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C R Kleeman

Publications and source records attributed to C R Kleeman.

At least 19 recordsLinked to original sources
PubMed1992

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.

Calcium↗
PubMed1992

Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells.

We have recently shown that two mechanisms are involved in the regulation of pHi in the osteoblastic phenotype cell line UMR-106 (Na(+)-H+ antiporter and a Na(+)-independent Cl(-)-HCO 3(-)-OH- exchanger). In the present work, we used the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein as well as isotope fluxes to investigate the influence of second messengers on the activity of these transporters. Elevation in intracellular calcium concentration [( Ca2+]in) in UMR-106 cells (measured by fura-2 fluorescence) is followed by stimulation of the Cl(-)-HCO3- exchanger, leading to cytosolic acidification. Subsequently, cell alkalinization, mediated by the Na(+)-H+ exchanger, restores pHi to its resting value. An acute reduction in [Ca2+]in abruptly stops the activity of the anion exchanger while having no influence on the activity of the Na(+)-H+ exchanger. The stimulatory effect of Ca2+in on the anion exchanger is dose dependent and is abrogated by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide and calmidazolium. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) brought about by forskolin, 8-bromo-cAMP, or prostaglandin E2 leads to inhibition of activity of both the Na(+)-H+ antiporter and the anion exchanger. The suppressive effect of cAMP on Cl(-)-HCO3- exchange could be overcome by elevating [Ca2+]in. We conclude that 1) Ca2+in and cAMP can influence pHi in osteoblasts by altering the activities of pHi regulatory mechanisms and 2) the effect of Ca2+in is probably mediated by calmodulin.

Anions↗
PubMed1992

1,25(OH)2D3 blunts hormone-elevated cytosolic Ca2+ in osteoblast-like cells.

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.

Calcitriol↗
PubMed1991

Voltage-dependent phosphate transport in osteoblast-like cells.

Phosphate ion (Pi) in sufficient concentrations is crucial for bone mineralization. The osteoblast (OB) may be responsible for the transport of Pi into the bone interstitium, where mineralization occurs. We previously characterized a Na(+)-dependent Pi transporter (NaPi) in the osteoblastic UMR-106-01 cell line. In the present study, the alteration of Na(+)-dependent Pi transport by changes in membrane potential was investigated. Depolarizing the cells with increasing concentrations of ambient K+ and valinomycin resulted in a progressive decline in Na(+)-dependent Pi uptake to a maximum of 28% at a membrane potential of -18 mV compared to control Na(+)-dependent Pi uptake at a membrane potential of approximately -60 mV. Hyperpolarizing the cells with SCN- increased Na(+)-dependent Pi uptake over control by 50% at an SCN- concentration of 70 mM. Determination of membrane potential by using the fluorescent probe, DiSC3(5), showed that the addition of Pi to cells in Na(+)-containing medium resulted in a small depolarization. These data show that NaPi activity can be altered by membrane potential changes and that the initiation of Na(+)-dependent Pi uptake is associated with depolarization of the plasma membrane of UMR-106-01 cells. Taken together, the cotransport of Na+ and Pi results in the movement of a net positive charge into the cell.

Benzothiazoles↗
PubMed1991

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Acute phosphate depletion dissociates hormonal stimulated second messengers in osteoblast-like cells.

The acute effect (24 h) of either phosphate depletion or phosphate surfeit on hormonal stimulated signal transduction systems was studied in the osteoblastic cell line UMR-106. Elevation of intracellular Ca2+ ([Ca2+]in), induced by different calciotropic hormones (PTH, prostaglandin E2, endothelin) was blunted by acute phosphate depletion, whereas at high inorganic phosphate (Pi) concentrations the rise in [Ca2+]in was augmented. Basal [Ca2+]in was not altered by either Pi depletion or Pi excess. The effect of acute phosphate depletion on hormonal mediated [Ca2+]in rise was not observed in the absence of extracellular Ca2+ suggesting that under these conditions, the release of Ca2+ from intracellular stores, is not affected. Also, nonhormonal calcium entry pathways such as depolarization-activated calcium channels or protein kinase C-activated Ca2+ channels were not affected by acute phosphate depletion. cAMP accumulation in the cells, either through receptor or nonreceptor-mediated mechanisms, increased under low Pi conditions and decreased as Pi concentration in the culture media was progressively increased from 0 to 2 mM during 24 h of incubation. Changes in Pi concentration had no effect on basal cAMP generation by the cells. The facilitative effect of acute Pi depletion on agonist-induced cAMP accumulation could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). PTH receptor binding assessed with [Nle8 Nle18 Tyr34] bovine PTH (1-34) NH2 was not altered by phosphate depletion. We conclude that exposure of osteoblasts to different Pi environments modulates the second messenger responses to hormones in a reciprocal fashion so that acute phosphate depletion down-regulates [Ca2+]in signals while augmenting cAMP generation and vice versa. Inasmuch as bone resorption processes can be modulated by Ca2+ and cAMP the data presented herein suggest that the altered bone resorptive response to calciotropic hormones (e.g. PTH), under surfeit or deficit of phosphate, is mediated by changes in [Ca2+]in and cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions.

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.

Animals↗
PubMed1989

Classification of prostaglandin receptors based on coupling to signal transduction systems.

