Isodihydrocapsiate stimulates plasma glucose uptake by activation of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is implicated as a key factor in controlling whole body homeostasis, including fatty acid oxidation and glucose uptake. We report that a synthetic structural isomer of dihydrocapsiate, isodihydrocapsiate (8-methylnonanoic acid 3-hydroxy-4-methoxy benzyl ester) improves type 2 diabetes by activating AMPK through the LKB1 pathway. In L6 myotube cells, phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and glucose uptake were significantly increased, whereas these effects were attenuated by an AMPK inhibitor, compound C. In addition, increased phosphorylation of AMPK and ACC by isodihydrocapsiate was significantly reduced by radicicol, an LKB1 destabilizer, suggesting that increased glucose uptake in L6 cells with isodihydrocapsiate treatment is predominantly accomplished by a LKB1-mediated AMPK activation pathway. Oral administration of isodihydrocapsiate to diabetic (db/db) mice reduced blood glucose levels by 40% after a 4-week treatment period. Our results support the development of isodihydrocapsiate as a potential therapeutic agent to target AMPK in type 2 diabetes.     

Diphenylene iodonium stimulates glucose uptake in skeletal muscle cells through mitochondrial complex I inhibition and activation of AMP-activated protein kinase

NADPH oxidase inhibitors such as diphenylene iodonium (DPI) and apocynin lower whole body and blood glucose levels and improve diabetes when administered to rodents. Skeletal muscle has an important role in managing glucose homeostasis and we have used L6 cells, C(2)C(12) cells and primary muscle cells as model systems to investigate whether these drugs regulate glucose uptake in skeletal muscle cells. The data presented in this study show that apocynin does not affect glucose uptake in skeletal muscle cells in culture. Tat gp91ds, a chimeric peptide that inhibits NADPH oxidase activity, also failed to affect glucose uptake and we found no significant evidence of NADPH oxidase (subunits tested were Nox4, p22phox, gp91phox and p47phox mRNA) in skeletal muscle cells in culture. However, DPI increases basal and insulin-stimulated glucose uptake in L6 cells, C(2)C(12) cells and primary muscle cells. Detailed studies on L6 cells demonstrate that the increase of glucose uptake is via a mechanism independent of phosphoinositide-3 kinase (PI3K)/Akt but dependent on AMP-activated protein kinase (AMPK). We postulate that DPI through inhibition of mitochondrial complex 1 and decreases in oxygen consumption, leading to decreases of ATP and activation of AMPK, stimulates glucose uptake in skeletal muscle cells.     

Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells

G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.     

Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes

alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.     

Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC

2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidativechain from ATP production, preventing oxidative metabolism. Theconsequent increase in energy demand is, however, contested by cellsincreasing glucose uptake to produce ATP via glycolysis. In L6 skeletalmuscle cells, DNP rapidly doubles glucose transport, reminiscent of theeffect of insulin. However, glucose transport stimulation by DNP doesnot require insulin receptor substrate-1 phosphorylation and iswortmannin insensitive. We report here that, unlike insulin, DNP doesnot activate phosphatidylinositol 3-kinase, protein kinaseB/Akt, or p70 S6 kinase. However, chelation of intra- andextracellular Ca²⁺ with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM in conjunction with EGTA inhibited DNP-stimulated glucoseuptake by 78.9 ± 3.5%. BecauseCa²⁺-sensitive, conventionalprotein kinase C (cPKC) can activate glucose transport in L6 musclecells, we examined whether cPKC may be translocated andactivated in response to DNP in L6 myotubes. Acute DNP treatment led totranslocation of cPKCs to plasma membrane. cPKC immunoprecipitated fromplasma membranes exhibited a twofold increase in kinase activity inresponse to DNP. Overnight treatment with 4-phorbol 12-myristate13-acetate downregulated cPKC isoforms , , and  and partiallyinhibited (45.0 ± 3.6%) DNP- but not insulin-stimulatedglucose uptake. Consistent with this, the PKC inhibitorbisindolylmaleimide I blocked PKC enzyme activity at theplasma membrane (100%) and inhibited DNP-stimulated2-[³H]deoxyglucoseuptake (61.2 ± 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC- inhibitorLY-379196 partially inhibited DNP effects on glucose uptake (66.7 ± 1.6%). The results suggest interfering with mitochondrial ATPproduction acts on a signal transduction pathway independent from thatof insulin and partly mediated byCa²⁺ and cPKCs, of which PKC-likely plays a significant role.

     

Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes

The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCalpha) to the increase or reduction of mitochondrial Ca2+ uptake (PKCzeta and PKCbeta/PKCdelta, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia

Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK) activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+)/Calmodulin-dependent protein kinase kinase (CaMKK), which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO), a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG)-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV) in rats 30 min before 2DG ICV injection (40 μmol) to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol) did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.     

Does protein kinase C require Ca2+ for activation?

Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.     

Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.     

