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Stimulation of muscarinic cholinergic receptors on rat parotid acinar cells causes a rapid production of inositol phosphates, with the key metabolic event being the breakdown of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Here a high-performance liquid chromatographic technique was used to measure the effects of intracellular lithium ions on the amount of various inositol phosphates produced. When acini were stimulated maximally with acetylcholine (ACh), the sum of all inositol phosphates produced followed a monoexponential function with a production rate constant for Ins(1,4,5)P3 of 0.07 +/- 0.01 solidus/sec. The presence of 23 mM LiCl intracellularly reduced the production rate constant of Ins(1,4,5)P3 induced by ACh to 0.03 +/- 0.01 solidus/sec, resulting in a decrease in the Ins(1,4,5)P3 production as well as in the magnitude of the rise in the intracellular free Ca2+ concentration. The lithium ion (Li+) did not affect the rate of conversion of Ins(1,4,5)P3 to either inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate. The rate of the inositol phosphate production after the addition of the Ca2+ ionophore ionomycin was unaffected by intracellular Li+ (23 mM), which implies that the action of Li+ was at the muscarinic cholinergic receptor, on G-protein or on the interactions between G-proteins and phospholipase C. Thus, in the early events after receptor stimulation with ACh, Li+ causes a reduction in the concentration of the cellular messengers Ins(1,4,5)P3 and Ca2+.

Three genes encode the small-conductance Ca(2+)-activated K(+) channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC(50) value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis-quinolinium cyclophane UCL 1684 blocked hSK1 with an IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1.

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1-4 phosphate groups, resulting in release of Ca2+ from intracellular stores. Stimulation with the alpha-adrenergic agonist phenylephrine, however, causes a release of Ca2+ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca2+ rise was completely inhibited by 100 microM ryanodine. Adrenaline (causing activation of both alpha- and beta-adrenergic receptors) induces a Ca2+ release with inositol phosphate synthesis identical to that occurring in the beta-adrenergic response. Thus, the signalling pathway for alpha-adrenergic stimulation occurs via a path different from that which releases Ca2+ via muscarinic cholinergic and beta-adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5'-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] cause release of Ca2+ from intracellular stores. The Ca2+ release evoked by cADP-ribose, but not by Ins(1,4,5)P3, was abolished by 100 microM ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca2+ signalling. When the intracellular free Ca2+ concentration ([Ca2+]i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca2+]i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.

In permeabilized lacrimal acinar cells, cyclic ADP-ribose (cADP-ribose) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) release Ca2+ in a dose dependent manner from distinct thapsigargin-sensitive Ca2+ pools. Ryanodine specifically blocks the Ca2+ response to cADP-ribose, whereas heparin strongly reduces the response to Ins(1,4,5)P3 application. GTP causes a rapid Ca2+ release by a ryanodine- and heparin-insensitive mechanism and potentiates Ins(1,4,5)P3 but not cADP-ribose evoked Ca2+ release. It is estimated that cADP-ribose can release 16 mumol Ca2+/l cells, whereas Ins(1,4,5)P3 can mobilize 55 mumol Ca2+/l cells. The results suggest that cADP-ribose and Ins(1,4,5)P3 release Ca2+ from distinct internal stores and that a third Ca2+ pool exists which can selectively interact with the Ins(1,4,5)P3-sensitive Ca2+ store by a GTP-mediated process.

Stimulation of rat lacrimal acinar cells with ATP and acetylcholine (ACh) induced a rapid accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its degradation products, resulting in an initial release of Ca2+ from intracellular stores. However, after pretreating the acini with U73122 no increase in the intracellular free Ca2+ concentration ([Ca2+]i) or Ins(1,4,5)P3 production was observed. A short pretreatment with the phorbol ester 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) significantly attenuated the ATP- and ACh-induced increase in [Ca2+]i and overall inositol phosphate production. In contrast, staurosporine enhanced Ins(1,4,5)P3 and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] production and [Ca2+]i above control values in ATP- and ACh-stimulated cells. Stimulation of phospholipase C by ionomycin-evoked changes in [Ca2+]i were unaltered by pretreatment with staurosporine and PMA. The data show that a change in protein kinase C activity during cell stimulation affects the inositol phosphate metabolism and thereby the cellular Ca2+ signalling processes in lacrimal acinar cells.

The correlation between acetylcholine induced changes in the intracellular free, Ca2+ concentration ([Ca2+]i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca2+ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50-100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca2+]i and changes in the cellular Ins(1,4,5)P3 content, and it is concluded that in the individual cells Ins(1,4,5)P3 is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P3 of 2.83 x 10(-6) cm2/s (Allbritton et al., 1992, Science, 258, 1812-1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 microns where Ins(1,4,5)P3 is released in the center following a monoexponential function with a rate constant of 4 s-1. Assuming that Ins(1,4,5)P3 concentrations of 1 or 5% of the maximum value is able to release Ca2+, we calculated that Ca2+ waves can appear at a rate of 100 or 40 microns/s. The present data are consistent with Ins(1,4,5)P3 being a cellular messenger, that by diffusion, initiates the Ca2+ release from the cellular pools within the first fraction of a second.