Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Thyrotropin-releasing hormone and phorbol esters stimulate sphingomyelin synthesis in GH3 pituitary cells. Evidence for involvement of protein kinase C

Previous studies demonstrated that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 14525-14530). Since phosphatidylcholine may serve as the precursor for sphingomyelin synthesis, studies were performed to assess the effect of protein kinase C on sphingomyelin synthesis. The potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated time- and concentration-dependent incorporation of 32Pi into the head group of sphingomyelin in cells short term labeled with 32Pi and resuspended in medium without radiolabel. TPA (10(-7) M) increased incorporation at a rate 1.4-fold of control after 2 h; EC50 congruent to 2 x 10(-9) M TPA. This correlated closely to TPA-induced phosphatidylcholine synthesis; EC50 congruent to 9 x 10(-10) M TPA. TRH (10(-7) M), which activates protein kinase C via a receptor-mediated mechanism, similarly stimulated 32Pi incorporation into sphingomyelin at a rate 1.5-fold of control; EC50 congruent to 5 x 10(-10) M TRH. This correlated closely with TRH-induced phosphatidylcholine and phosphatidylinositol synthesis; EC50 congruent to 2 x 10(-10) and 1.5 x 10(-10) M TRH, respectively. In cells short term labeled with [3H]palmitate, TRH induced a time- and concentration-dependent reduction in the level of [3H]ceramide and a quantitative increase in the level of [3H]sphingomyelin. Compositional analysis of the incorporated [3H]palmitate revealed that TRH increased radiolabel into both the sphingoid base and the fatty acid moieties of sphingomyelin. Similarly, TRH increased incorporation of [3H] serine into sphingomyelin to 145 +/- 8% of control after 3 h. TPA also stimulated these events. Like the effect of TRH on phosphatidylcholine synthesis, TRH-induced sphingomyelin synthesis was abolished in cells "down-modulated" for protein kinase C. In contrast, TRH-induced phosphatidylinositol synthesis still occurred in these cells. These studies suggest that protein kinase C stimulates coordinate synthesis of phosphatidylcholine and sphingomyelin. This is the first report of stimulation of sphingomyelin synthesis via a cell surface receptor.     

Discrepancy in stoichiometry of steady-state muscle [Pi] and [PCr] induced by exercise

Phosphorus nuclear magnetic resonance was used to quantify the relations between metabolic phosphates, intracellular pH, and work rate in forearm muscle of six adult men over a range of work rates from 1.0 to 3.5 W. Three work rates were studied in each of four sessions (either 1.0, 2.0, and 3.0 or 1.5, 2.5, and 3.5 W), with measurements made before and during each bout, thereby permitting the partition of the variance attributable to rest, work-dependent, and time-dependent metabolic functions by regression analysis. There were no time-dependent changes in either [ATP] or intracellular [H+] as assessed during the rest intervals between bouts of exercise. In contrast, the total nuclear magnetic resonance (NMR)-visible phosphorus pool (TVPP) decreased with time, with both phosphocreatine (PCr) and inorganic phosphate (Pi) contributing significantly to TVPP reduction. Muscle [ATP] was unchanged by work at all intensities. Intracellular [H+] increased moderately and proportionately to work rate. [PCr] decreased and [Pi] increased in proportion to work rate, with the work-dependent coefficient for PCr consumption approximately 1.5 times that of Pi production. Neither Pi line width nor motion artifact accounted for the decrease in TVPP, so the reduced Pi accumulation in exercise may represent its sequestering in some NMR-invisible muscle pool and/or loss to the blood. Whatever the process involved, it is proportional to work rate and persists for at least 10-15 min after exercise.     

The Gibbs-Donnan near-equilibrium system of heart

The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.     

Estrogen-induced changes in high-energy phosphate metabolism in rat uterus: 31P NMR studies

Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found to be the following: creatine phosphate (CP), 2.1 +/- 0.2; nucleoside triphosphates, mainly adenosine 5'-triphosphate (ATP), 4.6 +/- 0.4; phospho monoesters, primarily sugar phosphates (SP), 5.4 +/- 0.7; and inorganic phosphate (Pi), 0.8 +/- 0.4. Adenosine 5'-diphosphate (ADP) concentration was estimated to be approximately 40 mumol/kg wet weight from the assumed equilibrium of the creatine kinase reaction. The concentration of CP, and to lesser extent ATP and SP, declined within the first 1.5-3 h after injection of 17 beta-estradiol, returned to control values between 6 and 12 h, and then increased, reaching maximal concentrations at 24 h. From the fractions of the total soluble ATP in free and Mg2+-bound forms, [free Mg2+] in the untreated uterus was estimated to be 0.2-0.4 mmol/kg wet weight. An increase in [free Mg2+] in the uterus was detected 1.5 h after estrogen injection. A subsequent parallel increase in the ratio of ATP to CP concentrations suggests that estrogen can also affect the apparent creatine kinase equilibrium by modulating [free Mg2+].     

