Effect of L-propranolol on the binding,internalization and degradation of 125I-low density lipoproteins by human skin fibroblasts

The effect of L-propranolol on the receptor mediated uptake of human LDL by fibroblasts was investigated. When L-propranolol was tested between 10^?6 and 10^?4 M at a ¹²⁵I LDL concentration ranging from 6.25 to 198 μg/ml, an increase in binding, internalization and degradation of the labelled lipoproteins was demonstrated with a maximal effect respectively of 52%, 680% and 105% observed with L-propranolol at 10^?4 M and a lipoprotein concentration of 37.5 μg/ml. The effect occurs rapidly and is seen 2 hrs after the addition of the drug. Lower drug concentrations, although less effective, still produced significant increase in lipoprotein uptake and degradation. Procaine, a local anesthetic, used at 10^?4 M has no effect on ¹²⁵I-LDL binding and internalization. D-propranolol, another local anesthetic and L-propranolol enantiomer, is also without effect. This, excluding a drug induced stabilization of the membrane as the responsible mechanism of action, indicates that stereospecificity is important.     

Effects of AY 9944 on low density lipoprotein metabolism in cultured human fibroblasts

Treatment of cultured human fibroblasts with the hypocholesterolemic drug AY 9944 resulted in a marked increase in low density lipoprotein internalization and degradation for concentrations up to 5 X 10(-6)M. Low density lipoprotein binding was less affected. Concentrations above 5 X 10(-6)M resulted in a relative decrease in low density lipoprotein degradation, whereas binding and internalization plateaued. The stimulation of low density lipoprotein internalization took place within the first hours of incubation of cells with the drug, which suggests a direct effect on the cell membrane. Such phenomenon could account at least partially for the hypocholesterolemic effect of the drug, besides its inhibitory effect on 7-dehydrocholesterol reductase.     

Phorbol esters inhibit low density lipoprotein processing by cultured human fibroblasts

A 24 h pretreatment of MRC5 fibroblasts with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a marked decrease in low density lipoprotein (LDL) internalization and degradation; the maximal effect (about 55% decrease) was observed for 10(-7) M TPA. LDL binding was reduced about 35-40%. A significant decrease (about 25%) in LDL internalization was observed after a 2 h incubation of cells with the drug, but longer incubation times (4-6 h) led to a greater effect. Another tumor promoter, phorbol 12,13-dibutyrate decreased LDL internalization by about 35%, whereas the non-tumor promoting 4 alpha-phorbol 12,13-didecanoate had no effect. The protein kinase C inhibitor alpha-cobrotoxin partially antagonized the inhibitory effect of TPA on LDL internalization. The non-phorbol tumor promoter mezerein, another protein kinase C activator, decreased LDL uptake by about 50%. Finally, it was found that TPA had no significant effect on the affinity of the receptor for the LDL. These results suggest a role for protein kinase C in the LDL pathway in cultured human fibroblasts.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Inhibitio of Na transport by prostacyclin (PGI2) in rabbit cortical collecting tubule

The effects of prostacyclin (PGI₂) on transepithelial potential difference (PD) and sodium transport were examined in rabbit cortical collecting tubules (CCT) perfused in vitro. Addition of PGI₂ (10^?6M) to the bathing medium, which was bubbled with 95% O₂ – 5% CO₂, caused a reversible decrease in PD averaging 49±9.4 (SE)%. Maximal effect was evident between 5–10 min. After addition of PGI₂ and PD returned spontaneously towards control values within 30 min., corresponding to the rapid degradation of PGI₂. In a more alkaline bathing solution achieved by bubbling with 100% O₂, in which the degradation of PGI₂ is known to be delayed markedly, the decrease in PD by PGI₂ was continuous and dose-dependent, with half-maximal and maximal effects achieved at 10^?7 M and 10^?5 M, respectively. Neither 10^?8 M PGI₂ nor vehicle alone exerted significant effects on PD. 6-keto-PGF_1α (10^?5M), believed to be the major metabolite of PGI₂, had no effect on PD. Lumen-to-bath flux of Na decreased with PGI₂ from 9.0 to 5.6 pEq/cm/sec (n=4, p<0.005), although bath-to-lumen flux did not change significantly. In summary, PGI₂ caused a dose-dependent decrease in PD of rabbit CCT and inhibited Na absorption in this segment in vitro. These results suggest that PGI₂ may play an important role in regulating Na transport in CCT.     

Effects of colchicine on insulin binding to isolated rat hepatocytes

The effects of colchicine, an inhibitor of microtubule polymerization, on the maintenance of steady state binding of insulin to isolated hepatocytes was studied. Colchicine (10^?5M) produced a 35–45% decrease in binding in presence of insulin (10^?8M) at 37°C. This decrease in binding was time and temperature dependent. The decrease was also dependent on the amount of insulin bound to the cell. The results suggest that colchicine may prevent the maintenance of steady state binding of insulin by impairing transfer of newly synthesized or recycled receptor from within the cell to the plasma membrane.     

Rapid degradation of [3H]leucine-enkephalin by intact neuroblastoma cells.

