Changes In Intracellular Concentration Research Articles

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes.

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilized oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilized eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilization. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilized oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilization but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.

Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions.

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin. Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+,K(+)-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K(+)-ATPase, which acts upstream of the bcl-2 protein.

Autologous Fat Grafting: A Study of Residual Intracellular Adipocyte Lidocaine Concentrations after Serial Rinsing with Normal Saline

Introduction: With evolution of sophisticated lipocontouring techniques increased interest has developed in utilization of vacuum harvested autologous fat cells for grafting purposes. Transplantation of viable adipocytes for contour augmentation, enlargement, or filling of defects has clinically become more important. Based on evidence that lidocaine solution may alter the metabolic activity, evaluation of the effects of multiple rinsing was undertaken. Materials and Methods: Gas chromatography was utilized to measure changes in intracellular concentration of lidocaine from closed syringe harvested adipocytes following serial rinsing with equal volumes of normal saline in preparation for fat grafting procedures. Results: Use of standard tumescent local anesthesia as a medium for harvesting mature fat cells via closed syringe techniques resulted in substantial absorption of lidocaine. Statistically significant reduction of intracellular lidocaine was accomplished via serial rinsing with normal saline solution. It was concluded that a minumum of three rinses utilizing equal volume of saline to autologous fat achieved significant and substantial reduction of intracellular lidocaine levels, but did not result in complete removal. Discussion: The importance of thorough rinsing and removal of cellular debris from harvested fat cell grafts prior to transfer is widely recognized to improve efficacy and predictability. This study proves that serial rinsing of fat cells harvested with closed syringe system using a tumescent fluid technique does statistically reduce the retention of intracellular lidocaine. It is believed that high concentrations of retained intracellular lidocaine may inhibit the metabolic activity and impact the survivability of autologous fat grafts.

Regulation of a volume-sensitive anion channel in rat pancreatic beta-cells by intracellular adenine nucleotides.

1. The patch-clamp technique in the whole-cell configuration was used to measure the effects of intracellular adenine nucleotides on activity of the volume-sensitive anion channel in single, isolated rat pancreatic beta-cells. 2. In the absence of intracellular nucleotides, swelling of cells with a hypertonic pipette solution failed to activate the conductance. Addition of ATP over the range 2-10 mM maintaining the same degree of hypertonicity caused a progressive activation of the conductance. An increase in ATP produced a similar activation of the conductance in non-swollen cells, albeit with reduced current amplitudes. 3. Activation of the conductance was also observed in the presence of ATPgammaS, adenylyl imidophosphate (AMP-PNP), ADP, diadenosine tetraphosphate and GTPgammaS. Neither ADP nor GDPbetaS inhibited activation of the conductance by ATP. 4. It is concluded that activity of the beta-cell volume-sensitive anion channel can be modulated by changes in intracellular concentrations of ATP within the physiological concentration range by a mechanism that does not require nucleotide hydrolysis. Activity of the channel does not appear to be modulated by a G protein-coupled mechanism.

Cyclic nucleotide gated channels.

Cyclic nucleotide-gated (CNG) channels are ion channels which are activated by the binding of cGMP or cAMP. The channels are important cellular switches which transduce changes in intracellular concentrations of cyclic nucleotides into changes of the membrane potential and the Ca2+ concentration. CNG channels play a central role in the signal transduction pathways of vision and olfaction. Structurally, the channels belong to the superfamily of pore-loop cation channels. They share a common domain structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and Eag-like K+ channels. In this chapter, we give an overview on the molecular properties of CNG channels and describe the signal transduction pathways these channels are involved in. We will also summarize recent insights into the physiological and pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient mouse models and human channelopathies.

