Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.     

Mitochondria adjust Ca(2+) signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca(2+) signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca(2+) signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca(2+) entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca(2+) machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca(2+) imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca(2+) signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca(2+) handling system directing more Ca(2+) into mitochondria via microdomains of high [Ca(2+)] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca(2+)-ATPase-mediated Ca(2+) uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca(2+) fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Analysis of the effect of the alpha 1-adrenostimulator mezaton on plasma renin activity in healthy people of varying age]

30 healthy people aged 20-34, 60-74 and 75-89 (10 subjects in each group) were examined to study the changes in plasma renin activity (PRA), the sympathetic nervous system tone, indices of central and renal haemodynamics after intramuscular injections of alpha 1-adrenostimulator mezaton (phenylephrine) in dose of 0.15 mg/kg of body mass. The pharmacological activity of postsynaptic alpha 1-adrenoreceptors was found to induce more considerable and prolonged increase of PRA in older persons. The increase of PRA was reliably correlated with a decrease of renal blood flow indices and was combined with an increase of sympathetic tone. As to the authors' opinion a more pronounced constrictor response of afferent renal arterioles to alpha 1-adrenoreceptors activation of vessels is the cause of more considerable changes of PRA in older age groups.     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

Role of SH Groups in the Functioning of Ca2+-Transporting ATPases Regulating Ca2+ Homeostasis and Exocytosis

Using a ^Ca2+-sensitive fluorescent indicator, Fura-2/AM, and a metallochromic dye, arsenazo, we measured the intracellular concentration of Ca²⁺ ([Ca²⁺] _i ) and the content of total calcium in isolated acinar cells of the rat submandibular salivary gland. It was shown that the influence of a mercaptide-forming compound, sodium p-chloromercuribenzoate (pChMB), increased both the [Ca²⁺] _i and content of total calcium but did not change the intensity of exocytosis. Such a situation is probably related to the fact that pChMB inhibits plasmalemmal Ca²⁺-ATPase (PMCA). The absence of changes in the exocytotic activity can be explained as follows: the influence of a pChMB-induced significant increase in the [Ca²⁺] _i is neutralized due to the functioning of Ca²⁺-ATPases of the endoplasmic reticulum (SERCA), which pump Ca²⁺ into the store. Incubation of a microsomal fraction with pChMB resulted in suppression of the specific PMCA and SERCA activities with apparent constants of inhibition (I ₅₀) 245 and 52 M, respectively. Dithiothreitol (DTT, 0.1 mM) increased the PMCA and SERCA activities (probably facilitating the access of substrate to the active centers of ATPases at the expense of a decrease in the number of disulfide bonds, which is followed by changes in the conformation of intracellular hydrophilic loops of their molecules). Dithiothreitol also recovered the suppression of PMCA and SERCA activities induced by pre-incubation with pChMB (by 45 and 32%, respectively); these activities did not, however, reach the initial levels. A probable interpretation of this fact is that DTT shields from the action of pChMB only superficial but not sterically less accessible SH groups. Limited proteolysis of the microsomes by -chymotrypsin decreased the specific PMCA and SERCA activities by 16 and 60%, respectively. Incubation of the microsomes in an -chymotrypsin-containing medium (15 sec) with subsequent addition of 150 M pChMB exerted almost no influence on the PMCA activity, whereas the SERCA activity dramatically increased (by 146%). This fact allows us to suggest that -chymotrypsin is capable of eliminating the inhibitory effect of pChMB on the SERCA activity; the mechanism of this effect remains unknown. Therefore, functionally important SH groups are present in the catalytic and active centers of both PMCA and SERCA; superficial SH groups dominate in the PMCA molecules, whereas SERCA is controlled by more deeply localized SH groups.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Molecular Mechanisms of Cadmium-Induced Nonspecific Mitochondrial Permeability

In mitochondria obtained from the rat liver, we confirmed the ability of Cd²⁺ to induce the development of nonspecific mitochondrial permeability (NMP). A kinetic analysis of this process was performed. We demonstrated that the cadmium-induced NMP is mediated by activation of megachannels (mitochondrial transition permeability pores) and is realized with the participation of porin, the ADP/ATP antiporter, and cyclophilin D. A key event in the process of induction of the mitochondrial pore opening is interaction of Cd²⁺ with just the ADP/ATP antiporter.     

Methodical approaches to identification of transmembrane current of sodium-calcium exchange

The methodical approaches allowing one to record transmembrane currents related to coupled Na⁺−Ca²⁺ exchange through the cell membrane are considered. The techniques are based either upon changes of the Na⁺ or/and Ca²⁺ concentration gradients, or upon shifts of the membrane potential. The advantages and disadvantages of these techniques applied to different objects, as well as the authors' own experiments on secretory cells of the salivary glands ofChironomus larvae, are discussed.