Neomycin was used to assess the involvement of Ins (1,4,5)P, in the CaZ+ release from the endoplasmic reticulum induced by the bile acid taurolithocholate.In saponin-penneabilized rat hepatocytes, neomycin via its ability to bind Ins (1,4,5)P, abolished the release of Ca*+ Induced by added Ins (1,4,5)P,. In contrast, it did not alter the Ca*+ release initiated by the bile acid. In intact cells, neomycin had no effect on the [Ca2+], rises promoted by taurolithocholate and vasopressin. It is suggested that the effect of taurolithocholate in liver is not mediated by Ins (1,4,5)P, but results from a primary action on endoplasmic reticulum.
The development of hormone-mediated Ca2+ signals was analysed in polarized doublets, triplets and quadruplets of rat hepatocytes by video imaging of fura2 fluorescence. These multicellular models showed dilated bile canaliculi, and gap junctions were observed by using an anti-connexin-32 antibody. They also showed highly organized Ca2+ signals in response to vasopressin or noradrenaline. Surprisingly, the primary rises in intracellular Ca2+ concentration ([Ca2+]i) did not start randomly from any cell of the multiplet. It originated invariably in the same hepatocyte (first-responding cell), and then was propagated in a sequential manner to the nearest connected cells (cell 2, then 3, in triplets; cell 2, 3, then 4 in quadruplets). The sequential activation of the cells appeared to be an intrinsic property of multiplets of rat hepatocytes. (1) In the continued presence of hormones, the same sequential order was observed up to six times, i.e. at each train of oscillations occurring between the cells. (2) The order of [Ca2+]i responses was modified neither by the repeated addition of hormones nor by the hormonal dose. (3) The mechanical disruption of an intermediate cell slowed down the speed of the propagation, suggesting a role of gap junctions in the rapidity of the sequential activation of cells. (4) The same multiplet could have a different first-responding cell for vasopressin or noradrenaline, suggesting a role of the hormonal receptors in the sequentiality of cell responses. It is postulated that a functional heterogeneity of hormonal receptors, and the presence of functional gap junctions, are involved in the existence of sequentially ordered hormone-mediated [Ca2+]i rises in the multiplets of rat hepatocytes.
The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.
Arginine vasopressin elicits elaborate Ca 2+ signals in the liver (intercellular Ca 2+ waves), the functional implications of which are not understood. Waves propagate across hepatocyte plates following a lobular gradient in V1a vasopressin receptor density. Here, we report that changes in this receptor distribution control Ca 2+ wave propagation and bile flow. Although basal circulating vasopressin levels do not play a major role in the regulation of V1a receptor expression, increases in vasopressin concentration within physiological limits for 24 h can abolish the lobular gradient in V1a receptor, as assessed by spectrofluorimetry, videomicroscopy, binding studies, and RNase protection assays. In animals in which the V1a receptor gradient was abolished, intercellular Ca 2+ waves were impaired due to the equalization of Ca 2+ responses in the various zones of the lobule. In the isolated perfused liver, the early increase in vasopressininduced bile flow observed in control rats was much smaller if the V1a receptor density gradient was abolished. These findings suggest that V1a vasopressin receptor distribution controls intercellular Ca 2+ wave propagation and bile flow. The control of hormone receptor distribution in a tissue by an agonist may turn the signaling and function of this agonist on or off.Key words: intercellular signaling • cellular • heterogeneity • receptor • density • bile flow • vasopressin rginine vasopressin (AVP), in addition to having well-known antidiuretic effects, is involved in the regulation of many other functions, including liver processes such as ureogenesis, glycogenolysis, and neoglucogenesis (1-3). In the rat, the physiological impact of AVP stimulation in the liver is still a matter of debate, despite the fact that hepatocyte is one of the cell types richest in V1a AVP receptors (4). AVP, by activating the V1a receptor, induces increases in intracellular Ca 2+ concentration ([Ca 2+ ] i ), which may propagate among hepatocytes as intercellular waves, the complex mechanisms of which are beginning to be understood (5-7). In freshly isolated multicellular systems of hepatocytes and in the intact perfused liver, oriented Ca 2+ waves start in one cell and propagate towards the other cells on AVP stimulation (5,6,(8)(9)(10)(11)(12). We have recently suggested that this propagation follows a A gradient in sensitivity to the agonist. Each hepatocyte has its own density of hormone receptors, which results in a slight shift in phase of agonist-induced [Ca 2+ ] i increases with respect to neighboring cells. Intercellular calcium waves thus appear to be receptor-oriented in hepatocytes, a configuration comparable with cell-cell triggering in cardiac pacemaker cells (6, 7). This heterogeneity of Ca 2+ mobilization in adjacent hepatocytes results from an in situ gradient in the number of hormone receptors across the liver cell plate, as has been shown for the V1a vasopressin receptor, the density of which declines from the perivenous (PV) zone to the periportal (PP) area (6,9,13). The functional...
