular ATP and Mg2+, but the mechanisms remain unknown. Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/threonine phosphatase inhibitors and is inhibited by prior depletion of either ATP with antimycin A, or mg2+ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosphatase-inhibitor-treated cells were resistant to subsequent ATP or Mg2+ depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg2+ depletion leads to dephosphorylation of membrane proteins in cAMP-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg2+ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators. The relative effectiveness of okadaic acid (EC50 approximately 630 nM) and calcyulin A (EC50 approximately 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg2+ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.

Adenosine Triphosphate↗

PubMed1993

Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogues, and adducts.

The inhibitory effect of ethacrynic acid (EA) and a variety of its derivatives on Na-K-2Cl cotransport in avian erythrocytes was investigated. The most potent compound tested was the adduct of EA with L-cysteine, with an IC50 of 7.2 x 10(-7) M. EA itself, dihydro-EA, EA-D-cysteine, and adducts of EA with other sulfhydryl (-SH) compounds were much less potent. The mechanism of action of EA and EA-L-cysteine differed in several respects: 1) EA-L-cysteine acted more rapidly than EA (half times of < 1 and 4 min, respectively, at 37 degrees C); 2) the action of EA-L-cysteine was reversible by washing, whereas that of EA was not; and 3) the degree of inhibition by EA-L-cysteine varied with medium [K], whereas that of EA did not. The inhibitory effects of both EA-L-cysteine and EA were affected by medium [Na] and [Cl]. We conclude that EA-L-cysteine does not "deliver" EA to transport-related -SH residues or act as an alkylating agent but has some stereospecific effect on cotransport that is a property of the entire molecule. EA does appear to inhibit cotransport by alkylating -SH residues, as closely related compounds lacking the ability to covalently react with such groups were reversible, and other -SH reagents (e.g., N-ethylmaleimide) also inhibited cotransport. EA, EA-L-cysteine, and EA-D-cysteine all inhibited [3H]bumetanide binding to membranes from activated avian erythrocytes at concentrations similar to those that inhibited cotransport. It is possible that the EA and bumetanide types of diuretics interact with closely apposed sites on the Na-K-2Cl cotransporter.

Animals↗

PubMed1993

Signal transduction events mediated by the BDNF receptor gp 145trkB in primary hippocampal pyramidal cell culture.

The trkB gene encodes a tyrosine kinase receptor, gp145trkB, for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). To understand the role of gp145trkB in the nervous system, we have investigated its expression in embryonic rat hippocampal pyramidal cell cultures and examined the effects of BDNF on signal transduction in the primary neurons. The expression of trkB transcripts was established by PCR analysis and in situ hybridization. In addition to gp145trkB, the pyramidal neuronal cultures expressed transcripts specific for the NT-3 receptor gp145trkC, but not for the high-affinity NGF receptor gp140trk or for p75LNGFR, a low-affinity receptor for all known members of the NGF family of neurotrophins including the gp145trkB ligands, BDNF and NT-4. The presence of gp145trkB receptors in the primary neuronal cultures was confirmed by immunocytochemical analysis in which > 90% of the cells stained with affinity-purified polyclonal antibodies to gp145trkB. Immunoblots using this antibody revealed a single approximately 140 kDa protein in both adult hippocampus and pyramidal cultures. Addition of recombinant BDNF to these cultures induced the tyrosine phosphorylation of gp145trkB, as determined by antiphosphotyrosine staining of gp145trkB immunoprecipitates. Moreover, BDNF treatment activated the microtubule-associated protein (MAP) kinases, as determined by an increase in MAP2 phosphorylation in vitro. Both the 41 and 44 kDa forms of MAP kinase were activated by BDNF. BDNF also increased c-fos expression in over 90% of the cells. These results indicate that gp145trkB does not require p75LNGFR to form a functional receptor for BDNF in hippocampal pyramidal neurons.

3T3 Cells↗

PubMed1992

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: selectivity and kinetic properties of ion binding sites.

The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport-activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [3H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na+ and K+ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The Km for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na+ and K+ binding sites. Na+ and K+ were partially replaceable, with the selectivity of the Na+ site being Na+ greater than Li+ greater than NH4+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ also supported a small amount of specific binding when substituted for Na+. The selectivity of the K+ site was K+ approximately Rb+ greater than NH4+ greater than Cs+; N-methyl-D-glucamine+, choline+ and tetramethylammonium+ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li+ could partially substitute for Na+ in supporting cotransport, but other monovalent cations were completely inactive. The order of potency at the K+ site was NH4+ greater than K+ approximately Rb+ greater than Cs+ much greater than other monovalent cations. The effect of Cl- on bumetanide binding was biphasic, being stimulatory at low [Cl-] but inhibitory at high [Cl-]. As this implies the existence of two Cl- binding sites (termed ClH and ClL for the "high-" and "low-" affinity sites, respectively) each phase was examined individually. Cl- binding to ClH could be described by a rectangular hyperbola with a Km of 2.5 mM, while kinetic analysis of the inhibition of bumetanide binding at high [Cl-] revealed that it was of a noncompetitive type (Ki = 112.9 mM). The selectivity of anion binding to the two sites was distinct. ClH was highly selective with Cl- greater than SCN- greater than Br-; F-, NO3-, ClO4-, MeSO4-, gluconate- and SO4(2-) were inactive. The efficacy of anion inhibition of binding to ClL was ClO4- greater than I- greater than SCN- greater than NO3- greater than Cl-; F-, MeSO4-, gluconate-, and SO4(2-) were inactive. Thus, ClH is much more selective than ClL and largely accounts for the specificity of the system with respect to anion transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed1991

Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.

Animals↗

PubMed1991

Glutamate-stimulated protein phosphorylation in cultured hippocampal pyramidal neurons.

The regulation of second-messenger production and protein phosphorylation by glutamate has been investigated in primary cultures of pure hippocampal pyramidal neurons. Embryonic rat pyramidal neurons were prepared according to the procedures of Bartlett and Banker (1984) and studied 1-21 d after plating. Glutamate caused a transient increase in intracellular free [Ca2+], determined with fura-2, in the presence of 1.26 mM extracellular Ca2+, but not in 50 nM free Ca(2+)-containing solution. Glutamate also transiently increased cellular diacylglycerol content in both normal and low-[Ca2+] media. Neurons were prelabeled with 32P-orthophosphate to label intracellular ATP, then stimulated with glutamate (100 microM). A rapid transient incorporation of 32P into primarily three proteins of 120, 87, and 48 kDa was found by analysis of two-dimensional gels. At 30 sec after glutamate stimulation, 32P incorporation into the 87-kDa and 48-kDa proteins peaked (240% and 170% basal levels, respectively), and by 2 min, phosphorylation of the 87-kDa protein had returned to basal levels, while that of the 48-kDa protein decreased but remained above control levels. The phosphorylation of these proteins appeared to be mediated by protein kinase C (PKC) because all three showed an increase in phosphorylation after phorbol ester treatment of cultures. Phosphate incorporation was accompanied by an acidic shift in the isoelectric point of both 87- and 48-kDa proteins. Glutamate stimulation resulted in phosphorylation in the presence and absence of Ca2+ influx. Antibody recognition and biochemical characteristics indicated that the 87-kDa phosphoprotein is the PKC substrate MARCKS (myristoylated, alanine-rich C-kinase substrate). The 48-kDa protein, though very similar to GAP-43, was not recognized by specific antibodies raised against GAP-43. These results suggest that glutamate stimulates the transient generation of second messengers that activate PKC in hippocampal neurons, resulting in a significant increase in the phosphorylation of three specific proteins.

Adenosine Triphosphate↗

PubMed1990

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of

Animals↗

PubMed1990

[3H]bumetanide binding to avian erythrocyte membranes. Correlation with activation and deactivation of Na/K/2Cl cotransport.

The relationship between Na/K/2Cl cotransport activation in duck erythrocytes and binding of the diuretic [3H]bumetanide to isolated membranes from stimulated cells has been assessed. Cotransport was activated by either cAMP-dependent (norepinephrine) or -independent (fluoride, hypertonicity) pathways. Membranes isolated from unstimulated cells possessed no specific bumetanide binding. In the presence of norepinephrine, cotransport and saturable binding rose in parallel, reaching a maximum after 5-7 min. In membranes from maximally stimulated cells the K1/2 and Bmax for bumetanide binding were 100 nM and 1.7 pmol/mg protein, respectively. The diuretic binding properties of these membranes were characteristic of interactions of ligands with the Na/K/2Cl cotransporter: specific binding required the presence of all three cotransported ions (Na, K, and Cl), and the rank order of potency for diuretic competition with bumetanide for binding sites was benzmetanide greater than bumetanide greater than furosemide. The appearance of specific bumetanide binding was also seen in membranes from erythrocytes activated by non-cAMP-dependent stimuli, with an excellent temporal correlation between cotransport activation and diuretic binding. On removal of all stimuli both cotransport and bumetanide binding declined in parallel. Duck erythrocytes treated with norepinephrine in a solution containing 15 mM K+ swell to a new stable cell volume after 60 min, during which time cotransport becomes inoperative. Bumetanide binding to both whole cells and isolated membranes paralleled the decline in cotransport activity. It is concluded that bumetanide binding to isolated membranes faithfully reflects the state of activation of the Na/K/2Cl cotransporter in intact cells under a variety of conditions.

Animals↗

PubMed1990

Nerve growth factor-induced down-regulation of calmodulin-dependent protein kinase III in PC12 cells involves cyclic AMP-dependent protein kinase.

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A126-1B2 compared to wild-type cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the "activation ratio" (-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1990

Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.

Animals↗