Ionic transport in macula densa cells

Recent work has provided substantial insights into functional characteristics of macula densa (MD) cells. Microelectrode and patch-clamp experiments on the rabbit isolated thick ascending limb (TAL)/glomerulus preparation have shown that MD cells possess a furosemide-sensitive Na:K:2Cl cotransporter, an apical 41-pS K+ channel, and a dominant basolateral Cl- conductance. Increasing luminal fluid [NaCl] ([NaCl]L) results in furosemide-sensitive cell depolarization due to a rise in intracellular [Cl-] that stimulates basolateral electrogenic Cl- efflux. Intracellular pH (pHi) measurements show the presence of an apical Na:H exchanger that couples transepithelial Na+ transport to pHi. Experimental results and thermodynamic considerations allow estimation of intracellular [Na+] and [Cl-] ([Na+]i, [Cl-]i) under different conditions. When the Na:K:2Cl cotransporter is equilibrated (or in the presence of furosemide), [Na+]i and [Cl-]i are low (approximately 6 to 7 mM), whereas when the cotransporter is fully activated, [Na+]i and [Cl-]i increase substantially to approximately 70 and 20 mM, respectively. Finally, luminal addition of NH4+ produces cell acidification that depends on NH4+ apical transport rate through the Na:K:2Cl. Using a simple transport model for NH4+, the initial NH4+ influx rate in MD cells is comparable to the corresponding flux in TAL. This challenges the idea that MD cells have a low transport activity but supports our findings about large changes in intracellular concentrations as a function of [NaCl]L.

An experimental study on the mechanism of anisodamini hydrobromidum in treating acute ischemic renal failure

In order to explore the mechanism of anisodamini hydrobromidum (654-2) in treating acute ischemic renal failure, the model of acute ischemic renal failure in white New Zealand rabbits was established to dynamically observe and statistically analyze the intracellular concentration changes of free calcium ([Ca2+]i) and inositol triphosphate (IP3). The results showed that the levels of [Ca2+]i and IP3 in acute renal failure group were higher than those in control group (P < 0.01). However, the levels of [Ca2+]i and IP3 in 654-2 treated group were significantly lower than those in acute renal failure group (P < 0.001). It was concluded that 654-2 could alleviate Ca(2+)-overload in renal histocytes in acute ischemic renal failure. The protective mechanism is associated with intracellular reduction of IP3.

The effects of sodium substitution on currents determining the resting potential in guinea‐pig ventricular cells

It has recently been shown that a sodium background current, ib,Na, exists in cardiac muscle cells whose effect is to depolarize the membrane so that the resting potential, Vm, is positive to the potassium equilibrium potential, EK. In ventricular cells, where ib,Na is smallest, Vm is about 10 mV positive to EK (EK = -87 mV at 37 degrees C). Yet, replacement of Na+ ions by large impermeant cations does not cause the expected hyperpolarization. We have studied this problem in guinea-pig myocytes using a single microelectrode recording technique in combination with a rapid external solution switch. Cells depolarized < or = 0.5 mV from potentials between -80 and -73 mV and hyperpolarized up to 5 mV from potentials between -73 and -64 mV when 70 mM choline chloride or N-methyl-D-glucamine chloride were used to replace 70 mM Na+ in the bathing solution. Replacement by 70 mM lithium chloride, however, only caused hyperpolarization in very depolarized cells when the voltage change was much smaller. The changes were complete almost as soon as the solution change, i.e. within 250 ms, indicating that the actions are attributable to the external solution change rather than to secondary changes in intracellular concentrations. Patch clamp recording was used to investigate the mechanism involved. These experiments showed that the presence or absence of the inward rectifier current iK1 determines in which direction Na+ removal acts. In the absence of iK1 the changes are attributable to removal of ib,Na, whereas in the presence of iK1 the changes resemble the i(V) relation for iK1, implying that Na+ regulates iK1 in a way that can mask the changes in ib,Na. These results explain why removal of Na+ does not lead to hyperpolarization in ventricular cells as would be expected if changes in ib,Na were solely responsible. Computer reconstruction shows that the effects may be attributed to actions of sodium removal on the conductance and gating of iK1.

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway.

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.

Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Use of secreted alkaline phosphatase as a reporter of gene expression in mammalian cells.

The cDNA encoding secreted alkaline phosphatase (SEAP; ) is a powerful tool for investigating the function of known or presumed enhancer/promoter elements in transfected cells. As a reporter of gene expression, SEAP differs from intracellular reporters such as chloramphenicol acetyltransferase (CAT) and firefly luciferase in that SEAP is secreted from transfected cells. SEAP can thus be assayed using a small portion of the culture medium. The SEAP gene encodes a truncated form of human placental alkaline phosphatase (PLAP), an enzyme which normally resides on the cell surface. The SEAP gene lacks sequences encoding the membrane anchoring domain, thereby allowing the expressed protein to be efficiently secreted from cells. Levels of SEAP activity detected in the culture media are directly proportional to changes in intracellular concentrations of SEAP mRNA and protein (). SEAP has the unusual properties of being extremely heat-stable and resistant to the phosphatase inhibitor L-homoarginine. Therefore, endogenous alkaline phosphatase activity can be eliminated by treatment of samples at 65°C and incubation with this inhibitor. The secreted nature of SEAP provides several advantages for the use of this enzyme as a genetic reporter: 1. Preparation of cell lysates is not required. 2. The kinetics of gene expression can be easily studied using the same cultures by repeated collection of the medium. 3. Transfected cells are not disturbed for measurement of SEAP activity, and remain intact for further investigations such as RNA and protein analysis. 4. Background from endogenous alkaline phosphatase activity in the culture media is almost absent. 5. Sample collection and assay can be automated using 96-well microtiter plates.

