Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl

Nitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KCl. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KCl is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2 mM N-nitro-L-arginine methyl ester (L-NMAE), and 10 mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KCl under the depolarization conditions with 45 mM KCl. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.     

Differential Response of Adenylate Cyclase and ATPase Activities After Chronic Ethanol Exposure of PC12 Cells

The direct effects of chronic ethanol administration on adenylate cyclase, Na,K-ATPase, and Mg-ATPase activities in a cell containing neuronal characteristics were investigated using PC12 pheochromocytoma cells. Exposure of PC12 cells to 0, 75, and 150 mM ethanol for 4 days caused a dose-dependent increase in the stimulation of adenylate cyclase by in vitro ethanol without altering activation of the enzyme by GTP, NaF, MnCl2, or 2-chloroadenosine. Conversely, a 4-day treatment with 150 mM ethanol increased Na,K-ATPase and Mg-ATPase activities without altering the inhibitory effects of in vitro ethanol. The increase in Na,K-ATPase activity was associated with an increase in Vmax without any change in the Km for KCl. Chronic ethanol exposure also increased the amount of [3H]ouabain specifically bound to PC12 cell membranes. Except for the increase in Mg-ATPase activity, the above results were also observed when chronic ethanol treatment was carried out in the presence of pyrazole. Although ethanol slowed PC12 cell growth, observed changes were not due to an ethanol-induced reduction in cellular density. A 4-day exposure of a nonneuronal cell line (Madin Darby canine kidney cell) to 150 mM ethanol did not alter adenylate cyclase or ATPase activities. The present study indicates that the direct effects of chronic ethanol exposure of a neuronal-like cell involve an increase in the density of sodium pumps per cell and an enhanced sensitivity of adenylate cyclase to activation by ethanol.     

Potentiation of nerve growth factor(NGF)-mediated neurite outgrowth in high K+ medium is associated with increased binding of iodinated NGF in PC12 cells

Nerve growth factor(NGF)-mediated neurite outgrowth of PC12 pheochromocytoma cells was potentiated in medium containing high concentrations of extracellular K+. The binding of iodinated NGF to the cells was also enhanced by raising the concentration of K+ in medium up to 100 mM; the enhancement was saturated at 50 mM K+. Although the mechanism by which NGF-mediated neurite outgrowth is potentiated in high K+ medium remains to be largely unknown, high K+-induced alterations in the NGF binding are suggested to play a role in this phenomenon.     

Lysis of Escherichia coli mutants by lactose.

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.     

Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex

Effects of taurine on Ca⁺⁺ binding to microsomes isolated from rat cerebral cortex were investigated in a medium containing various concentrations of KCl and/or NaCl. Calcium binding to microsomes was inhibited in a dose-dependent fashion by taurine in the incubation medium containing 5 mM KCl and 115 mM NaCl, while there was no inhibition in the medium containing 115 mM KCl and 5 mM NaCl. Taurine also decreased Ca⁺⁺ binding in the medium containing 70 mM KCl without NaCl. A similar tendency toward inhibition of the Ca⁺⁺ binding was observed in the medium with 5 mM or 120 mM KCl without NaCl. Taurine did not influence the Ca⁺⁺ binding in the medium containing different concentrations of NaCl without KCl, or in the medium from which KCl and NaCl were omitted. Isethionate, glycine, γ-aminobutyric acid, β-alanine and L-leucine did not significantly alter the Ca⁺⁺ binding to microsomes in the medium containing 70 mM KCl without NaCl. Thus it would appear that taurine may modulate the binding of calcium to microsomes in conditions which resemble the state of depolarization, while it is inactive in the normal resting state. This effect is apparently specific to taurine amongst a series of putative “inhibitory” amino acids.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and differentiation of primary tracheal epithelial cells in culture: regulation by extracellular calcium

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (greater than 0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucus-secreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (greater than 0.6 mM). In contrast, MTE cells cultured in low calcium medium (less than 0.15 mM) were squamous-like with prominent tonofilaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.     

