Methylamine, but not ammonia, is hypophagic in mouse by interaction with brain Kv1.6 channel subtype

Abstract

Ammonia and methylamine (MET) are endogenous compounds increased during liver and renal failure, Alzheimer's disease, vascular dementia and diabetes, where they alter some neurobehavioural functions probably acting as potassium channel blockers. We have already described that potassium channel blockers including tetraethylammonium (TEA), ammonia and MET are hypophagic in mice. Antisense oligonucleotides (aODNs) against Shaker-like Kv1.1 gene abolished the effect of TEA but not of ammonia and MET. The central effects elicited in fasted mice by ammonia and MET were further studied. For MET, an ED(50) value 71.4+/-1.8 nmol mouse(-1) was calculated. The slope of the dose-response curves for these two compounds and the partial hypophagic effect elicited by ammonia indicated a different action mechanism for these amines. The aODNs pretreatments capable of temporarily reducing the expression of all seven known subtypes of Shaker-like gene or to inactivate specifically the Kv1.6 subtype abolished the hypophagic effect of MET but not that of ammonia. Reverse transcription-polymerase chain reaction, Western blot and immunohistochemical results indicate that a full expression in the brain of Kv1.6 is required only for the activity of MET, and confirms the different action mechanism of ammonia and MET.

Figure 1

Dose-related reduction of food intake in 12 h starved mice after the i.c.v. injection of MET or NH₃, as measured 60 min after food readministration. Each point represents the mean±s.e.m. of at least 10 mice.

Figure 2

Effect of increasing concentrations (i.c.v. injection at days 1, 4 and 7) of aODN₁₋₇ or dODN₁₋₇ on MET-induced food consumption in 12 h starved mice, as measured 60 min after food readministration. Each point represents the mean±s.e.m. of at least 10 mice. ^*P<0.01 in comparison with dODN₁₋₇-pretreated mice.

Figure 3

Effect of aODN₂, aODN₃, aODN₄, aODN₅, aODN₆, aODN₇ and related dODN_s (controls) (9 nmol per single i.c.v. injection at days 1, 4 and 7) on MET-induced food consumption in 12 h fasted mice, as measured 60 min after food readministration. Each point represents the mean±s.e.m. of at least 10 mice. ^*P<0.01 in comparison with dODN₆-pretreated mice.

Figure 4

Recovery of the hypophagic response of i.c.v.-administered MET (226 nmol) in aODN₆- or dODN₆- (controls) (9 nmol per single i.c.v. injection at days 1, 4 and 7) pretreated mice. At 7 days after the last aODN injection, the inhibitory effect of the aODN₆ was completely lost. ^*P<0.01 in comparison with dODN₆-pretreated mice taken as controls. Each point represents the mean±s.e.m. of at least 10 mice.

Figure 5

(a) Lowering of Kv1.6 over β-actin mRNA levels in brain homogenates as detected by RT–PCR. (b) Reduction of phenotypic expression of the 80 kDa Kv1.6 protein level in brain homogenates obtained from aODN₆-treated mice. (c) Left panel, Kv1.6 immunoreactivity in the LAH of hypothalamic neurons of dODN₆- (controls) pretreated mice; evidence for a significant reduction of immunostaining in brain sections from aODN₆-pretreated mice (right panel).