Oxidative Stress-Induced Increase in Intracellular Ca2+ and Ca2+-Induced Increase in Oxidative Stress: An Experimental Model Using Dissociated Rat Brain Neurons

In oraer to study the oxidative stress-induced change in intracellular concentration of Ca2+ ([Ca2+]i) and Ca2+-induced oxidative stress, effects of hydrogen peroxide and ionomycin, a calcium ionophore, on rat cerebellar neurons were examined using a flow cytometer and fluorescent dyes: fluo-3 for monitoring [Ca2+]i; 2',7'-dichlorofluorescin, for reactive oxygen species; and 5-chloromethylfluorescein, for cellular nonprotein thiols. Oxidative stress induced by hydrogen peroxide dose-dependently increased [Ca2+]i and decreased the content of nonprotein thiols. Ionomycin increased oxidative metabolism and decreased the content of nonprotein thiols. Results suggest that oxidative stress induces an increase in [Ca2+]i while an increase in [Ca2+]i increases oxidative stress in neurons.

Clofibric acid increases the undirectional chiral inversion of ibuprofen in rat liver preparations.

1. The formation of (S)-ibuprofen from (R)-ibuprofen was monitored in perfused rat livers and in rat hepatocytes and the rate constants calculated. 2. Pre-treatment of animals for three days with clofibric acid markedly increased the (R)-to-(S) inversion of ibuprofen in both preparations. In contrast, clofibric acid did not elicit such a reaction with flurbiprofen, an analogue that does not undergo inversion under control conditions. 3. An increase in the chiral inversion was also seen when clofibric acid was added to the perfusion medium or to hepatocyte suspensions. In the latter system this increase was shown to be concentration dependent. 4. Pre-treatment of rat combined with addition of clofibric acid to the perfusion medium produced a cumulative stimulation of (R)-to-(S) inversion of ibuprofen. 5. Clofibric acid added to hepatocyte suspensions transiently increased intracellular concentrations of coenzyme A whereas (R)-ibuprofen transiently decreased CoA concentrations. The two effects cancelled each other upon co-incubation of the two compounds. 6. It is postulated that the metabolic interaction observed between clofibric acid and (R)-ibuprofen may be due to changes in intracellular concentrations of CoA.

Changes in intracellular concentration of amino acids in human saphenous vein during preparation for coronary artery bypass grafting

Earlier research on human skeletal and cardiac muscle has shown a fall in the intracellular free amino acid pool, particularly the non-essential amino acids, during surgery. Surgical preparation of the human saphenous vein for coronary artery by pass grafting has been shown to provoke metabolic damage and may therefore induce changes in cellular amino acids which may have long-term implications for the performance of the grafted vein. This work investigates the hypothesis that surgical preparation of saphenous vein induces changes in amino acids. Changes in the intracellular free amino acid pool were monitored, using high-performance liquid chromatography (HPLC), in both freshly isolated and surgically prepared human saphenous veins during coronary artery bypass grafting. In order to asses the extent of metabolic damage incurred by surgical preparation, adenosine triphosphate (ATP) levels were also measured. A substantial fall in intracellular free amino acid was associated with surgical preparation of human saphenous vein for coronary artery bypass grafting (190.8 +/- 25 to 106 +/- 18 mumol.g-1 protein, n = 10, P < 0.05). Although the fall was seen in most amino acids detected, it was marked and statistically significant (P < 0.05) for taurine (30.6 +/- 3.1 to 9.0 +/- 2.2 mumol.g-1 protein), glutamate (43.6 +/- 5.6 to 18.2 +/- 4.5 mumol.g-1 protein), glutamine (12.8 +/- 1.6 to 3.3 +/- 1.2 mumol.g-1 protein) and asparagine (9.6 +/- 1.6 to 4.1 +/- 1.0 mumol.g-1 protein). The fall in tissue taurine which would be largely due to transport showed a strong positive correlation with the fall seen in glutamate, glutamine and asparagine. ATP levels significantly decreased as a result of surgical preparation (279 +/- 44 to 96 +/- 21 nmol.g-1 wet weight, P < 0.05). The fall in ATP was accompanied by an increase in alanine/glutamate ratio which may reflect increased glutamate-alanine transaminase activity and therefore metabolic changes. The metabolic damage associated with surgical preparation of human saphenous vein for coronary artery bypass grafting is accompanied by a fall in the intracellular free amino acid pool which is marked and statistically significant for glutamate, taurine, glutamine and asparagine. The fall is likely to be due to transport metabolism may also contribute. This may have important implications for the functional recovery of the grated vein.

