Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P _3A -type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H ⁺ -ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H ⁺ accumulation and subsequent K ⁺ efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H ⁺ -ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H ⁺ -ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events.

Background : Gastrointestinal motility modulatory factors include substances of the intestinal content, such as polyamines and trace amines (TAs), the focus of this study. Methods : The amines of food, intestinal content and from faecal bacteria of Swiss mice were determined by HPLC and functionally characterised in isolated distal ileum and medial colon rings. Results : Mouse food and intestinal content contain polyamines (spermidine>putrescine>spermine) and TAs (isoamylamine>cadaverine). Intestinal bacteria mainly produce putrescine and cadaverine. The amines inhibited the spontaneous motility of the ileum (0.1-3 mM) and colon rings (0.01-3 mM, with lower IC 50 ), with: spermine~isoamylamine~spermidine. Spermine inhibition was tetrodotoxin (TTX)-insensitive, while isoamylamine was TTX-sensitive, suggesting neural control. Mainly in the ileum, isoamylamine (3 mM) elicited acute effects modified by TTX, atropine and propranolol, and suppressed by spermine (3 mM), not being localized at the smooth muscle level. The amines assayed (3 mM), except putrescine and cadaverine in the ileum and isoamylamine in the colon, antagonised acetylcholine (ACh, 0.1 mM)-elicited phasic contractions. Isoamylamine and spermine in colon relaxed KCl (100 mM)-elicited tonic contractions, suggesting an effect on smooth muscle, but did not justify the suppression of motility caused by spermine and isoamylamine. Conclusions : Polyamines and TAs of the intestinal content might act on chemosensors and modulate intestinal peristalsis.

In yeast, we reported the critical role of K⁺-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H⁺-ATPase. In the present study, the K⁺ channel Tok1p was identified as the K⁺ channel-mediating K⁺-efflux, as indicated by the protective effect of extracellular K⁺ (≥30 mM), K⁺-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K⁺-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH₄Cl or nigericin which released a percentage of K⁺ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (~0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H⁺-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K⁺ channel, Tok1p, allowing an electrogenic K⁺-efflux. These ionic events, cytosolic accumulation of H⁺ followed by K⁺-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.