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The purpose of this study was to compare Na+/H+ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of22Na into isolated MVM vesicles in the presence of a H+ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214±28 vs. 49±9 pmol/mg protein per 30 s, respectively, means±SEM,n=8, 6,P<0.001). The22Na uptake by MVM, but not by BM, was reduced in the absence of a H+ gradient and in the presence of 500 µM amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited22Na uptake with an EC50 of 0.13±0.05 µM (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC50 of 3.01±0.85 µM (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pHi) from an acid load using a H+-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na+ and the presence of amiloride (500 µM) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

1. The intestine has been implicated as a site of increased glucose utilization by the antihyperglycaemic drug, metformin. This study makes a quantitative assessment of this effect. 2. Glucose utilization by the intestine and hind limb region was determined by arterial-venous glucose difference adjusted for blood flow rate in fasted rats receiving a hyperglycaemic hyperinsulinaemic infusion. 3. Intrajejunal administration of metformin, 250 mg kg-1, increased glucose disposal during the infusion procedure, associated with increased glucose utilization in the intestine by 69% and in the hind limb region by 40%. 4. Metformin, 250 mg kg-1, increased glucose disappearance during an intravenous glucose tolerance test. This was accompanied by increased uptake of tritiated 2-deoxy-D-glucose into the intestinal mucosa to a greater extent than into skeletal muscles (per unit wet weight of tissue). 5. The results demonstrate that the intestinal mucosa is a quantitatively important site of increased glucose utilization during the blood glucose-lowering effect of metformin.

1. Insulin-dependent diabetic BB/S rats with little or no endogenous insulin were used to determine whether insulin is required for the acute antihyperglycaemic effect of metformin (dimethylbiguanide). 2. Metformin (250 mg kg-1, intrajejunally) did not lower the hyperglycaemia in BB/S rats in the absence of exogenous insulin, but metformin increased by 69% (P < 0.05) the blood glucose-lowering effect of exogenous insulin. 3. Metformin (250 mg kg-1, intrajejunally) improved glucose disposal in rats with a normal insulin response to an intravenous glucose challenge. Plasma glucose disappearance was increased from 0.7 +/- 0.1 to 2.5 +/- 0.1% min-1 (P < 0.05). 3. When the insulin response to glucose was suppressed with somatostatin and diazoxide, metformin improved glucose disposal to a similar extent to that in rats with a normal insulin response. Plasma glucose disappearance was increased from 0.24 +/- 0.02 to 1.0 +/- 0.1% min-1 (P < 0.01). 5. The results indicate that insulin is required for the acute antihyperglycaemic effect of metformin, but the extent of this effect is not proportional to the prevailing insulin concentration.

2-Nitropropane (2-NP) is an industrial chemical with hepatotoxic and genotoxic properties. It exists in chemical equilibrium with propane-2-nitronate, which is much more genotoxic than 2-NP. In this work the link between toxicity and metabolism of 2-NP and its nitronate was investigated. To that end 2-NP or propane-2-nitronate were incubated with murine hepatic microsomes at concentrations of up to 10 mM, and generation of nitrite was measured as product of metabolic oxidation of the two species. Under the acidic reaction conditions of the colorimetric nitrite assay propane-2-nitronate decomposed chemically to nitrite. Therefore an ion-pair HPLC assay at neutral pH was developed which enabled determination of nitrite formed from the nitronate. The rate of metabolic nitrite generation from propane-2-nitronate was 5-10-fold that obtained with 2-NP. Metabolism of either species to nitrite was dependent on the presence in the incubate of viable microsomes and of NADPH, and it was inhibited in the presence of carbon monoxide or the cytochrome P-450 inhibitor SKF525A. Acetone could also be measured as a metabolite of 2-NP. Optical difference spectra were recorded in mixtures of propane-2-nitronate with liver microsomes from phenobarbital-pretreated rats. The spectral dissociation constant was found to be 30 mM, which compares with 10 mM reported for 2-NP. 2-NP and propane 2-nitronate were incubated with mouse hepatocytes in suspension and cytotoxicity was determined by measurement of leakage of cellular lactate dehydrogenase into the medium. Both species were hardly toxic, as concentrations of 20 mM were required to elicit significant damage to the cells. The results demonstrate that propane-2-nitronate, like 2-NP, undergoes microsomal oxidative denitrification, probably catalysed by cytochrome P-450. Metabolism of both species occurs at markedly different rates, but the difference in metabolism is not reflected by a difference in hepatocytotoxic potential.