Changes in intracellular concentration of amino acids in human saphenous vein during preparation for coronary artery bypass grafting

Changes in intracellular concentration of amino acids in human saphenous vein during preparation for coronary artery bypass grafting

High-performance liquid chromatography of the fluorescent dyes Fura-2 and Mag-Fura stability in organic solvents

The fluorescent dye indicators Fura-2/AM and Mag-fura are used to estimate changes in intracellular concentrations of calcium and magnesium, respectively. HPLC, coupled to fluorescence and ultraviolet detectors, indicated that these dyes are unstable when dissolved in methanol and are especially unstable in glass containers compared to plastic. Both dyes were very stable in acetonitrile and dimethyl sulfoxide. The glass vials used in these studies appear to contain an unidentified substance which causes changes in the spectral intensities of these fluorescent dyes in the presence of methanol but not other solvents. With Fura-2/AM, there was an initial two fold increase in fluorescence during the first four hours in methanol and glass with no change in retention time, peak shape or ultraviolet absorption. With Mag-fura, no increase in fluorescence was observed but instead there was a reduction of more than 90% in both fluorescence and ultraviolet absorption within 30 min, indicating that significant decomposition occurs under these conditions. These results confirm previous studies which suggest that acetonitrile is preferable to methanol for sample preparation and chromatographic analysis of Fura-2 and Mag-Fura. These data also indicate that glass vials contain an extractable substance which markedly enhances the spectral intensities of Fura-2/AM.

High-Resolution Proton NMR Studies of Lymphocyte Extracts

Anatomic imaging is now a well-developed application of magnetic resonance. Greater capabilities for physiologic characterization should become possible by concomitant application of spectroscopic methods. High-resolution in vitro spectroscopy must first provide a framework upon which in vivo and diagnostic interpretation may be based. Biochemical profiles consisting of quantitation of extracted aqueous metabolites and lipids of particular cells or organs establish an in vitro glossary for what may be found in the intact cell or living subject. A large variety of amino acids, intermediary metabolites, membrane precursors, and nucleotides are detectable in extracts of human peripheral blood lymphocytes, and significant changes in intracellular concentrations have been monitored after lectin-induced activation. Corresponding changes in lipid profile have also been noted. An increasing variety of other cells and tissues are being similarly characterized. Despite its limitations, NMR analysis possesses the unique prospect of providing a noninvasive and nondestructive source of biochemical information.

Effects of lovastatin on hepatic fatty acid metabolism.

The in vitro and in vivo effects of lovastatin on fatty acid metabolism were studied in isolated rat hepatocytes. When added in vitro to cell incubations, lovastatin stimulated de novo fatty acid synthesis and acetyl-CoA carboxylase activity, whereas fatty acid synthase activity was unaffected. Lovastatin depressed palmitate, but not octanoate, oxidation. This may be attributed to the lovastatin-induced increase in intracellular malonyl-CoA levels, as no concomitant change of carnitine palmitoyltransferase I (CPT-I) specific activity was detected. Lovastatin had no effect on the synthesis and secretion of triacylglycerols and phospholipids in the form of very low density lipoproteins (VLDL). When rats were fed a diet supplemented with 0.1% (w/w) lovastatin for one week, both acetyl-CoA carboxylase activity and de novo fatty acid synthesis were reduced compared to pair-fed controls, whereas fatty acid synthase activity was unaffected. Palmitate oxidation was enhanced in the lovastatin-fed group. There was an increase in CPT-I activity but no change in intracellular concentration of malonyl-CoA. Lovastatin feeding had no significant effect either on the esterification of exogenous palmitic acid into both cellular and VLDL triacylglycerols and phospholipids or on hepatic lipid accumulation. The in vitro and in vivo effects of lovastatin were not significantly different between periportal and perivenous hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intracellular amino acid concentrations, cyclic AMP, and dexamethasone on lysosomal proteolysis in primary cultures of perinatal rat hepatocytes.

