Drug-sensitive ecto-ATPase in human leukocytes

An ecto-ATPase has been described in human leukocytes. The enzyme is present in both granulocytes and lymphocytes, exhibiting higher activity and substrate affinity in the latter cells, K_m values ranged from 0.06 mM in lymphocytes to 0.3 mM in the unresolved leukocyte fraction. Enzymatic activity had a pH optimum in the physiological range, required Mg²⁺, and was insensitive toward ouabain and Ca²⁺. Potent inhibitors of ecto-ATPase were tricyclic antidepressants and substituted phenothiazines, to which the enzyme in lymphocytes was most susceptible. These drugs also decreased the phagocytic ability of leukocytes. However, while drug inhibition of the enzyme was reversible by washing, the effect on phagocytosis was not.     

Conjugated normetanephrine in human and rat plasma and erythrocytes

Enzymatic hydrolysis was applied to the deconjugation of normetanephrine (NMN) in plasma and red blood cell lysate. By this procedure, in human plasma 77% of total NMN circulated in sulfate-conjugated form, while in rat plasma 63% was in glucuronidated form. Total NMN in human lysate was significantly higher than in plasma (P > 0.001) and was mostly in the free form, indicating that red blood cells may play an important role in metabolism of norepinephrine. Enzymatic hydrolysis is superior to the standard method by acid hydrolysis plus heat since: (1) more conjugated NMN is hydrolyzed in human plasma and (2) a smaller sample is needed for hydrolysis.     

alpha 2-Adrenergic receptors in human polymorphonuclear leukocyte membranes

Human polymorphonuclear cell membranes contain alpha 2-adrenergic receptors which are measured by binding of the alpha 2-adrenergic antagonist [3H]yohimbine. The alpha 1-adrenergic antagonist [3H]prazosin showed no specific binding. High and low affinity sites were detected which had Kd values of 2.38 +/- 0.4 and 139 +/- 12 nM, respectively, and which bound maximally 4.82 +/- 0.9 and 81 +/- 9 fmoles of [3H]yohimbine/mg membrane protein. The high and low affinity sites were also detected by competition studies with phentolamine, epinephrine and norepinephrine and by dissociation kinetics of bound [3H]yohimbine. [3H]Yohimbine binding was stereospecifically inhibited by (-)- and (+)-epinephrine and norepinephrine. [3H]Yohimbine binding to intact cells showed about 500 high affinity sites per cell (Kd 0.5 nM) and approximately 4000 lower affinity sites per cell (Kd 3-4 nM). Yohimbine enhanced the (-)-norepinephrine stimulation of cAMP production in intact cells.     

Erythromycin binding to human serum

Erythromycin binding to human serum was measured under conditions of binding equilibrium. The binding is sensitive to pH changes, decreasing at acid pH. Over a great range of serum dilution, the bound fraction is semilogarithmically related to serum concentration. Binding is shown to be completely reversible. With increasing erythromycin concentration a specific part of binding is saturable and specifically displaceable by erythromycin in excess, whereas a nonspecific part linearly increases with total concentration. Erythromycin is specifically bound to a single class of noninteracting binding sites with an apparent dissociation constant K_d=5.9 μM (38°C). The kinetic and thermodynamic parameters at 25° are: K_d = 8.4 μM, ΔH° = +4.4 × 10³ cal per mole, ΔG° = ?6.9 × 10³ cal per mole, ΔS°= +38 e.u.     

Monoamine oxidase in human platelets: Kinetics and methodological aspects

The kinetic properties of human platelet monoamine oxidase (MAO) were examined in 20 apparently healthy controls. The mean value (±S.D.) of the maximum velocity (V) was found to be 5.36 ± 1.97 pmoles of product formed/10° platelets/min and the Michaelis-Menten constants were for phenethylamine (K^PEA_m) 14.6 ± 8.20 μM and for oxygen (K_m^O₂)254 ± 125 μM, when assayed in 0.1 M phosphate buffer, pH 7.4. The relation between the value of the corresponding apparent constants was studied. Inhibition of the enzyme activity was seen at 20 μM of PEA and 180 μM of oxygen. The enzyme kinetics were also studied at different pH. Two pK values were found, pK₁ = 6.65 and pK₂ = 6.95. The influence of homogenization on the MAO activity was compared with the activity in the undisrupted platelet. At PEA concentrations below 10 μM higher MAO activities were found in the intact cell. A 15 per cent loss of activity was detected in platelet samples after storing at ?20° for three and a half years.     

