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Vasopressin receptor distribution in the liver controls calcium wave propagation and bile flow¹

Unité de Recherche U.442, Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, 91405 Orsay, France; and
^* Unité de Recherche U.469, Institut National de la Santé et de la Recherche Médicale, 34094 Montpellier, France

²Correspondence: Unité de Recherche U.442, Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, bât. 443, 91405 Orsay, France. E-mail: thierry.tordjmann{at}ibaic.u-psud.fr.

SPECIFIC AIMS

A gradient in hormone receptor density in the liver lobule is responsible for the spatial organization of hormone-induced intercellular calcium waves. In this study, we show that the loss of this receptor distribution abolished intercellular propagation of Ca²⁺ signals and address the hypothesis that these receptor-oriented waves can modulate bile flow.

PRINCIPAL FINDINGS

1. In vivo regulation of the V1a AVP receptor distribution in the liver lobule
We measured a 50% decrease in sinusoidal AVP concentration across the liver lobule due to a V1a receptor-specific uptake. This concentration gradient was not responsible for the gradual distribution of V1a receptors in the lobule, as demonstrated in rats treated with a V1a-specific antagonist and in Brattleboro rats deficient in AVP secretion. Although basal circulating AVP does not exert any major control on V1a AVP receptor expression in the liver lobule, increased AVP concentration (AVP-treated rats) elicits an abolition of the V1a receptor gradient in the lobule. This was demonstrated by binding experiments with radiolabeled antagonist of the V1a receptor, by Rnase protection assay with a V1a receptor-specific riboprobe, and by spectrofluorometry on hepatocytes isolated from periportal (PP) and perivenous (PV) areas of the lobule.

2. Abolition of the V1a AVP receptor gradient in the liver cell plate results in the suppression of intercellular Ca²⁺ waves
Because the in situ gradient in the number of V1a receptors across the liver cell plate is responsible for the directional propagation of intercellular Ca²⁺ waves, we analyzed AVP-elicited Ca²⁺ responses in hepatocytes isolated from rats lacking the V1a receptor gradient (AVP-treated rats). If the sensitivities of adjacent hepatocytes to AVP were similar, the propagation of any receptor-oriented Ca²⁺ wave would be precluded. In this situation, the latencies and oscillation frequencies (two kinetic variables fundamentally correlated with hepatocyte sensitivity) of AVP-induced Ca²⁺ responses would be similar in the adjacent cells. We therefore measured these two variables by videomicroscopy, using fura2-loaded hepatocytes (‘total’ hepatocyte population) from AVP-treated and control rats, after AVP stimulation. We then compared the variances of the two hepatocyte populations (AVP-treated and control). Figure 1a shows the distribution of hepatocyte latencies in response to AVP (0.3 nM) in a typical experiment (1 of 4). Latencies were more homogeneous in cells from AVP-treated rats than in cells from control rats, as assessed by Student’s t tests with the mean variances in each experiment (P<0.001, n=4). The mean variance was significantly smaller in the AVP group (Fig. 1a ). Similar results were obtained for [Ca²⁺]_i oscillation frequencies in response to AVP (Fig. 1b ), as expected given the linear correlation between these two kinetic variables.

View larger version (26K): [in this window] [in a new window]   Figure 1. Hepatocyte Ca2+ responses are more homogeneous in AVP-treated rats. Analysis with videomicroscopy. Hepatocytes from control and AVP-treated rats were fura2-loaded and their Ca2+ responses analyzed by videomicroscopy. The latency and oscillation frequency of each cell were measured after AVP stimulation (0.3 nM). A distribution graph of both the cell latencies (a) and oscillation frequencies (b) was plotted for each experiment. One typical experiment representative of 4 is shown in each panel. For these 4 experiments, 13 µg AVP was given over 24 h to the treated group. *Mean variances for AVP-treated and control rats were significantly different (P=0.01). Mean numbers of cells ± SE for each rat are given. c) Hepatocyte [Ca2+]i time course under AVP (0.1 nM) stimulation from control (upper traces) and AVP-treated (lower traces) rats. [Ca2+]i are expressed as arbitrary units of fluorescence. One triplet of cells representative of n multiplets in 4 independent experiments is shown in each group of rats. For control rats, latencies (time lapsed after agonist addition) were 53, 61, and 68 s, respectively, for cells 1, 2, and 3. For AVP-treated rats, latencies of the 3 cells were identical, 88 s.

Differences in AVP sensitivity between adjacent cells were also analyzed in hepatocyte doublets and triplets. Abolition of the sensitivity gradient in AVP-treated rats resulted in very small cell-to-cell differences in latency (intercellular delays) after AVP (0.1 nM) stimulation (2.9±0.6 s, n=29 in 3 experiments), these differences being significantly smaller than those in control rats (10.6±2.9 s, n=16 in 3 experiments) (P<0.01). Representative traces of AVP-elicited Ca²⁺ responses in fura2-loaded hepatocytes from AVP-treated and control rats are shown in Fig. 1c . As a confirmation of data from Fig. 1a , b , latencies of Ca²⁺ responses after AVP addition were longer in cells from AVP-treated rats. Also, oscillation frequencies in the three connected cells from control rats (0.98±0.1 osc/min) were significantly higher than in the AVP-treated rat triplet (0.27 osc/min in the three cells). These results suggest the almost synchronous firing of all hepatocytes in the plate on AVP stimulation in AVP-treated animals, tending to abolish intercellular Ca²⁺ waves.

