Studies on the mechanism of action of antifertile PG in animal models

Antifertile effects of PGF2 alpha, PGE2, PGE1, sulprostone and other PGs were evaluated in different pregnancy models in rats, guinea pigs and rhesus monkeys and the underlying mechanisms of action were investigated. Quantitative and qualitative species differences and pregnancy stage dependency were recorded. Basic regulatory differences of the pregnant uterus seem to exist in these species. In early pregnant rats, abortifacient effects were based on luteolytic effects, independent of the PG used. The myometrium was found to be refractory to the injected PG as long as serum progesterone levels were kept high. By contrast, in guinea pigs after the luteoplacental shift of progesterone secretion (tested after day 40 p.c.) and in rhesus monkeys even before this shift (tested day 20 p.c.) abortifacient effects were found to be exerted by direct stimulation of the myometrium. Uterine stimulation was possible in the presence of any level of serum progesterone. The induction of uterine PG synthesis was probably of importance supporting the expulsion. The role of obvious tissue damage within the conceptus remained uncertain. In contrast to rats there seems to be a pre-existing PG-sensitivity of the pregnant myometrium in guinea pigs and primates. In guinea pigs sensitivity slightly increased for E- but not for F-type PG toward term. Oxytocin sensitivity was found to increase by a factor of more than 100 between days 23-63 of pregnancy. Time dependent changes in uterine receptivity to PG and oxytocin may be considered as a regulatory principle which might permit parturition to occur in the presence of progesterone as an evolutionary adaptation to a placental progesterone secretion which cannot be abolished. It was concluded that in the presence of already established gradual uterine responsiveness to PG (and Oxytocin) during gestation efficient blocking mechanisms for uterine PG-formation must exist in order to explain uterine quiescence. Almost complete resistance of pregnancy to oestrogen which exists in humans, monkeys and guinea pigs was considered as to be pharmacological evidence of such a mechanism. The principles of endocrine control of the myometrium and its pharmacology seem similar in guinea pig and primate pregnancy. The guinea pig might therefore provide a relevant model to study potential drug effects on the regulatory balance of the pregnant uterus and also to achieve a better understanding of human uterine physiology.     

Polymyxin B is an inhibitor of insulin-induced hypoglycemia in the whole animal model. Studies on the mode of inhibitory action

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.     

Studies on the mechanism of zinc uptake by human fibroblasts

The mechanisms of zinc uptake from a complete culture medium by human fibroblasts have been studied. The metal is accumulated in a biphasic pattern; an initial rapid phase followed by a slower linear phase. We suggest that the former represents binding to carriers or receptors on the cell surface followed by uptake to within the cell, or at least to a compartment inaccessible to proteolytic digestion. The uptake correlates well with estimates of the zinc requirement of a growing fibroblast. The process of uptake is saturable, with an apparent association constant of 1.1 X 10(7) M-1. Interestingly, there appears to be a very large number of binding sites, 2 X 10(7) per cell. No explanation for this observation is immediately apparent. The mechanism of uptake is not dependent on metabolic energy, or at least on ATP levels within the cell, but N-ethyl maleimide does block uptake in a dose-dependent manner. Weak bases and ionophores, apart from nigericin, do not affect uptake. The results suggest that zinc is not taken up by a receptor-mediated endocytic pathway as has been described for transferrin and iron.     