In the present study, we investigated the possible role of external Ca2+ in the rise of the cytosolic Ca+ concentration induced by the monohydroxy bile acid taurolithocholate in isolated rat liver cells. The results showed that: (a) the bile acid promotes the same dose-dependent increase in the cytosolic Ca' concentration (half-maximal effect at 23 pM) in hepatocytes incubated in the presence of 1.2 mM Ca" or 6 pM Ca2+; (b) taurolithocholate is able to activate the Ca2 +-dependent glycogen phosphorylase a by 6.3-fold and 6.0-fold in high and low Ca2+ media, respectively; (c) ['4C]taurolithocholate influx is not affected by external Ca2+, and 45Ca2+ influx is not altered by taurolithocholate. These results establish that the effects of taurolithocholate on cell Ca2 + do not require extracellular Ca2+ and are consistent with the view that monohydroxy bile acids primarily release CaZ+ from the endoplasmic reticulum in the liver.In liver cells as in a variety of other tissues, several hormones or neurotransmitters mobilise Ca2 + from the endoplasmic reticulum by using the intracellular messenger inositol 1,4,5-trisphosphate (InsP,) [l, 21. Other natural molecules, such as the monohydroxy bile acid taurolithocholate, also rapidly release Ca2+ from the endoplasmic reticulum of rat hepatocytes, probably by another mechanism [3 -51. In intact hepatocytes, these bile acids increase the cytosolic free Ca2 [4,6] and stimulate Ca2+ efflux [4,7]. Since these effects were maintained in the presence of the Ca2 + chelator EGTA [4] and since bile acids mobilise the same pool as h s P 3 in saponin-treated hepatocytes [4, 51, we proposed that the primary site of the action of these molecules is the internal pool. In view of the importance of this compartment in the regulation of cytosolic Ca2+ and also as a source of internal Ca2+ required for a number of cell functions [I, 2, 8 In view of these differences, it was important to investigate whether the efficiency of monohydroxy bile acids in inducing Ca2 + release from the internal pool depended on experimental conditions. The results show that taurolithocholate is able to activate the Ca2 ' -dependent glycogen phosphorylase a in rat hepatocytes incubated in low- [Caz+] taurolithocholate and Ca2 +-transport systems are independent. They suggest that monohydroxy bile acids do not require external Ca2+ to exert their action on cell Ca2+. MATERIALS AND METHODS Materials Preparation of' hepatocytesHepatocytes were isolated from female Wistar rats and maintained (2 x 106/ml) in an Eagle's medium containing 116 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl,, 1.2 mM MgCl,, 0.92 mM NaH2P04, 25 mM NaHCO,, 15 mg/ml gelatin, vitamins and amino acids, and 5.6 mM glucose [4]. It was gassed with 95% 0 2 / 5 % C 0 2 (pH 7.4) at 37°C. Cell viability, as estimated by trypan blue exclusion, was always greater than 98% and remained stable for 4-5 h. Quin2-loading of the cellsCells (2 ml, 0.4 x 106/ml) were loaded with 50 KM quin2/ AM in Eagle's medium for 150 s, then washed by centrifugation (50 x g for l min...
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