Human myofibroblastic hepatic stellate cells express Ca(2+)-activated K(+) channels that modulate the effects of endothelin-1 and nitric oxide.

BACKGROUND/AIMS: High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK(Ca) channels and what their role in the regulation of HSCs contractility is. METHODS: The presence of BK(Ca) channels in HSCs was assessed by the patch-clamp technique. The functional role of BK(Ca) channels was investigated by measuring intracellular calcium concentration ([Ca(2+)](i)) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK(Ca) channels. RESULTS: BK(Ca) channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK(Ca) channel blocker, increased both the sustained phase of [Ca(2+)](i) elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK(Ca) channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK(Ca) channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca(2+)](i) increase and cell contraction. CONCLUSIONS: Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.

In vitro and in vivo activation of rat hepatic stellate cells results in de novo expression of L-type voltage-operated calcium channels.

Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblast-like cells, which develop contractile properties and contribute to increased resistance to blood flow. We investigated whether this phenotypic activation includes changes in the expression of L-type voltage-operated Ca2+ channels (VOCC), which mediate Ca2+ influx and regulate cell contraction in vascular cell types. Rat HSCs were studied in the quiescent phenotype and after their activation in vitro (cultured on plastic for 14 days) and in vivo (isolated from rats with CCl(4)-induced cirrhosis). Patch-clamp studies showed Ca2+ currents through L-type VOCC in HSCs activated both in vitro and in vivo, whereas no currents were detected in quiescent HSCs. Moreover, binding studies with (3)H-isradipine, a specific L-type VOCC antagonist, showed a large number of binding sites in activated HSCs, while no specific binding was found in quiescent HSCs. Finally, messenger RNA (mRNA) encoding L-type VOCC was not detected in quiescent HSCs as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis, whereas it was present in activated HSCs. Stimulation of L-type VOCC with KCl resulted in a marked increase in [Ca2+](i) followed by cell contraction in HSCs activated both in vitro and in vivo, whereas no effects were observed in quiescent HSCs. We conclude that the activation of HSCs is associated with up-regulation of L-type VOCC that mediate Ca2+ influx and cell contraction. These results may be relevant to the pathogenesis of portal hypertension.

Human hepatic stellate cells secrete adrenomedullin: potential autocrine factor in the regulation of cell contractility.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are perisinusoidal pericytes which have receptors for vasoactive factors, such as endothelin-1, which can regulate cell contractility in an autocrine manner. It is unknown whether human HSCs have receptors for and are able to synthesize the vasodilator peptide adrenomedullin (ADM), a peptide produced by most contractile cells. METHODS AND RESULTS: Stimulation of HSCs with ADM resulted in a dose-dependent raise in cAMP concentration (radioimmunoassay) and markedly blunted the endothelin-induced increase in [Ca2+]i and cell contraction, as assessed in cells loaded with fura-2 using a morphometric method. The existence of the receptor CRLR for ADM and their associated proteins RAMP-1 and RAMP-2 was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, activated human HSCs spontaneously secreted ADM in the culture medium in a time-dependent manner. ADM secretion was markedly enhanced by tumour necrosis factor-alpha and interleukin-1beta. Specific mRNA for ADM (RT-PCR and Northern blot) was detected in HSCs and increased after incubation of cells with cytokines. CONCLUSIONS: Human HSCs have functional receptors for ADM, the stimulation of which blunts the contractile effect of endothelin-1. Cultured human HSCs secrete ADM in baseline conditions. This secretion is markedly increased by cytokines. These results suggest that ADM can regulate HSCs' contractility in an autocrine manner.

Angiotensin II induces contraction and proliferation of human hepatic stellate cells.

BACKGROUND & AIMS: Circulating levels of angiotensin II (ANGII), a powerful vasoconstrictor factor, are frequently increased in chronic liver diseases. In these conditions, hepatic stellate cells (HSCs) proliferate and acquire contractile properties. This study investigated the presence of receptors for ANGII and the effects of ANGII in human HSCs activated in culture. METHODS: The presence of ANGII receptors was assessed by binding studies. The effects of ANGII on intracellular calcium concentration ([Ca(2+)](i)), cell contraction, and cell proliferation were also assessed. RESULTS: Binding studies showed the presence of ANGII receptors of the AT1 subtype. ANGII elicited a marked dose-dependent increase in [Ca(2+)](i) and cell contraction. Moreover, ANGII stimulated DNA synthesis and increased cell number. All these effects were totally blocked by losartan and reduced by nitric oxide donors or prostaglandin E(2). The effects of ANGII were barely detectable in quiescent cells (2 days in culture), suggesting that phenotypic transformation of HSCs is associated with a marked increase in the effects of ANGII. CONCLUSIONS: ANGII induces contraction and is mitogenic for human-activated HSCs by acting through AT1 receptors. These results suggest that activated HSCs are targets of the vasoconstrictor action of ANGII in the intrahepatic circulation.

Prolonged cholestasis after acute paraquat poisoning through skin absorption.

Ingestion of paraquat is the most common cause of fatal pesticide poisoning. Liver involvement in acute paraquat poisoning is self-limited and usually consists of cholestasis. However, long-term hepatic effects after paraquat exposition have not been described up to now, probably because of the high mortality rate of this acute poisoning. We report the case of an agricultural worker who developed persistent cholestasis after an episode of acute paraquat poisoning through skin absorption.

Atrial natriuretic peptide antagonizes endothelin-induced calcium increase and cell contraction in cultured human hepatic stellate cells.

Hepatic stellate cells (HSCs) participate in the regulation of hepatic microcirculation and have receptors for many vasoconstrictor factors. It is unknown whether HSCs have receptors for circulating vasodilators such as atrial natriuretic peptide (ANP). This study investigated the presence of ANP receptors in human HSCs and whether ANP antagonizes the effects of endothelin-1 in these cells. ANP receptors were assessed by binding and cross-linking studies, reverse-transcriptase polymerase chain reaction (PCR), and measuring intracellular cyclic guanosine monophosphate concentration. Intracellular calcium concentration ([Ca(2+)](i)) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method. Binding and cross-linking affinity experiments showed the existence of ANP receptors in human HSCs. PCR products with the expected length were obtained for guanylate cyclase A receptor, the physiological receptor of ANP, both in quiescent and activated human cells. ANP induced a dose-dependent increase in intracellular cyclic guanosine monophosphate concentration and blunted the increase in [Ca(2+)](i) elicited by endothelin-1. Most importantly, ANP markedly reduced cell contraction induced by endothelin-1. HSCs isolated from rats with carbon tetrachloride-induced cirrhosis showed a higher number of ANP receptors compared with HSCs isolated from normal rats, indicating that in vivo activation of HSCs is associated with an up-regulation of ANP receptors. These results indicate that human HSCs have receptors for ANP, the activation of which reduces the effects of endothelin-1 on [Ca(2+)](i) and cell contraction. ANP could participate in regulating the contractility of HSCs by antagonizing the effect of vasoconstrictors.

Contraction of human hepatic stellate cells activated in culture: a role for voltage-operated calcium channels.

BACKGROUND/AIMS: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca2+]i) and cell contractility. METHODS: Binding studies using [3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca2+]i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. RESULTS: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1+/-1.4x10(4) sites/cell with a Kd of 2.1+/-0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca2+]i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca2+]i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. CONCLUSION: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca2+]i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.