A wide spectrum of prostaglandins (PG) stimulate both the production of cyclic AMP and an increase in free cytosolic Ca2+ concentration [( Ca2+]i) in the osteogenic osteosarcoma cell line, UMR-106-01, which has characteristics compatible with osteoblasts. Using PG-stimulated determinations of the second messengers cyclic AMP and [Ca2+]i, a method for classification of PG receptors is presented. UMR-106-01 cells demonstrate three subclasses of PG receptors. One receptor interacts with PGF2 alpha, PGD2, and thromboxane B2 (TxB2) to increase [Ca2+]i. A second receptor binds PGE2, PGE1, PGI2, PGA2 and 6-oxo-PGF1 alpha to increase [Ca2+]i by stimulation of a second separate phospholipase C pool. A third receptor accepts PGE2, PGE1, PGA2, PGI2 and to a lesser extent PGF2 alpha, PGD2 and TxB2 to increase cyclic AMP. Such a classification system may be applicable to other cells responding to multiple PGs by inducing changes in cellular second messengers.

Adenylyl Cyclases↗
PubMed1989

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01.

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.

Calcium↗
PubMed1989

Properties of the depolarization-activated calcium and barium entry in osteoblast-like cells.

Measurements of free cystolic Ca2+ ([Ca2+]i) and Ba2+ ([Ba2+]i) concentrations with Fura 2 were used to identify and characterize the properties of a depolarization-activated Ca2+ and Ba2+ entry in the plasma membrane of osteoblast-like cells. The presence of this pathway was demonstrated in two osteoblastic cell lines, UMR-106 and MC3T3-E1 and osteoblasts isolated from rat long bone and rat neonatal calvariae. Subsequent characterization of the pathway was performed in the osteosarcoma cell line UMR-106. Depolarization of the cells with high medium K+ was followed by an increase in [Ca2+]i which was dependent on medium Ca2+. Ba2+ ions depolarized the cells and were transported by this pathway. Mg2+ ions interfered with Ca2+ and Ba2+ entry. At 140 mM KCl and 1 mM MgCl2, the pathway could be saturated with Ca2+ or Ba2+. The apparent affinity for Ca2+ was 0.78 mM and for Ba2+ 1.82 mM. Ca2+ or Ba2+ entry into the cells was blocked by low concentrations of nicardipine, diltiazem, verapamil, and La3+. In the absence of an increase in [Ca2+]i or [Ba2+]i, the pathway inactivated within about 5 min after depolarization. When [Ca2+]i or [Ba2+]i was allowed to increase, the pathway inactivated within about 20 s. These properties suggest that Ca2+ and Ba2+ entry are mediated by an L-type, depolarization-activated Ca2+ channel in osteoblasts. The activity of these channels changes little with an increase or decrease in cell volume. Thus, it is concluded that these pathways do not provide the Ca2+ entry pathway required for initiation of volume decrease by osteoblasts.

Animals↗
PubMed1989

Metabolic coma.

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Adult↗
PubMed1989

Modulation of osteoblast function by prostaglandins.

The naturally occurring prostaglandins (PGs) were studied with respect to their abilities to change free cytosolic Ca2+ concentrations ([Ca2+]i), adenosine 3',5'-cyclic monophosphate (cAMP) levels, and cell proliferation in the osteoblastic cell line, UMR-106-01, and primary cultures of osteoblasts prepared from neonatal rat calvariae. All PGs tested stimulated an increase in [Ca2+]i, which was mainly due to Ca2+ release from intracellular stores. Measurements of the 50% effective concentration for the different PGs show that the potency ranking for PG-evoked [Ca2+]i increase in these cells is F2 alpha greater than D2 much greater than E2 greater than TxB2 greater than E1 greater than I2 much greater than A2. The PGs also increase cAMP levels in osteoblasts. At the highest concentrations tested (10-25 microM), dose-response saturation of cAMP production was observed only by PGE2 and PGE1. The potency rank for PG-stimulated cAMP increase was E2 greater than E1 much greater than A2 greater than I2 greater than F2 alpha greater than D2 greater than TxB2. Measurements of the effect of the PGs on thymidine uptake showed that low concentrations of PGF2 alpha and PGD2 had either no effect or stimulated proliferation of osteoblast-like cells. Relatively low concentration of PGE2, PGE1, and PGA2 inhibited proliferation. The potency ranking for PG-mediated inhibition of cell proliferation was identical to that found for PG-stimulated cAMP production. We conclude that all the naturally occurring PGs tested can activate the two signal transduction systems in osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1988

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells.

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Selective modification of the kinetic properties of Na+/H+ exchanger by cell shrinkage and swelling.

The effect of volume perturbation on the interaction of Na+ and H+ with the intracellular and extracellular faces of the Na+/H+ exchanger was studied in UMR-106 cells, a rat osteosarcoma cell line. Osmotic shrinkage of the cells stimulated the activity of the Na+/H+ exchanger. Kinetic analysis of this stimulation demonstrated that in hyperosmotically stressed cells, the apparent affinities for intracellular H+ and intracellular Na+ are modified in opposite directions. While there is an increased apparent affinity for protons from 0.275 +/- 0.03 to 0.107 +/- 0.025 microM in isotonic and hypertonic conditions, respectively, the apparent affinity for intracellular Na+ decreases from 83 +/- 9 to 126 +/- 6 mM under the same conditions. Osmotic swelling induced a decreased exchanger activity which appeared to involve reduction in Vmax only without changes in the apparent affinities of either H+i or Na+i. We conclude that: 1) osmotic shrinkage and swelling modify the kinetic behavior of the Na+/H+ exchanger in different modes; 2) in hyperosmotically stressed cells, the interactions of intracellular H+ and Na+ are modified in a selective mode. The described phenomenon may serve as a general mechanism for activation of the exchanger by various stimuli.

Animals↗
PubMed1988

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger.

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.

Amiloride↗
PubMed1987

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106.

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.

Calcium↗