Transport studies with brush border membrane vesicles: choice of experimental parameters

1. We have studied different parameters, in their effects on a transport system chosen as a model: the Na+-phosphate symporter of the renal brush border membrane. 2. Ionic strength was found to be a critical factor in the retention capacity of the filter. 3. When high ionic strength solutions containing 150 mM NaCl or KCl were used, less than 8% of the membrane proteins were lost through filtration. 4. Lowering the ionic strength by replacing NaCl or KCl by 300 mM mannitol, however, caused a 52% loss of protein. 5. Addition of 15 mM NaCl to this low ionic strength solution was sufficient to restore full retention of the vesicles by the filter. 6. The presence of arsenate, a competitive inhibitor, in the stop solution did not improve the retention of phosphate by the vesicles in high ionic strength media, but caused a pronounced temperature dependent loss of the vesicle content, as a function of time of incubation in low ionic strength solutions. 7. Addition of 5 mM phosphate in the stop solution caused a 31 and 37% loss for KCl and NaCl stop solutions, respectively, while no effect was observed for the mannitol stop solution. 8. The presence of HgCl2 gave a 32% stimulation for the mannitol solution and a 35 or 22% inhibition for the KCl or NaCl solutions. 9. Addition of NaCl in the stop solution caused an overaccumulation of 75%, after 60 sec of incubation at 25 degrees C. 10. Phosphate transport by renal vesicles is thus highly affected by the composition of the stop solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular potassium can simulate the role of serum as an activator of uridine uptake by quiescent hamster cells in culture

Replacement of ~100 mM of sodium chloride in the extracellular medium of quiescent hamster fibroblasts (Nil 8 and BHK cells) by potassium chloride causes an increase in the rate of uridine uptake. This increase is identical with that achieved by addition of 10% serum to the same cultures. The effects of serum and KCl are not additive. The dependence of the rate of uridine uptake on extracellular KCl concentration is of a sigmoid nature. The time course of the activation process is similar to that of serum activation of uridine uptake in the same cells. The high rate of uridine uptake persists for at least 30 min after return to an extracellular medium containing a high concentration of sodium.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common

In response to an external stimulus, neuronal cells release neurotransmitters from small synaptic vesicles and endocrine cells release secretory proteins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle membranes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense core granule membranes, monoclonal antibodies bound to beads were used to immunoisolate organelles containing the synaptic vesicle antigens. [3H]norepinephrine was used to label both chromaffin granules purified from the bovine adrenal medulla and rat pheochromocytoma (PC12) cells. Up to 80% of the vesicular [3H]norepinephrine was immunoisolated from both labeled purified bovine chromaffin granules and PC12 postnuclear supernatants. In PC12 cells transfected with DNA encoding human growth hormone, the hormone was packaged and released with norepinephrine. 90% of the sedimentable hormone was also immunoisolated by antibodies to all three proteins. Stimulated secretion of PC12 cells via depolarization with 50 mM KCl decreased the amount of [3H]norepinephrine or human growth hormone immunoisolated. Electron microscopy of the immunoisolated fractions revealed large (greater than 100 nm diameter) dense core vesicles adherent to the beads. Thus, large dense core vesicles containing secretory proteins possess all three of the known synaptic vesicle membrane proteins.     

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca++ medium (0.09 mM) as measured by incorporation of [3H]thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC50 of about 10 microM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 microM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number over control at 10 microM. These results are of importance since they suggest Ca++ may influence the effect of retinoids on keratinocytes.     

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.     

Somatostatin Release from Rat Cerebral Cortex Synaptosomes

Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin-like immunoreactivity (SRIF-LI) was measured by means of a radioimmunoassay procedure. High KCl (9-50 mM) concentration dependently evoked SRIF-LI release; the evoked overflow reached a plateau at 25 mM KCl and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KCl used. The 15 mM K(+)-evoked release of SRIF-LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K(+)-evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 microM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF-LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1-50 microM) induced release of SRIF-LI in a concentration-dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF-LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium-deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that greater than 90% of the SRIF-LI released either during the spontaneous outflow or by 15 mM KCl was represented by SRIF-14 (SRIF-28(14-28]. These values reflected the ratio SRIF-14/SRIF-28 found in synaptosomes at the end of the experiments.     

Role of calcium in the potentiating effect of phorbol ester on KCl-induced vasocontraction

The mechanism of the potentiating effect of phorbol ester on potassium-induced contraction in rat aorta was investigated. The contractile response to KCl in the medium containing 0.5 mM CaCl2 was significantly increased by pretreatment with 10(-8) M phorbol 12-myristate 13-acetate (PMA), but not with 10(-7) M 4 alpha-phorbol. The dose-response curve to calcium in 30 mM KCl-induced contraction was shifted to the left by PMA pretreatment and the EC50 value (the concentration producing a half maximal response) of calcium was significantly lower in aorta pretreated with PMA than in the control. On the other hand, calcium influx stimulated by 30 mM KCl was not changed by PMA pretreatment. Both the contractile response and the corresponding calcium influx induced by 30 mM KCl were abolished by preincubation with 10(-6) M verapamil for 45 min. These results suggest that activation of protein kinase C potentiates the contractile response to KCl by increasing the sensitivity of the intracellular contractile apparatus for calcium.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.