We have studied factors regulating the rate of protein degradation in cultured hepatocytes obtained from 17-day-old fetal, 7-day-old suckling, and 20-day-old weanling rats. At all three stages of development 60-70% of protein degradation was sensitive to inhibition by amino acids and 3-methyladenine, an inhibitor of macroautophagy, indicating a major role of the lysosomes in proteolysis under these conditions. A combination of dibutyryl cyclic AMP and dexamethasone strongly stimulated proteolysis in hepatocytes from weanling, but not from fetal and suckling rats. The stimulatory effect of these compounds was eliminated at high amino acid concentrations in the culture medium. Cultured perinatal hepatocytes responded to exposure to dibutyryl cyclic AMP and dexamethasone by de novo synthesis of mRNA for carbamoyl-phosphate synthase and for phosphoenolpyruvate carboxykinase, demonstrating that the developmental change in the effect of dibutyryl cyclic AMP and dexamethasone on proteolysis was due to a developmental change in the regulation of proteolysis. An analysis of the changes in intracellular amino acid concentrations in response to variations in the extracellular amino acid concentrations at all three stages of development showed that of all amino acids that could be identified, only Ile, Leu, Lys, Phe, and Tyr are implicated as possible regulators of hepatic proteolysis. Dibutyryl cyclic AMP and dexamethasone did not affect the intracellular concentrations of these amino acids, showing that hormonal regulation of proteolysis is not mediated by changes in intracellular concentrations of these amino acids. It is concluded that the lack of sensitivity of the proteolytic system to catabolic hormones in the period around birth, combined with higher circulating plasma amino acid concentrations, are mechanisms contributing to the low rate of intrahepatic proteolysis in vivo in the perinatal period and thus to the rapid growth of the liver in this period.

Cerebral taurine transport is increased during streptozocin-induced diabetes in rats.

Taurine is a cerebral osmolyte whose intracellular content changes in parallel with plasma osmolality. We conducted experiments to assess whether cerebral taurine transport is modified during chronic hyperglycemia. Rats with STZ-induced diabetes were studied after 1 wk of sustained hyperglycemia. Cerebral taurine uptake in synaptosomes (metabolically active nerve terminal vesicles) was measured using a rapid filtration technique. The synaptosomes were isolated by homogenization of the brain and purification on discontinuous Ficoll gradients (n = 8 synaptosome preparations). Diabetic rats (n = 13) displayed a 15-25% increase in synaptosomal taurine uptake compared with normoglycemic control animals (n = 12) at all time points assayed between 5 and 120 min. Thus, after a 30-min incubation, cerebral taurine uptake increased from a control level of 3.53 +/- 0.23 to 4.10 +/- 0.24 mumol/mg protein (n = 10) in hyperglycemic rats, P less than 0.03. The magnitude of the plasma-to-brain cell taurine gradient was unchanged in diabetic animals. The intrasynaptosomal taurine concentration (approximately 2 microM) and taurine efflux from the synaptosomes were no different in hyperglycemic versus control rats; efflux amounted to less than 2.5% of the uptake value at corresponding time points. Maximal brain taurine uptake under both control and experimental hyperglycemic conditions required the presence of external Na+ and Cl-. Synaptosomal taurine transport was reduced by competing beta-amino acids such as beta-alanine, beta-aminoisobutyric acid, and hypotaurine (P less than 0.01). Addition of oubain and the anionic binding site inhibitors, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'diisothio-cyanatostilbene-2,2'-disulfonic acid, also decreased cerebral taurine uptake under normoglycemic and hyperglycemic conditions (P less than 0.01). The increased synaptosomal taurine uptake by diabetic rats was not a result of generalized membrane dysfunction because glycine transport was not elevated in hyperglycemic rats. The enhanced transport rate was attributable to a 35 and 81% increase in the Vmax of the high- and low-affinity taurine transporters, respectively (P less than 0.01), without significant change in the Km of the carrier systems. Treatment of hyperglycemic rats (n = 5) with ultra-long-acting insulin to normalize the serum glucose concentration restored synaptosomal taurine uptake to the level observed in normoglycemic controls. The effect of insulin was attributable to correction of hyperglycemia, because addition of insulin (500 mU/ml) to the in vitro assay system did not alter synaptosomal taurine uptake.(ABSTRACT TRUNCATED AT 400 WORDS)