Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H(+) and Ca(2+) are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a "coaxial" or "concentric" inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca(2+)] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca(2+)-selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca(2+)-electrodes were 14.9 +/- 1.3 and 5.3 +/- 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity-dependent, extracellular pH, and [Ca(2+)] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed.
Synchronous neural activity causes rapid changes of extracellular pH (pH e ) in the nervous system. In the CA1 region of the hippocampus, stimulation of the Schaffer collaterals elicits an alkaline pH e transient in stratum radiatum that is limited by extracellular carbonic anhydrase (ECA). When interstitial buffering is diminished by inhibition of ECA, the alkalosis is enhanced and NMDA receptor (NMDAR)-mediated postsynaptic currents can be augmented. Accordingly, the dendritic influx of Ca 2ϩ elicited by synaptic excitation may be expected to increase if ECA activity were blocked. We tested this hypothesis in the CA1 stratum radiatum of hippocampal slices from juvenile rats, using extracellular, concentric pH-and Ca
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.
SummaryCannabinoid receptors (CBRs) belong to the G protein-coupled receptor superfamily, and activation of CBRs in salivary cells inhibits agonist-stimulated salivation and modifies saliva content. However, the role of different CBR subtypes in acinar cell physiology and in intracellular signalling remains unclear. Here, we uncover functional CB 1 Rs and CB 2 Rs in acinar cells of rat submandibular gland and their essential role in saliva secretion. Pharmacological activation of CB 1 Rs and CB 2 Rs in the submandibular gland suppressed saliva outflow and modified saliva content produced by the submandibular gland in vivo. Using Na + -selective microelectrodes to record secretory Na + responses in the lumen of acini, we observed a reduction in Na + transport following the activation of CBRs, which was counteracted by the selective CB 1 R antagonist AM251. In addition, activation of CB 1 Rs or CB 2 Rs caused inhibition of Na + -K + -ATPase activity in microsomes derived from the gland tissue as well as in isolated acinar cells. Using a Ca 2+ imaging technique, we showed that activation of CB 1 Rs and CB 2 Rs alters [Ca 2+ ] cyt signalling in acinar cells by distinct pathways, involving Ca 2+ release from the endoplasmic reticulum (ER) and store-operated Ca 2+ entry (SOCE), respectively. Our data demonstrate the expression of CB 1 Rs and CB 2 Rs in acinar cells, and their involvement in the regulation of salivary gland functioning.
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