Equilibrium constants under physiological conditions for the reactions of choline kinase and the hydrolysis of phosphorylcholine to choline and inorganic phosphate.

The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.     

The distribution of inorganic phosphate and malate between intra- and extramitochondrial spaces. Relationship with the transmembrane pH difference

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.     

Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR.

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.     

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.     

Does the inositol tris/tetrakisphosphate pathway exist in rat heart?

Appearance of two isomers of inositol trisphosphate (InsP3) was observed when [3H]inositol prelabelled rat heart ventricles were stimulated for 10 and 30 s with noradrenaline. In contrast, inositol tetrakisphosphate (InsP4) could not be detected. However the existence of the inositol tris/tetrakisphosphate pathway was demonstrated by studying [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) metabolism in a soluble fraction of rat heart. There, [3H]Ins-1,4,5-P3 was phosphorylated to form [3H]Ins-1,3,4,5-P4. Raising [Ca2+] from 1 nM to 1 microM increased InsP3 kinase activity by 2-fold (EC50 for Ca2+ approx. 56 nM). This effect appeared to be due to an increase of the apparent Vmax of the enzyme while the apparent Km was unchanged.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.     

The roles of ion fluxes in skeletal muscle fatigue

Intense muscle contractions result in large changes in the intracellular concentrations of electrolytes. The purpose of this study was to examine the contributions of changes in intracellular strong ions to calculated changes in steady-state membrane potential (Em) and muscle intracellular H+ concentration ([H+]i). A physicochemical model is used to examine the origin of the changes in [H+]i during intense muscle contraction. The study used the isolated perfused rat hindlimb intermittently stimulated to contract at high intensity for 5 min. This resulted in significant K+ depletion of both slow (soleus) and fast (white gastrocnemius, WG) muscle fibers and a release of K+ and lactate (Lac-) into venous perfusate. The major contributor to a 12- to 14-mV depolarization of Em in soleus and WG was the decrease in intracellular K+ concentration ([K+]i). The major independent contributors to [H+]i are changes in the concentrations of strong and weak ions and in CO2. Significant decreases in the strong ion difference [( SID]i) in both soleus and WG contributed substantially to the increase in [H+]i during stimulation. In WG the model showed that the decrease in [SID]i accounted for 35% of the increase in [H+]i (133-312 nequiv/L; pHi = 6.88-6.51) at the end of stimulation. Of the main contributors to decreased [SID]i, increased [Lac-]i and decreased [K+]i contributed 40 and 60%, respectively, to increased [H+]i, whereas a decrease in [PCr2-]i contributed to reduced [H+]i. It is concluded that decreased muscle [K+]i during intense contractions is the single most important contributor to reduced Em and increased [H+]i. Depletion of PCr2- simultaneous to the changes in [Lac-]i and [K+]i prevents larger increases in [H+]i and helps maintain the intracellular acid-base state.     

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

Erythroid differentiation factor stimulates hydrolysis of polyphosphoinositide in Friend erythroleukemia cells

We examined an early action of erythroid differentiation factor (EDF), a polypeptide which induces differentiation of Friend murine erythroleukemia (MEL) cells. (Eto et al., Biochem. Biophys. Res. Commun. 142: 1095-1103, 1987). In MEL cells, EDF caused a rapid and transient increase in cytoplasmic concentration of free calcium, [Ca2+]c. EDF increased [Ca2+]c even in the absence of extracellular calcium. When [3H]inositol-labeled MEL cells were incubated with EDF, EDF rapidly increased radioactivity in inositol trisphosphate, bisphosphate and monophosphate. EDF also increased [3H] diacylglycerol in [3H]arachidonate-labeled MEL cells. These results indicate that EDF increases [Ca2+]c by stimulating hydrolysis of polyphosphoinositide.     

How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.