Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.     

Identification of the inhibition type of hexokinase and pyruvate kinase activity by Ca2+]

Ca2+ inhibitory effect on hexokinase (HK) and pyruvatkinase activities is studied. Kinetic analysis of the inhibition reaction is carried out to determine the inhibition type. Non-competitive inhibition with respect to reaction activator, Mg2+, is found for HK and PK. On the basis of graph analysis data for both reactions the values of the activatory constant (KA) and the inhibitory constant (Ki) are calculated. The experimental results are discussed with respect to possible regulatory effect of Ca2+ on glycolysis cycle.     

Stimulation of pyruvate kinase phosphatase activity by insulin in isolated rat hepatocytes

Addition of insulin (10(-8)M) to hepatocytes, incubated either in the absence or in the presence of a suboptimal concentration of glucagon, caused the reactivation of pyruvate kinase and simultaneously provoked a transient stimulation of pyruvate kinase phosphatase activity (40-70% over control values). The stimulatory effect of insulin on pyruvate kinase phosphatase activity was dose-dependent (ED50 = 1 to 2 X 10(-11)M) and persisted after Sephadex G-25 filtration or ammonium sulfate precipitation of hepatocytes extracts. Our results demonstrate that insulin exerts a short-term regulation on hepatic pyruvate kinase phosphatase activity.     

Insulin reverses effects of starvation on the activity of pyruvate dehydrogenase kinase in cultured hepatocytes.

In tissue culture of hepatocytes, insulin (0.1-1 munits/ml for 4 h) reversed completely the effects of starvation of rats to decrease the activity of pyruvate dehydrogenase (PDH) complex and to increase the activities of PDH kinase and PDH kinase activator protein. It had no effect in hepatocytes from fed rats. Significant effects of insulin were detected with 0.01 munit/ml after 4 h, and in 1-2 h with 1 munit/ml.     

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.     

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna^H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna^H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Role of pyruvate kinase in non-specific changes of carbon photosynthetic metabolism, caused by the photophosphorylation inhibition]

The decrease in the level of NADP reduction in chloroplasts under injections of cofactors of pseudocyclic FMN photophosphorylation (vitamin K3 and methylviologen) into pea, tomato and cucumber leaves resulted in the decrease of 14CO2 autotrophic accumulation rate and in the change of distribution of assimilated carbon among the assimilation products. The inhibition of synthesis of labelled sugars and the increase of the content of 3-phosphoglyceric and glyceric acids in the labelled low molecular fraction were observed in all the experiments. Similar changes in the distribution of synthesized in Kalvin cycle labelled products, associated with the inhibition of its reduction unit, were observed under the effect of photophosphorylation uncoupling agents. However, the decrease of ATP/NADP ratio in chloroplasts resulted in the simultaneous increase of fixed 14CO2 incorporation into alanine. The role of pyruvate kinase in "alanine" effect, characteristic of non-specific changes of carbon photosynthetic metabolism, is discussed on the basis of the authors' previous data on the effect of phosphorylation on glycolysis reactions and on the basis of results of ADP introduction into leaf cuts.     

The role of phosphorylation in the α-adrenergic-mediated inhibition of rat hepatic pyruvate kinase

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40–50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2–3-fold increase in the phosphate content of the enzyme. These changes were blocked by the α-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the α-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by α-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.     

Apparent inhibition of pyruvate kinase by phosphocreatine and phosphoarginine.

1. Addition of a non-dialysable, heat-labile and acid-precipitable factor which was not absorbed on DEAE-cellulose column, could restore the sensitivity of the chromatographed muscle pyruvate kinase from Marphysa sanguinea towards phosphocreatine inhibition. 2. This factor, being non-specific as it acts on pyruvate kinase isozymes from different sources, demonstrated high creatine kinase activity. 3. High concentrations of ADP, creatine or replacement of ADP with IDP/UDP or high pH abolished the inhibition indicating that the inhibition was mediated through creatine kinase by depleting ADP. 4. Apparent inhibition of phosphocreatine was related to the relative activities of 3 intracellular enzymes--pyruvate kinase, creatine kinase and adenosine triphosphatase.     

Lasalocid immediately and completely prevents the myocardial damage caused by coronary ischemia reperfusion in rat heart

Lasalocid, a specific mobile membrane ionophore for calcium, dopamine and norepinephrine was assayed in its capacity to reduce or maintain unaltered the cardiovascular function in conditions of imminent myocardial injury. In experiments of coronary blockade and reperfusion carried out in rat heart, it was found that when administered from 5 to 30 minutes prior to the induction of coronary blockade, at a concentration of 2 mg/kg of body weight, the ionophore immediately, simultaneously, and completely interrupts the blood pressure decay, cardiac frequency increase, electrical ventricular tachycardia and fibrillation, as well as the fall of mitochondrial oxidative phosphorylation and decay of mitochondrial oxygen uptake provoked by the induced myocardial injury. It appears that the molecular mode of action of the lasalocid is associated with its unique ability to transport both calcium and the catecholamines, dopamine and norepinephrine, across mitochondrial and bimolecular lipid membranes, as well as through synaptic cell membrane terminals from rat heart, myocardial fibers of the heart and heart chromaffin membrane vesicles. It is suggested that for the potential medical use of lasalocid to detain incoming ischemic myocardial damage, there exists a need to develop a personal electronic device able to simultaneously monitor, detect, and inform on the very early and simultaneous signs of cardiac alterations of electrical, mechano-chemical, metabolic and hydraulic nature, all which precede heart failure and to administer the lasalocid.

     

L-Phenylalanine inhibition of muscle pyruvate kinase

The allosteric inhibition of M_l-type pyruvate kinase from rabbit skeletal muscle by phenylalanine is reciprocally dependent on Mg²⁺ and phosphoenolpyruvate concentrations . At pH 8, phenylalanine acts as a competitive inhibitor with respect to Mg²⁺ and phosphoenolpyruvate, and vice versa. Phenylalanine introduces sigmoidicity into the dependence of the reaction velocity on [Mg²⁺]. In vitro kinetic analysis indicates that phenylalanine inhibition of muscle pyruvate kinase is unlikely to have regulatory significance in vivo.     

Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.     

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.