Body position and the neuroendocrine response to insulin-induced hypoglycemia in healthy subjects

Changes in body fluid distribution are known to influence neuroendocrine function. The aim of the present study was to test the hypothesis that changes in plasma volume affect the counterregulatory neuroendocrine response to hypoglycemia. The tests were performed in 12 subjects in two situations: 'head-up' (+60 degrees head-up tilt standing for 30 min and hypoglycemia in sitting position afterwards) and 'leg-up' (leg-up position for 30 min and hypoglycemia in leg-up position afterwards) in a random order. Insulin-induced hypoglycemia was adjusted to 2.7 mmol/l for 15 min by glucose infusion. Plasma volume was greater by 2.2% (p < 0.001) in leg-up and lower by 9.6% (p < 0.001) in head-up position compared to the basal value in sitting position. Head-up position was associated with increases in ACTH, aldosterone, norepinephrine levels and plasma renin activity (p < 0.01). Leg-up position resulted in decreases in plasma growth hormone and epinephrine concentrations (p < 0.05). Except epinephrine, the neuroendocrine response to hypoglycemia, if any, was mild. Hypoglycemia failed to activate ACTH release after head-up position. Body fluid redistribution did not modify hormonal changes during insulin hypoglycemia. In conclusion, we suggest that body position and accompanying plasma volume changes do not appear to affect neuroendocrine and counterregulatory responses to moderate, short duration hypoglycemia in healthy subjects.     

Studies on the singlet oxygen scavenging mechanism of human macular pigment

It is thought that direct quenching of singlet oxygen and scavenging free radicals by macular pigment carotenoids is a major mechanism for their beneficial effects against light-induced oxidative stress. Corresponding data from human tissue remains unavailable, however. In the studies reported here, electron paramagnetic resonance (EPR) spectroscopy was used to measure light-induced singlet oxygen generation in post-mortem human macula and retinal pigment epithelium/choroid (RPE/choroid). Under white-light illumination, production of singlet oxygen was detected in RPE/choroid but not in macular tissue, and we show that exogenously added macular carotenoids can quench RPE/choroid singlet oxygen. When the singlet oxygen quenching ability of the macular carotenoids was investigated in solution, it was shown that a mixture of meso-zeaxanthin, zeaxanthin, and lutein in a ratio of 1:1:1 can quench more singlet oxygen than the individual carotenoids at the same total concentration.     

Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase.

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.     

Studies on the mechanism of radiation-induced structural disorganization of human erythrocyte membranes

The structural changes of human erythrocyte membranes after X-irradiation were investigated with the aid of fluorescent probes. It was found that the fluorescence characteristics (intensity, polarization and the dissociation constant) of 1-anilino-8-naphthalene sulphonate (ANS) bound to X-irradiated (up to 40 Gy) membranes were quite different from those in unirradiated ones. Sulphydryl (SH)-oxidizing reagents showed the same effects as X-rays on the ANS fluorescence. In addition, pretreatment of the membranes with SH reagents completely blocked the radiation-induced fluorescence changes. These results demonstrated that the initial cause of the radiation effect on membranes is the oxidation of membrane SH groups. There were two different steps in the development of the radiation effect on membrane structure; one is the radiation chemical reaction of SH groups, which is independent of the post-irradiation incubation temperature, and the other is markedly influenced by the temperature, particularly between 12 and 26 degrees C. Therefore it was concluded that structural disorganization of the membranes, including rearrangement of membrane components, might take place following exposure to radiation. This was supported by the fact that treatment with detergents mimicked the effect of X-irradiation. The reaction of OH and/or O2- from the aqueous environment was shown to be responsible for the membrane effect of radiation.     

Urinary cyclic 3',5'-adenosine monophosphate (cAMP) in diabetic subjects with and without symptomatic hypoglycemia and in normal subjects with insulin-induced hypoglycemia.

Urinary cAMP was investigated in diabetic subjects. It was found normal in patients on oral antidiabetics and on insulin in good or poor control. Patients with insulin-induced symptomatic hypoglycemia, however, excreted increased amounts of cAMP in the urine. To demonstrate the significance of hypoglycemia we induced hypoglycemia by i.v.-injection of 0.15 U insulin/kg body weight in six normal subjects. An increase of urinary cAMP was found even during the first 15 minutes with maximal hypoglycemia. Determination of urinary cAMP seems to be helpful in the diagnosis of unrecognized hypoglycemia and counterregulation.