Enkephalin, a brain peptide with morphine-like activity, is rapidly degraded by N4TG1 neuroblastoma cells which contain opiate receptors. The enzymic activity is temperature dependent and it is maximal at 37°C with an apparent Km of 5 × 10^?5M. The enzyme can be inhibited by bacitracin and puromycin with apparent Ki values of 3.2 × 10^?5M and 2.3 × 10^?7M, respectively. Digesting intact cells with trypsin greatly diminishes the ability of the cells to degrade enkephalin and suggests that the degradative enzyme is probably localized at the cell surface. Opiates such as morphine and naloxone as well as leu- and met-enkephalin and D-Ala²-substituted analogs at very high concentrations (10^?5M) have no effect upon enzymic activity despite the fact that they totally block binding of the labeled enkephalin to receptors. The data strongly suggest that there is no correlation between receptor occupancy and rate of enkephalin degradation.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Endogenous regulators of benzodiazepine recognition sites

The method used to prepare crude synaptic membranes (CSMs) from rat brain affects the results obtained for the binding characteristics of ³H-diazepam and the GABA-induced stimulation of ³H-diazepam to CSM. In freshly prepared membranes (rich in GABA and other endogenous inhibitory factors), the K_D for ³H-diazepam is approximately 10 nM and the threshold dose of GABA needed to stimulate this binding is approximately 10^?5M. Removal of GABA resulted in an increase in the K_D values for ³H-diazepam binding. In contrast removal of endogenous inhibitory factors (by treatment of the membranes with Triton X-100) resulted in a decrease of the K_D values. In the Tritron X-100 treated membranes the threshold dose of GABA (10^?8M) required to stimulate ³H-diazepam binding is in the range of the high affinity component of ³H-GABA binding. Addition of crude preparations of endogenous inhibitor to these membranes increased the K_D of ³H-diazepam and inhibited the GABA-induced stimulation of ³H-diazepam binding.     

Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells

[³H]Epinephrine binding to isolated purified rat liver plasma membrane is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125μg. Rat liver plasma membranes stored at ?70 °C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes.Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A₂ (phosphatide acylhydrolase EC 3. 1. 1. 4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phopholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively.In the presence of 10^?3M Mg²⁺ ions, increasing concentrations of GTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10^?5M GTP, representing an inhibition of 52% of the control. In a Mg²⁺-free system, epinephrine binding was unaffected by GTP. However, in a Mg²⁺-free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10^-3 M reduced epinephrine binding to 28% of the control. GTP (10^?5M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane.[³H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40–200 μg protein).GTP (10^?5M) and ATP (10^?4M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10^?2?10^?3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [³H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [³H]epinephrine binding.Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [³H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

BINDING OF [99mTc]CHONDROITIN SULFATE TO SCAVENGER RECEPTORS ON HUMAN CHONDROCYTES AS COMPARED TO BINDING OF OXIDIZED [125I]LDL ON HUMAN MACROPHAGES

ABSTRACT

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with ^99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37°C for 2?h revealed the presence of high-affinity binding sites for CS with a K_d of 2.3 × 10^?9?mol/L and a B_max of 5.0 × 10⁸. Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 ± 19.5% and of the number of CS binding sites/cell by 62.0 ± 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [¹²⁵I]-LDL binding we found a K_d of 0.45 × 10^?8?mol/L and a B_max of 0.14 × 10⁶ on quiescent monocytes and for [¹²⁵I]-(ox)LDL binding a K_d of 1.8 × 10^?8?mol/L and a B_max of 1.3 × 10⁶ using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein–proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

Reciprocal changes in fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase activity in response to glucagon and epinephrine

The effect of hormones on the enzymes responsible for the synthesis (fructose-6-P,2-kinase) and degradation (fructose-2,6-Pase) of fructose-2,6-P₂ was examined in isolated rat hepatocytes. Glucagon (10^?11 M), epinephrine (10^?5 M), or calcium (2.4 mM) and A23187 (10^?5 M) administration to hepatocytes produced simultaneous activation of fructose-2,6-Pase and inactivation of fructose-6-P,2-kinase within 2 minutes. The effect of epinephrine on these two enzymes was dependent on the presence of Ca⁺⁺. These results suggest that the level of fructose-2,6-P₂ is controlled by recriprocal changes in fructose-2,6-Pase and fructose-6-P,2-kinase activities.     

Regulation of fructose 2,6-P2 concentration in isolated hepatocytes

The effect of hormones on fructose-2,6-P₂ level and fructose-6-P,2-kinase activity was examined using rat hepatocytes. The dose response curve shows the half-maximal effect of glucagon on fructose-2,6-P₂ occurs at 3 X 10^?13 M glucagon, whereas the half-maximal effect on cyclic AMP occurs at 3 × 10^?0 M. The decrease in fructose-2,6-P₂ parallels the decrease in fructose-6-P,2-kinase activity. Incubation of cells with dibutryl cyclic AMP and cyclic AMP results in a 2- to 3-fold decrease in fructose-2,6-P₂. Epinephrine (10^?5 M) mediates a 2-fold decrease in fructose-2,6-P₂; isoproterenol has no effect. These results suggest that regulation of fructose-6-P,2-kinase is complex, involving cyclic AMP-dependent and -independent mechanisms.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Determination of glucose,urea and penicillin using enzyme-pH-electrodes

Conventional hydrogen ion glas electrodes have been used for the preparation of enzyme-pH-electrodes by either entrapping the enzymes within polyacrylamide gels around the electrode or as liquid layer trapped within a cellophane membrane. The enzymes were glucose oxidase, urase and penicillinase.The pH response to glucose concentration was about linear within 10^?1–10^?3 M glucose and for urea linear within 5·10^t—–5·10^?5M. The pH response to penicillin was about linear in the range from 10^?3–10^?2 M resulting in a pH shift of 1.4 units; reproduceable pH response was obtained down to concentrations of 3·10^?5 M.Studies as to the effect of buffer using an urease–pH-electrode showed a buffer concentration of 10^?2 M a substantial shift of about one pH-unit in the range of 10^?4 to 10^?2 M urea. Both urease- and penicillinase–pH-electrodes were tested as to stability showing no decrease in pH response except at high substrate concentration (1·10^?2 M) over a period of 2–3 weeks kept at room temperature.