Characterization of Mg2+ inhibition of mitochondrial Ca2+ uptake by a mechanistic model of mitochondrial Ca2+ uniporter

Ca(2+) is an important regulatory ion and alteration of mitochondrial Ca(2+) homeostasis can lead to cellular dysfunction and apoptosis. Ca(2+) is transported into respiring mitochondria via the Ca(2+) uniporter, which is known to be inhibited by Mg(2+). This uniporter-mediated mitochondrial Ca(2+) transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg(2+) inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg(2+) and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg(2+) inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca(2+) uptake. The model also appropriately depicts the inhibitory effect of Mg(2+) on the uniporter function, in which Ca(2+) uptake is hyperbolic in the absence of Mg(2+) and sigmoid in the presence of Mg(2+). The model suggests a mixed-type inhibition mechanism for Mg(2+) inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca(2+) handling to understand the mechanisms by which Ca(2+) mediates signaling pathways and modulates energy metabolism.     

Ultraviolet enhances the sensitivity of pancreatic cancer cells to gemcitabine by activation of 5' AMP-activated protein kinase

Although gemcitabine is recognized as the standard drug for the treatment of advanced pancreatic cancer, the clinical outcome is not satisfactory. We recently reported that relatively high dose ultraviolet-C (UV-C; 200 J) inhibits cell growth by desensitization of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells. In the present study, we investigated the combination effects of low dose UV-C (10 J) and gemcitabine on apoptosis and cell growth in these cells. UV-C enhanced gemcitabine-induced suppression of cell viability. In addition, the combination use clearly induced apoptosis, while neither UV-C nor gemcitabine alone did. Concurrently, combination use caused the decrease in the EGFR protein level and reduced EGF-induced activation of Akt pathway, subsequently resulting in accumulation of β-catenin. The order of the treatment with UV-C and gemcitabine did not affect their synergistic effects on apoptosis and cell growth. Interestingly, combination use synergistically induced phosphorylation of 5′ AMP-activated protein kinase (AMPK) alpha at Thr172 and acetyl-CoA carboxylase at Ser79 as a downstream molecular target of AMPK. AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-riboside, induced apoptosis and suppressed cell growth in these cells, thus suggesting that combination effects of UV-C and gemcitabine is due to the activation of AMPK. Together, our findings could provide a new aspect of pancreatic cancer therapy.     

Ca2+ influx-independent synaptic potentiation mediated by mitochondrial Na(+)-Ca2+ exchanger and protein kinase C

Activity-dependent modulation of synaptic transmission is an essential mechanism underlying many brain functions. Here we report an unusual form of synaptic modulation that depends on Na+ influx and mitochondrial Na(+)-Ca2+ exchanger, but not on Ca2+ influx. In Ca(2+)-free medium, tetanic stimulation of Xenopus motoneurons induced a striking potentiation of transmitter release at neuromuscular synapses. Inhibition of either Na+ influx or the rise of Ca2+ concentrations ([Ca2+]i) at nerve terminals prevented the tetanus-induced synaptic potentiation (TISP). Blockade of Ca2+ release from mitochondrial Na(+)-Ca2+ exchanger, but not from ER Ca2+ stores, also inhibited TISP. Tetanic stimulation in Ca(2+)-free medium elicited an increase in [Ca2+]i, which was prevented by inhibition of Na+ influx or mitochondrial Ca2+ release. Inhibition of PKC blocked the TISP as well as mitochondrial Ca2+ release. These results reveal a novel form of synaptic plasticity and suggest a role of PKC in mitochondrial Ca2+ release during synaptic transmission.     

Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake

Angiotensin II elicits cytosolic and mitochondrial Ca2+ signal in H295R adrenocortical cells. We found that Ca2+ uptake rate and peak values in small mitochondrial regions both depend on the colocalization of these mitochondrial regions with GFP-marked endoplasmic reticular (ER) vesicles. The dependence of the Ca2+ response on this colocalization is abolished by SB202190 and PD169316, inhibitors of p38 MAPK, as well as by transfection with siRNA against p38 MAPK mRNA. The same manoeuvres result in an increased ratio of global mitochondrial to global cytosolic Ca2+ response, indicating that inhibition of p38 MAPK is followed by enhanced mitochondrial Ca2+ uptake. alpha-Toxin and TNFalpha, agents which similarly to angiotensin II increase the phosphorylation of p38, failed to affect mitochondrial Ca2+ uptake, indicating that activation of p38 MAPK is necessary but not sufficient for the inhibition of Ca2+ uptake. Bisindolylmaleimide, an inhibitor of the conventional and novel-type protein kinase C isoforms also evokes enhanced mitochondrial Ca2+ uptake, whereas G?6976 that inhibits the conventional isoforms only failed to exert any effect. These data show that angiotensin II attenuates Ca2+ uptake predominantly into mitochondria that do not colocalize with ER, by a mechanism involving p38 MAPK and a novel-type PKC.     

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C

The influence of infrared laser pulses on intracellular Ca²⁺ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo‐4 was used to measure Ca²⁺ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo‐4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca²⁺ transient with similar dose‐response characteristics in hippocampal neurons and glioblastoma cells (half‐maximal effective energy density EC₅₀ of around 58 J.cm^?2). For both type of cells, the source of the infrared‐induced Ca²⁺ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP₃‐induced Ca²⁺ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP₃ cell signaling cascade has many potential applications including the development of optoceutical therapeutics.      

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.     

Autoregulation of prostaglandin E2-induced Ca2+ influx in osteoblast-like cells: inhibition by self-induced activation of protein kinase C.

In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.