Preferential accumulation of lithium in the dense bodies of human platelets

From 50 to 73% of the lithium contained in platelets of patients receiving oral therapy with lithium carbonate was released by brief thrombin treatment. Similarly, about 50% of the lithium in platelets of normal volunteers incubated with lithium chloride was thrombin-releasable. The data indicate that an amount of lithium approximately equal to 10% of the calcium content was sequestered in the dense bodies (amine storage organelles) of human platelets. Electron microprobe analysis of dense bodies suggests that the addition of lithium did not change the phosphorus content but produced a loss of about 10% of the dense-body calcium. Nevertheless, synthetic solid analogues of the dense-body core incubated with lithium chloride did not sequester lithium preferentially over potassium and failed to exchange calcium for lithium. Thus, the mechanism responsible for the observed changes in platelet dense bodies may be related to selective membrane permeability properties rather than to binding of lithium to nucleotides or pyrophosphate in the dense-body core.     

Activities of purine-metabolizing enzymes in human colon carcinoma cell lines and xenograft tumors

The activities of eleven enzymes of purine metabolism have been determined in extracts of four human colon carcinoma cell lines (clones A and D of the heterogeneous DLD-1 line, HCT-15 and DLD-2) in culture and as xenografts in nude mice. Activities of the enzyme adenine phosphoribosyltransferase (APRT), hypoxanthine phosphoribosyltransferase (HPRT), adenosine kinase, 5'-deoxy-5'-methylthioadenosine (MTA) phosphorylase, adenylate (AMP) kinase, guanylate (GMP) kinase, and nucleoside diphosphokinase (NDP kinase) changed little from line to line in vitro. Adenosine deaminase (ADA) activity varied more than 5-fold between HCT-15 and clone A cells. Purine nucleoside phosphorylase (PNP) activity was about 3-fold higher in DLD-2 cells than in the other three lines. Guanine deaminase (guanase) activity was 4- to 6-fold higher in clone A and D cells than in HCT-15 and DLD-2 cells. Xanthine oxidase was not detected in any of the lines grown in vitro or in vivo. The activity of APRT was highest in clone D xenografts. HPRT was lowest in DLD-2 tumors. ADA activity in DLD-2 tumors was more than 2-fold elevated over that for clone A and HCT-15 xenografts. PNP activity in clone A and HCT-15 xenografts was lower than that for clone D or DLD-2 tumors. Guanase was lower in HCT-15 and DLD-2 tumors than in those of clones A and D. Clone D tumor GMP kinase activity was elevated more than 4-fold above that for the other three tumors. The activity of NDP kinase was highest in clone D tumors. MTA phosphorylase activity was similar in all four xenografts. In general, the activity of a given enzyme was similar in cells growing in monolayer culture and as xenografts in nude mice (37/44 comparisons). Enzyme activities were also measured in normal human colon and compared to those in the xenografts. Where significant differences were seen, the values for normal tissue were almost always lower than those of the tumors. The striking similarities between the in vitro and in vivo enzymic profiles for each of these four human colon carcinoma lines indicate that, at least on this basis, responses of these cells to purines and purine analogs in the two-dimensional monolayer culture system should be predictive of in vivo responses.     

Influence of psychoactive and nonpsychoactive cannabinoids on chromatin structure and function in human cells

The influences of two naturally occurring psychoactive cannabinoids (Δ⁹-tetrahydrocannabinol and Δ⁸-tetrahydrocannabinol), of a psychoactive cannabinoid metabolite (11-OH-Δ⁹-THC) and of a nonpsychoactive cannabinoid (cannabinol) on the composition, metabolism and structure of human (HeLa cell) chromatin were examined. The effects of all four cannabinoids on the composition and synthesis of chromosomal proteins were studied by pulse-labeling with [³H]leucine. The relative amounts of both histones and nonhistone chromosomal proteins remained unaffected, but a decrease in [³H]leucine incorporation into both histones and nonhistone proteins was observed. Post-translational acetylation and phosphorylation of chromosomal proteins were studied in cannabinoid-treated cells by pulse-labeling with [³H]acetate and [³²P], Variations were observed in [³H]acetate incorporation into histones and [³²P] incorporation into both histones and nonhistone chromosomal proteins. Pancreatic DNase I digestion of chromatin from cannabinoid-treated HeLa cells failed to demonstrate any drug-induced alteration in DNA-protein interactions.     

