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

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

Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells

We examined the nature and regulation of the inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of L-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases in L-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 microM) and A-23187 (1 and 3 microM) failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells. We concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.     

Regulation of intracellular calcium homeostasis in Trypanosoma cruzi. Effects of calmidazolium and trifluoperazine

Trypanosoma cruzi epimastigotes maintained an intracellular free calcium concentration of about 0.15 microM, as measured with the fluorescent indicator Fura-2. The maintenance of low [Ca2+]i is energy-dependent since it is disrupted by KCN and FCCP. When the cells were permeabilized with digitonin, the steady-state free Ca2+ concentration in the absence of ATP was about 0.7 microM. The additional presence of ATP resulted in a steady-state level close to 0.1-0.2 microM which compares favorably with the concentration detected in intact cells. Intracellular Ca2+ uptake at high levels of free Ca2+ (greater than 1 microM) was due to energy-dependent mitochondrial uptake as indicated by its FCCP-sensitivity. However, as the free Ca2+ concentration was lowered from 1 microM, essentially all uptake was due to the ATP-dependent Ca2+ sequestration by the endoplasmic reticulum as indicated by its stimulation by ATP, and its inhibition by sodium vanadate. High concentrations of the calmodulin antagonist trifluoperazine, inhibited both the Ca2+ uptake by the endoplasmic reticulum and by the mitochondria, while calmidazolium released Ca2+ from both compartments. In addition, trifluoperazine and calmidazolium inhibited respiration and collapsed the mitochondrial membrane potential of T. cruzi, thus indicating non-specific effects unrelated to calmodulin.     

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.     

Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.

Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.     

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.     

Stage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators

The effects of Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 microM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 microM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, diltiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythrocyte invasion by merozoites. Fluorescence microscopy with the cationic fluorescent dye rhodamine 123 revealed that nicardipine, TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although all Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture. Involvement of calcium- and cAMP-dependent pathways.

Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.     

Regulation of the human organic cation transporter hOCT1

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO-hOCT1) and in human embryonic kidney cells (HEK293-hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)) as substrate. hOCT1 expressed in CHO-cells was inhibited by protein kinase A (PKA) activation (1 microM forskolin, -58 +/- 6%, n = 12), calmodulin inhibition (0.1 microM calmidazolium, -68 +/- 3%, n = 6; 10 microM ophiobolin A, -48 +/- 10%, n = 7), calmodulin-dependent kinase II inhibition (1 microM KN62, -78 +/- 4%, n = 12), and inhibition of p56(lck) tyrosine kinase (10 microM aminogenistein, -35 +/- 7%, n = 12). The apparent affinities for TEA(+) were lower in CHO-hOCT1 than in HEK293-hOCT1, while those for TPA(+) and quinine were almost identical; the rank order of EC(50) values (TPA(+) > quinine > TEA(+)) was independent of the expression system. EC(50) values for TEA(+) in CHO-hOCT1 or HEK293-hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin-dependent kinase II, and p56(lck) tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.     

Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 microM calmidazolium, 12 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and 17.5 microM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 microM, 125 microM, 300 microM and 500 microM, respectively. A weak analogue of W7 inhibited only 20% at 1000 microM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes. The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.     

Regulation of a voltage-sensitive release mechanism by Ca(2+)-calmodulin-dependent kinase in cardiac myocytes

A role for Ca(2+)-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca(2+)-induced Ca(2+) release (CICR). VSRM contractions and Ca(2+) transients were absent in cells dialyzed with standard pipette solution but present when 2-5 microM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca(2+) current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 microM of tetracaine, but not 100 microM of Cd(2+), whereas CICR was inhibited by Cd(2+) but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by approximately 50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells.     

The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification

The (Ca2+ + Mg2+) ATPase of dog heart sarcolemma (Caroni, P., and Carafoli, E. (1980) Nature 283, 765-767) has been characterized. The enzyme possesses an apparent Km (Ca2+) of 0.3 +/- 02 microM, a Vmax of Ca2+ transport of 31 nmol of Ca2+/mg of protein/min, and an apparent Km (ATP) of 30 microM. It is only slightly influenced by monovalent cations and is highly sensitive to orthovanadate (Ki = 0.5 +/- 0.1 microM). The high vanadate sensitivity has been used to distinguish the sarcolemmal and the contaminating sarcoplasmic reticulum Ca2+-dependent ATPase in heart microsomal fractions. Calmodulin has been shown to be present in heart sarcolemma. Its depletion results in the transition of the Ca2+-pumping ATPase to a low Ca2+ affinity; readdition of calmodulin reverses this effect. The Na+/Ca2+ exchange system was not affected by calmodulin. The results of calmodulin extraction can be duplicated by using the calmodulin antagonist trifluoperazine. The calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column. One major (Mr = 150,000) and 3 minor protein bands could be eluted from the column with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The major protein band (72%) has Ca2+-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+-dependent reaction.     

Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni(2+)-stimulated calmodulin-independent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-stimulated phosphatase activity also recovered from the inhibition by calmidazolium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.     

Protein kinase G activation of K(ATP) channels in human-cultured prostatic stromal cells

In this study, we identify and investigate the role of protein kinase G (PKG) in cells cultured from human prostatic stroma. Cells were used for immunocytochemistry, contractility or K(+) fluorescent imaging studies. All cultured prostatic stromal cells showed PKG immunostaining. Phorbol 12,13 diacetate (PDA, 1 microM) elicited contractions from human-cultured prostatic stromal cells that could be blocked by both the L-type Ca(2+) channel blocker, nifedipine (3 microM), and the protein kinase C inhibitor, bisindolylmaleimide (1 microM). The nitric oxide donor, sodium nitroprusside (SNP, molar pIC(50) 5.16+/-0.17) and the cGMP-phosphodiesterase inhibitor, zaprinast (50 microM), inhibited PDA (1 microM)-induced contractions. The PKG activator beta-phenyl-1, N(2)-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP, molar pIC(50) 6.96 +/- 0.25) also inhibited PDA (1 microM)-induced contractions. Glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not iberiotoxin (100 nM) or Rp-cAMP (5 microM), reversed this inhibition. In human-cultured prostatic stromal cells loaded with the K(+) fluorescent indicator, 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI), PET-cGMP (300 nM) caused a reduction in intracellular K(+) that was blocked by glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not by iberiotoxin (100 nM). These data are consistent with the hypothesis that, in human-cultured prostatic stromal cells, PKG inhibits contractility through the activation of K(ATP) channels.     

Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine

Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, Mr = 60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50 mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 microM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+-dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 microM and 2.5 microM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.     

Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.     

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.