Kinetic properties of the redox switch/redox coupling mechanism as determined in primary cultures of cortical neurons and astrocytes from rat brain

Abstract

We investigate the mechanisms underlying the redox switch/redox coupling hypothesis by characterizing the competitive consumption of glucose or lactate and the kinetics of pyruvate production in primary cultures of cortical neurons and astrocytes from rat brain. Glucose consumption was determined in neuronal cultures incubated in Krebs ringer bicarbonate buffer (KRB) containing 0.25-5 mM glucose, in the presence and absence of 5 mM lactate as an alternative substrate. Lactate consumption was measured in neuronal cultures incubated with 1-15 mM lactate, in the presence and absence of 1 mM glucose. In both cases, the alternative substrate increased the K(m) (mM) values for glucose consumption (from 2.2 +/- 0.2 to 3.6 +/- 0.1) or lactate consumption (from 7.8 +/- 0.1 to 8.5 +/- 0.1) without significant changes on the corresponding V(max). This is consistent with a competitive inhibition between the simultaneous consumption of glucose and lactate. When cultures of neurons or astrocytes were incubated with increasing lactate concentrations 1-20 mM, pyruvate production was observed with K(m) (mM) and V(max) (nmol/mg/h) values of 1.0 +/- 0.1 and 109 +/- 4 in neurons, or 0.28 +/- 0.1 and 342 +/- 54 in astrocytes. Thus, astrocytes or neurons are able to return to the incubation medium as pyruvate, a significant part of the lactate consumed. Present results support the reversible exchange of reducing equivalents between neurons and astrocytes in the form of lactate or pyruvate. Monocarboxylate exchange is envisioned to operate under near equilibrium, with the transcellular flux directed thermodynamically toward the more oxidized intracellular redox environment.

Cellular and Metabolic Origins of Flavoprotein Autofluorescence in the Cerebellar Cortex in vivo

“…The removal of glucose from the Ringer’s in the substitution experiment reduces the substrate availability for glycolysis in both neurons and astrocytes. Furthermore, in both the replacement and supplementation experiments, the increased lactate in the bath will inhibit glycolysis while providing lactate for use in the TCA cycle [63]. Therefore, these findings demonstrate that the vast majority of the onbeam light phase is due to oxidative metabolism and that neurons can sustain oxidative metabolism using lactate, which is in agreement with the ANLS hypothesis [16, 24, 56, 64–67].…”

Is lactate a volume transmitter of metabolic states of the brain?

“…In turn, any effects of changes of lactate on the NADH/NAD + ratio in one place lead to similar effects in widely distributed populations of cells (cf. Cerdán et al, 2006; Ramírez et al, 2007; Rodrigues et al, 2009). In fact, the transfer of lactate is so efficient that it is difficult to observe any differences of lactate concentrations across cell membranes in brain tissue (Gjedde and Marrett, 2001; Ido et al, 2001, 2004; Gjedde et al, 2002), such that significant cellular compartmentation of lactate is unlikely to exist under normal conditions, except briefly.…”

Metabolic and Hemodynamic Events after Changes in Neuronal Activity: Current Hypotheses, Theoretical Predictions and in vivo NMR Experimental Findings

“…Different experimental procedures in preparing the cell cultures might explain the different outcomes of the afore mentioned in vitro studies (Bak et al , 2006; Bouzier-Sore et al , 2006; Ramirez et al , 2007). From a more general perspective, a key component about cellular function that should be kept in mind is that electrical excitability is compromised under in vitro conditions because cell membranes are in an unusual environment.…”

“…Results by Ramirez et al (2007) suggested that ∼3.6 mM extra-cellular [Lac] is needed to inhibit neuronal glucose consumption by 50%. However, tissue [Lac] has been measured to increase no more than 1 μmol/g during stimulation (Dienel et al , 2002; Frahm et al , 1996; Madsen et al , 1999; Mangia et al , 2007a; 2007b; Prichard et al , 1991); even the increases in [Lac] measured in the extra-cellular medium did not overcome this limit (Hu and Wilson, 1997).…”