The competitive inhibition of glucose transport in human erythrocytes by compounds of different structures

By kinetic analysis we found that the transport protein for glucose in human erythrocyte membranes has different binding sites for competitive inhibitors. They all change the transport protein with the effect that it loses its affinity to glucose. Some of the competitive inhibitors alter the conformation of the transport protein, so that other ones cannot be boand. There are inhibitors, however, which do not affect the affinity of other competitive inhibitors. A schematic model of our assumption about the mechanism of the competitive inhibition of glucose transport is presented.     

Effect of immunosuppressive agents on human T and B lymphoblasts

We have studied the effects of various immunosuppressive drugs on the growth of human-derived T (MOLT-4) and B (MGL-8) lymphoblasts. In addition, we have examined whether the lymphotoxic effect of any of these drugs could be attributed to inhibition of either adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP). Results indicated that 1-beta-D-arabinofuranosylcytosine (Ara-C), methotrexate and chlorambucil were four to seven times more toxic for T than for B cells, while azathioprine, 6-thioguanine, 6-mercaptopurine, and 5-fluorouracil were highly toxic for both T and B cells. Cyclophosphamide and oxisuran were lymphotoxic only at concentrations exceeding 300 microM. Deoxyadenosine (50 microM), deoxyguanosine (10 microM) and deoxycoformycin (10 microM) failed to enhance T cell toxicity when individually combined with each drug. None of the drugs tested inhibited T or B lymphoblast ADA or PNP activity. With the exception of Ara-C, neither dATP nor dGTP accumulated in T lymphoblasts incubated in the presence of any of the drugs. We conclude that the cell culture system used in this investigation is useful for identifying lymphotoxic and T cell-specific immunosuppressive agents. However, none of the drugs studied appeared to function as an inhibitor of, or a competitive substrate for, either ADA or PNP.     

Gold suppression of human neutrophil function in vitro

The effect of myochrysine and Auranofin on leukocyte function were measured using quantitative leukocyte iodination. Both suppressed iodination at concentrations achieved in patients. Under conditions of leukocyte submaximum stimulation, enhanced gold suppression was observed. The active portion of Myochrysine appeared to be protein bound while the active portion of Auranofin appeared to be free. Preincubation experiments indicated suppression of the myeloperoxidase-halide system. Inhibition of this probable mediator of inflammation may be one of the modes of action of gold.     

Consequences of the NB transition of albumin for the binding of warfarin in human serum

The protein binding of warfarin in serum has been studied by means of circular dichroism and equilibrium dialysis. Evidence was found that the NB transition of albumin, occurring around physiological pH, takes place not only in solutions of pure albumin but also in serum. The protein binding of warfarin in serum is pH-dependent and increases with pH especially around physiological pH. This pH-dependent serum binding of warfarin can be reasonably explained by the NB transition of albumin.The effect of Ca²⁺ and Mg²⁺ on the protein binding of warfarin in serum is negligible at pH 7.4, whereas at this pH Cl^? increases the free-warfarin concentration by a competitive displacement.     

Prostaglandins and cannabis--IX. Stimulation of prostaglandin E2 synthesis in human lung fibroblasts by delta 1-tetrahydrocannabinol

Preliminary data [S. Burstein and S. A. Hunter, Biochem. Pharmac. 27, 1275 (1978)] showed that cannabinoids at levels of 1 microM or greater elevated the concentrations of prostaglandins in cell culture models. Further study [S. Burstein and S. A. Hunter, J. clin. Pharmac. 21, 240S (1981)] led to the suggestion that this effect was due to a stimulation of phospholipase A2 resulting in the release of free arachidonic acid which was then partly converted into the prostaglandin(s) normally synthesized by the particular target system. The present report gives detailed data on the cannabinoid-induced synthesis of prostaglandin E2 by te WI-38 fibroblast derived from human lung. The effect could be blocked by pretreatment with mepacrine, a phospholipase inhibitor, and aspirin, a cyclooxygenase inhibitor. These findings lend support to the hypothesis that some of the in vivo actions of the cannabinoids are due to modulations in prostaglandin synthesis at various tissue sites.     

Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions

Human and rat liver microsomal fractions exhibit non-linear Michaelis-Menten kinetics in the O-deethylation of both phenacetin and 7-ethoxycoumarin. Comparison of various models indicated that the data were best described by a biphasic plot, which could be interpreted in terms of two populations of cytochrome P-450. The K(m)'s of the high affinity phase of 7-ethoxycoumarin O-deethylase activity were 1.8 +/- 0.4 microM and 2.3 +/- 0.4 microM for human and rat respectively while the K(m)'s of the low affinity phase were 205 +/- 20 microM and 237 +/- 59 microM in the two species respectively. V(max) of the high affinity phase of human 7-ethoxycoumarin O-deethylase activity was 96.9 +/- 19.0 pmol mg(-1) min(-1) and the activity of the corresponding phase in the rat was 2.7 times greater. The activities of the low affinity phase were 10-15 times greater than the respective activity of the high affinity phase. Rat and human also had similar values for the K(m)'s of the two phases of phenacetin O-deethylase activity, around 5 microM for the high affinity phase and 300 microM for the low affinity phase. Total activity was very similar in the two species, 1500-1750 pmol mg(-1) min(-1) and the difference between the two phases of activity was only 2.5-fold in man and 10-fold in rat. Studies on the effects of the in vitro modifiers of monooxygenase activity alpha-naphthoflavone and metyrapone further supported the hypothesis that the two phases of O-deethylase activity represent two different forms or populations of cytochrome P-450.     

Interaction of the human leukocyte proteinases elastase and cathepsin G with gold, silver and copper compounds

Gold thiomalate and the corresponding silver and copper derivatives were investigated as inhibitors of the human leukocyte proteinases elastase and cathepsin G. The kinetic inhibition mechanism for gold- and silver thiomalate is of the hyperbolic non-competitive type with both enzymes and the inhibitory efficiency of the metals increases in the order Cu less than Ag less than Au. On the contrary, D-penicillamine derivatives of the three metals do not influence at all the activity of the two proteinases. Although gold thiomalate is the most efficient of the investigated metal compounds (Ki = 33 microM and 25 microM for elastase and cathepsin G, respectively), the hyperbolic nature of the inhibition imposes a serious limit to its practical usefulness since the maximum inhibitory action on both enzymes is about 40%. We suggest that, in order to act as inhibitor, a copper, silver or gold compound must be able to easily transfer the metal to the enzyme.     

The human blood platelet: A cellular model to study the degradation of thymidine and its inhibition

Intact platelets catabolize extracellular thymidine into thymine. Studies of the concentration dependent degradation of thymidine by intact platelets indicate a Michaelis mechanism with an apparent K_m of about 0.12mM and a V_max of 2.5 nmoles/min for 3 × 10⁸ platelets. This degradation process is inhibited by various nucleosides, pyrimidine bases and C-5 or C-6 substituted uracils. Cytidine, deoxycytidine, adenosine and deoxyadenosine seem to inhibit thymidine degradation by reducing the intracellular transport of thymidine. Uridine inhibits both the thymidine transport and the activity of the phosphorolytic enzyme, thymidine phosphorylase (EC 2.4.2.4). Some substituted uracils are specific inhibitors of thymidine phosphorylase activity. 6-Amino-5-bromouracil, the most active of them, either with acellular extracts or purified thymidine phosphorylase, is also the best inhibitor of thymidine degradation in intact human platelets. Platelets constitute a new model to study the efficiency of specific inhibitors on thymidine catabolism in an ‘human intact cell’ which contains only one pyrimidine nucleoside phosphorylase, the thymidine phosphorylase.