Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies

Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes

alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50 microM phenylephrine and 1 microM propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe into-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Cao exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1 microM prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca(2+)-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1 microM 4 beta-phrobol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC.(ABSTRACT TRUNCATED AT 400 WORDS