The mechanisms of saliva secretion in cockroach salivary glands have been investigated comprehensively which, in addition, are partly similar to those reported in mammalian salivary glands. Similar to the two-stage hypothesis of salivation in mammalian salivary glands, those in the cockroach secrete an isosmotic NaCl-rich primary saliva into the acinar lumen upon stimulation with dopamine. The primary saliva is then modified in the salivary ducts, resulting in a hyposmotic final saliva. Our knowledge about the homeostasis of the intracellular chloride concentration ([Cl^-]_i) is still fragmentary, despite its importance as major anion in living cells. This is mainly due to technical difficulties in chloride measurements performed with invasive Cl^--sensitive microelectrodes limited to large cells or non-invasive UV-excitable fluorescent dyes with high photobleaching and dye leakage rates resulting in limited quantification properties.

We applied the favourably approach of two-photon fluorescence lifetime imaging (2P-FLIM) for measuring [Cl^-]_i. In contrast to the fluorescence intensity, the fluorescence decay time is mostly independent of variations in dye concentration, providing a more reliable quantification of ion concentrations. 2P-excitation in the NIR range exhibits confocal detection and results in reduced global photobleaching and cell damages. Time-resolved measurements were realised with time-correlated single-photon counting and 2P-excitation was performed by a non-tuneable, but inexpensive and easily manageable fs-fiber laser.

The Cl^--sensitive fluorescent dye MQAE showed 2P-excitation when excited at 780nm after loading into the cockroach salivary glands. MQAE was dynamically quenched by chloride and the dependence of MQAE fluorescence decay time on [Cl^-]_i could be described by the Stern-Volmer equation when performing an in situ calibration. On this basis the resting [Cl^-]_i in acinar peripheral cells and duct cells could be determined to 392mM and 591mM, respectively. Thus, in both cell types [Cl^-]_i is maintained at higher concentrations than predicted for a passive distribution across the basolateral membrane, indicating active Cl^- accumulation. However, exposure to Cl^--free saline caused only a moderate [Cl^-]_i drop in both cell types, although [Cl^-]_i recovered rapidly after re-addition of physiological saline. Thus, the basolateral membrane of both cell types seems to exhibit a relatively low Cl^- permeability, at least under resting conditions. Since rapid Cl^- uptake seems to be possible, the influence of Cl^- transport mechanisms such as Na⁺-K⁺-2Cl^- cotransporter and Cl^-/HCO₃^- antiporter was tested pharmacologically. In addition, stimulation of saliva secretion by 1mM dopamine caused no significant [Cl^-]_i changes in acinar peripheral cells and duct cells. These results suggest a more complex scenario of [Cl^-]_i homeostasis in cockroach salivary glands whose clarification is the aim of recent studies.