3. Suppression of intercellular Ca²⁺ waves leads to impaired bile flow regulation
There has been speculation as to the role of intercellular Ca²⁺ waves in the regulation of bile flow. Peristaltic waves of canalicular contraction have indeed been reported to propagate from PV areas toward PP zones in live rats. It is tempting to correlate this observation with interhepatocyte Ca²⁺ waves because canalicular contraction is Ca²⁺ dependent. As oriented intercellular Ca²⁺ waves elicited by AVP are tightly correlated with the V1a receptor gradient along the cell plate, we looked at bile flow modulation by AVP in rats with and without a lobular gradient in hormone receptors. Basal bile flow was similar in livers isolated from control (1.41±0.08 µl/min/g liver, n=9) and 6.5 µg/24 h AVP-treated rats (1.35±0.09 µl/min/g liver, n=9). As previously reported, AVP stimulation in control rats induced a rapid, sharp increase in bile flow (66.4±8% of baseline) representing 0.51 ± 0.07 µl bile/g liver as calculated by integrating the area under the curve. It has been suggested that the early increase in bile flow corresponds to an outflow in response to AVP-induced canalicular contraction and that the subsequent cholestasis results from the persistence of this contraction at high AVP concentration. In AVP-treated rats, the early bile flow peak was significantly damped, its amplitude being reduced to 24.3 ± 3% of baseline levels and representing only 0.17 ± 0.03 µl bile/g liver (P<0.001). The subsequent inhibition of bile flow and the return to basal levels were similar in the two groups of rats. Several lines of evidence suggest that the decrease observed in the early choleresis resulted from changes in receptor distribution rather than a decrease in the total number of receptors. As the intensity of AVP stimulation decreases, so does the latency of the early bile flow peak increase, without any significant change in the induced choleresis as calculated by integrating areas under the curves in control rats. This shift in latency is not observed in AVP-treated rats.

Thus, changes in bile flow regulation in AVP-treated rat seem to be linked to loss of the receptor gradient rather than to a reduction in AVP stimulation. In this way, loss of the receptor gradient, implying loss of oriented intercellular Ca²⁺ waves, leads to impaired AVP-elicited bile flow modulation probably because canalicular peristalsis cannot occur directionally. To further link the early AVP-induced choleresis with receptor-oriented calcium waves, we measured bile flow in conditions in which intercellular calcium waves have been reported to be impaired, i.e., under ATP stimulation. Liver perfusion with 10 µM ATP has been reported to elicit nonoriented [Ca²⁺]_i increases that are randomly distributed over the lobule. ATP (10 µM) perfusion elicited a marked and transient cholestasis but, as expected, we did not detect any early choleresis. Thus, nondirectional Ca²⁺ signals appear to be functionally less efficient than spatially oriented Ca²⁺ waves to enhance bile flow.

CONCLUSIONS AND SIGNIFICANCE

These results show that a hormone can regulate the pattern of intra- and intercellular signals not only directly, through its concentration in the bloodstream, but also by controlling the distribution of the receptor in the tissue. This is of potential importance in the liver because intercellular Ca²⁺ waves are thought to be involved in several physiological processes, including bile flow regulation. At low circulating hormone concentrations, the receptor gradient across the lobule is preserved and unidirectional agonist-induced intercellular Ca²⁺ waves may drive canalicular peristalsis and increase bile flow. In contrast, if circulating hormone concentrations are high, the receptor gradient is lost because of preferential desensitization in the PV zone, and the lack of oriented intercellular Ca²⁺ waves may impair hormone-mediated bile flow regulation (Fig. 2 ). The pathophysiological significance of this phenomenon is unknown. However, in more general terms, the control of hormone receptor distribution in a tissue via agonist-mediated desensitization may determine whether intercellular signaling is switched on or off, thereby affecting the function regulated by the signal.

View larger version (43K): [in this window] [in a new window]   Figure 2. Schematic diagram of the hypothesized involvement of receptor-oriented intercellular Ca2+ waves in bile flow regulation. At low AVP concentration (upper panel), receptor-oriented intercellular Ca2+ waves result from a gradient in the number of surface hormone receptors along the hepatocyte plates. Agonist-induced Ca2+ responses initiate in the cell with the most hormone receptors. Successive firing of cells is spatially oriented by the gradient in agonist binding sites. Coordination between Ca2+ responses of adjacent cells is due to gap junctional transfer of a messenger (probably InsP3) and allows repetitive Ca2+ waves to occur. Intercellular unidirectional Ca2+ waves are thought to trigger waves of canalicular contraction. By elevating AVP concentration in a sustained manner (lower panel), the V1a receptor gradient is lost due to preferential desensitization of receptors in the PV area. A nearly simultaneous firing of adjacent hepatocytes resulting in a lack of intercellular Ca2+ waves is observed. Bile flow regulation is proposed to be impaired because of an alteration in wave-like propagation of canalicular contraction.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0659fje ; to cite this article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0659fje

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