Mecanismos de alcalinização intestinal de Lutzomyia longipalpis: vias de sinalização celular e mecanismos indutores de alcalinização

Abstract

Lutzomyia longipalpis is the main vector of Leishmania infantum in the Americas. Adults feed on sugar solutions for most of their lives, although females require blood for ovarian development. Blood ingestion causes physiological changes in the midgut, especially in the regulation of luminal pH, which increases from 6.0 (in a sucrose diet, actively maintained by V-ATPases) to approximately 8.1 when they ingest blood or solutions containing proteins. This intestinal alkalinization influences the enzymatic activity and development of Leishmania, involving three main mechanisms: release of CO₂ from ingested blood; hormonal signaling by intestinal endocrine cells; and the transport of amino acids coupled to the entry of H⁺ ions into enterocytes. The experiments performed in this work indicate that intestinal alkalinization in female L. longipalpis depends on the increase in osmotic pressure in the intestinal lumen. Feeding the insects with a solution containing PEG 8,000 promoted alkalinization in a concentration-dependent manner, being more intense with more concentrated or salt-rich solutions. Sucrose, mixed with the PEG solution, also potentiated this effect. Other molecules, such as Dextran, Cellobiose and PEG 100,000, used in other feeding experiments, induced similar responses. Furthermore, it was found that the application of hyperosmotic solutions on the basolateral surface of isolated intestines does not induce alkalinization, suggesting that the osmotic imbalance responsible for this process occurs between the intestinal lumen and the apical region of the enterocytes. It was also clear that the inhibition of aquaporins significantly interferes with intestinal alkalinization in L. longipalpis. It was demonstrated that neither the ingestion of latex microspheres nor mechanical stretching of the intestine are capable of inducing alkalinization, ruling out the hypothesis that intestinal distension during blood feeding is a trigger for this process. Unlike the hemolymph of blood-fed females, which induces alkalinization in isolated intestines, the hemolymph of females fed with PEG 8,000 does not present this effect. Furthermore, no differences in the concentrations of amino acids in the hemolymph were observed between the two groups, which excludes these compounds as adjuvants of alkalinization. Finally, it was possible to recover the alkalinizing molecule released by the intestines through an experiment that induces the influx of Ca²⁺ in enteroendocrine cells. Tests with application of insulin and serotonin in isolated intestines of L. longipalpis demonstrated that only insulin was capable of inducing intestinal alkalinization. Using mass spectrometry (MALDI/TOF) with the supernatant from intestinal stimulation with Ca²⁺ ionophore, two peptides were identified whose mass is compatible with known insulins, such as human and bovine insulin. Gene expression analyses confirmed the presence of transcripts of insulin-like peptides (ILPs) and their receptors (ILRs) in the midgut of females. To investigate the intracellular signaling pathways associated with PEG 8,000-induced alkalinization, assays were performed with TOR pathway inhibitors (rapamycin and galactosamine), which resulted in inhibition of the alkalinization process triggered by PEG 8,000 feeding, suggesting the participation of this signaling pathway in the process triggered by osmotic imbalance. Once it was demonstrated that intestinal alkalinization induced by PEG 8,000 involves activation of the TOR pathway, experiments were performed with inhibitors of the HCO₃⁻/Cl⁻ transporters (DIDS and NPPB, both at 0.1 mM), which completely blocked the alkalinization process. The use of H89 (10 µM), a PKA inhibitor, demonstrated that phosphorylation of these transporters is essential for the process. Furthermore, the participation of adenylate cyclase and cAMP was verified: the administration of a permeable cAMP analogue (2.8 mM) was able to reverse the blockade promoted by rapamycin, while the administration of DDA, an adenylate cyclase inhibitor, abolished the alkalinization, confirming the involvement of the cAMP/PKA pathway in the mechanism of action of PEG 8,000. Finally, through enzyme kinetics assays using the substrate BApNA (1 mM), it was demonstrated that feeding with PEG 8,000 increases the trypsinolytic activity in female L. longipalpis. It was evident that the onset of intestinal alkalinization does not depend on the chemical nature of the ingested molecule, but rather on its concentration in the food bolus, which generates an osmotic imbalance sufficient to activate the process. In an unprecedented way, the participation of insulin-like peptides (ILPs) and their receptors (ILRs) in this mechanism was demonstrated. These findings, integrated with pre-existing knowledge, will allow the formulation of a comprehensive descriptive model of intestinal alkalinization processes in L. longipalpis.