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.
Development of diabetic sensory polyneuropathy is associated with alterations in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We have shown previously that in a model of streptozotocin (STZ)-induced diabetes, the calcium signal is prolonged and calcium release from ryanodine-sensitive calcium stores down-regulated in neurons of the nociceptive system. The aim of the present study was a more detailed characterization of calcium homeostasis in primary (dorsal root ganglia, DRG) and secondary (dorsal horn, DH) nociceptive neurons in STZ-induced diabetes. Fluorescence video-imaging was used to measure free cytosolic [Ca2+] ([Ca2+]i) in lumbar nociceptive neurons of control and streptozotocin-diabetic rats. Resting [Ca2+]i rose progressively in these neurons with the duration of diabetes and calcium mobilization from the endoplasmic reticulum (ER) decreased during diabetes. The amplitude of calcium release from both ryanodine- and IP3-sensitive calcium stores induced by caffeine, ionomycin, ATP or glutamate was significantly (P
The present study examines the mode of action of eyestalk factors, in particular crustacean hyperglycaemic hormone (CHH), on known target tissues (muscle and hepatopancreas) in two decapod crustacean species, Nephrops norvegicus and Carcinus maenas. The possibility that receptors to CHH are coupled to an elevation of cytosolic Ca2+ derived from intracellular stores via activation of the phosphatidylinositol pathway was investigated. Using an inositol 1,4,5-trisphosphate (InsP3)-specific competitive binding assay, measurable levels of InsP3 were detected in hepatopancreas and muscle in both N. norvegicus and C. maenas. Incubation of these tissues in the presence of sinus gland extracts (two sinus gland equivalents) consistently increased InsP3. In addition, Carcinus CHH (10 nM) increased InsP3 levels in Carcinus hepatopancreas and muscle. Incubation of hepatopancreas from both species in 10 mM lithium saline increased basal levels of InsP3, suggesting a significant turnover of phosphoinositides in this tissue.
Review. Modern data about mechanisms of generation of the calcium signals in the exocrine acinar cells are presented. The mechanisms of inositol-1,4,5-trisphosphate- and Ca(2+)-induced Ca2+ release from intracellular stores of the acinar cells and mechanisms their influx from extracellular medium are described. The mechanisms which initiate Ca2+ oscillations and their role in the secretion of the fluid and enzymes by acinar cells are discussed.
1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.