Brigitte Berthon

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist

Calcium‐mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2‐loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline‐induced Ca2+ signals. Gap junction blockade with 18 α‐glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline‐induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell‐cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a ‘pacemaker‐like’ intercellular spread of Ca2+ waves.

Receptor‐oriented intercellular calcium waves evoked by vasopressin in rat hepatocytes

Agonist‐induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2‐loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D‐myo‐inositol 1,4,5‐trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4−. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin‐binding sites, vasopressin‐induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+‐dependent processes.

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

Arginine vasopressin elicits elaborate Ca2+ signals in the liver (intercellular Ca2+ waves), the functional implications of which are not understood. Waves propagate across hepatocyte plates following a lobular gradient in Via vasopressin receptor density. Here, we report that changes in this receptor distribution control Ca2+ 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 Ca2+ waves were impaired due to the equalization of Ca2+ responses in the various zones of the lobule. In the isolated perfused liver, the early increase in vasopressin‐induced 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 Ca2+ 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.

An improved digitonin‐collagenase perfusion technique for the isolation of periportal and perivenous hepatocytes from a single rat liver: Physiological implications for lobular heterogeneity

Morphological and functional heterogeneity of hepatocytes according to their position in the liver lobule has been known for many years. The digitonin‐collagenase perfusion technique is widely used to study hepatocyte heterogeneity and has yielded reliable data. However, with this procedure, periportal (PP) or perivenous (PV) hepatocytes are isolated from different livers, allowing only comparison between cell populations issued from two separate animals. To overcome this drawback, we have modified this technique by perfusing the two main rat liver lobes of a single animal in succession. The procedure involved alternate clamping of the median and the left lateral lobes, restricting digitonin infusion to one lobe via the portal vein, and to the other via the caudal vena cava. Lobe exclusion during digitonin perfusion, and zonal restriction of digitonin‐induced damage, were monitored using macroscopic and histological controls. We compared our results with previous data on PP and PV hepatocytes issued from two different livers using the conventional digitonin‐collagenase perfusion technique. First, we found that the cellular sensitivity to angiotensin II, a calcium‐mobilizing agonist, was 60% to 80% higher in PV than in PP hepatocytes, whereas, previously, no difference had been recorded. Second, we found that albumin messenger RNAs (mRNAs) were 35% more abundant in PP than in PV hepatocytes, whereas, previously, larger differences had been reported. Our results show that PP and PV hepatocytes may be isolated from a single liver using an improved digitonin‐collagenase perfusion technique. Furthermore, we suggest that zonal differences can be artificially masked or amplified when comparing PP and PV cell populations from two different livers, indicating that it is preferable to use a single liver for accurate zonal comparisons between hepatocytes.

Morphological characterisation of rat isolated hepatocyte multiplets

The stimulation of rat hepatocytes by Ca2+ mobilising agonists induces cytosolic free Ca2+ oscillations. We have recently reported that, in contrast to single cells, multiplets (doublet or triplet) of associated hepatocytes whose bile canaliculus is maintained, showed coordinated oscillations and that a Ca2+ wave propagated from one cell to another. In the present work, we have studied the cell polarity and junctional complexes of these hepatocyte multiplets using different antibodies specifically recognising protein markers of particular plasma membrane domains and cell junctions. Multiplets (30% of the cell preparation) were obtained by moderate collagenase digestion of rat hepatocytes.. After one hour adherence on collagen coated coverslips, only multiplets having bile canaliculus were studied. The lateral domain of the plasma membrane was visualized by a monoclonal antibody B 1 and the canalicular (apical) domain was studied by two different approaches: Iutilisation of an anti-DPP4 polyclonal antibody directed against dipeptidyl peptidase. an enzyme predominant in the apical domain of epithelial cells. 2specific labelling of F-actin by phalloidin (Bodipy 581/591) revealing a circle of F-a&n around the bile canaliculus. Two types of cell junction were also visualized: the tight junction (zonula occludens) decorated by an antibody directed against ZO-1, a high molecular weight polypeptide associated with tight junction, and the gap junction revealed by an anti-connexin 32 antibody These results obtained by different specific antibodies showed that, in contrast to single cells where molecules specific for cell polarity were distributed all over the cell surface, hepatocyte multiplets maintained their cell polarity and junctional complexes, The preservation of these structures are likely lo be essential to the Ca2+ wave propagation observed in these multiplets under hormonal stimulation.