Inflammatory macrophage to hepatocyte signals can be prevented by extracellular vesicle reprogramming.

Macrophage-derived extracellular vesicles (EVs) play key roles in intercellular communication. Within the liver, they have been linked to several inflammatory diseases including nonalcoholic fatty liver disease (NAFLD). In this study, we found that inflammatory macrophages cause injury to hepatocytes, in part by a cell-cell crosstalk phenomenon involving the secretion of EVs containing pro-inflammatory cargo. Incorporation of these inflammatory signals into EV requires the cleavage of the trafficking adaptor protein RILP, which, as previously shown, results from inflammasome-mediated caspase-1 activation. RILP cleavage can be blocked by overexpressing a dominant negative, non-cleavable form of RILP (ncRILP). EV preparations from ncRILP-expressing cells are, by themselves, sufficient to suppress inflammatory effects in hepatocytes. These results suggest that both direct RILP manipulation and/or supplying ncRILP-modified EVs could be used as a novel therapy for the treatment of inflammatory liver diseases.

Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western diet alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by single cell RNAseq and targeted KC ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor-mediated endocytosis and lipid metabolism, but most were predicted to be noninflammatory and antifibrotic with 1 minor KC cluster having a proinflammatory and extracellular matrix degradation gene signature. Infiltrating monocyte/macrophage clusters, in contrast, were predicted to be proinflammatory and profibrotic. In vivo, diphtheria toxin-based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK7, and Igf2bp3. Gene set enrichment analysis of whole-liver RNAseq from the KC-ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for the maintenance of hepatocyte differentiation. Infiltrating monocytes/macrophages are largely proinflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to the treatment of liver failure and fibrosis in ALD.

Rapid Development of Achalasia After SARS-CoV-2 Infection: Polymerase Chain Reaction Analysis of Esophageal Muscle Tissue.

INTRODUCTION: Achalasia has been linked to viruses. We have observed cases of rapid-developing achalasia post-coronavirus disease 2019 (COVID-19). METHODS: We aimed to prospectively evaluate esophageal muscle for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from patients with rapid-onset achalasia post-COVID-19 and compare them with achalasia predating COVID-19 and achalasia with no COVID-19. RESULTS: Compared with long-standing achalasia predating COVID-19 and long-standing achalasia with no COVID-19, the subjects with achalasia post-COVID-19 had significantly higher levels of messenger RNA for the SARS-CoV-2 nucleocapsid (N) protein, which correlated with a significant increase in the inflammatory markers NOD-like receptor family pyrin domain-containing 3 and tumor necrosis factor. DISCUSSION: SARS-CoV-2, the virus responsible for COVID-19, is a possible trigger for achalasia.

Hepatitis C virus infection increases autophagosome stability by suppressing lysosomal fusion through an Arl8b-dependent mechanism.

Autophagy is a conserved cellular process involving intracellular membrane trafficking and degradation. Pathogens, including hepatitis C virus (HCV), often exploit this process to promote their own survival. The aim of this study was to determine the mechanism by which HCV increases steady-state autophagosome numbers while simultaneously inhibiting flux through the autophagic pathway. Using the lysosomal inhibitor bafilomycin A1, we showed that HCV-induced alterations in autophagy result from a blockage of autophagosome degradation rather than an increase in autophagosome generation. In HCV-infected cells, lysosome function was normal, but a tandem RFP-GFP-LC3 failed to reach the lysosome even under conditions that activate autophagy. Autophagosomes and lysosomes isolated from HCV-infected cells were able to fuse with each other normally in vitro, suggesting that the cellular fusion defect resulted from trafficking rather than an inability of vesicles to fuse. Arl8b is an Arf-like GTPase that specifically localizes to lysosomes and plays a role in autophagic flux through its effect on lysosomal positioning. At basal levels, Arl8b was primarily found in a perinuclear localization and co-localized with LC3-positive autophagosomes. HCV infection increased the level of Arl8b 3-fold and redistributed Arl8b to a more diffuse, peripheral pattern that failed to co-localize with LC3. Knockdown of Arl8b in HCV-infected cells restored autophagosome-lysosome fusion and autophagic flux to levels seen in control cells. Thus, HCV suppresses autophagic flux and increases the steady-state levels of autophagosomes by increasing the expression of Arl8b, which repositions lysosomes and prevents their fusion with autophagosomes.

Hepatitis C virus promotes virion secretion through cleavage of the Rab7 adaptor protein RILP.

Hepatitis C virus (HCV) is an enveloped RNA virus that modifies intracellular trafficking processes. The mechanisms that HCV and other viruses use to modify these events are poorly understood. In this study, we observed that two different RNA viruses, HCV and Sendai, cause inhibition of ras-related protein Rab-7 (Rab7)-dependent endosome-lysosome fusion. In both cases, viral infection causes cleavage of the Rab7 adaptor protein RILP (Rab interacting lysosomal protein), which is responsible for linking Rab7 vesicles to dynein motor complexes. RILP cleavage results in the generation of a cleaved RILP fragment (cRILP) missing the N terminus of the molecule. Although RILP localizes in a perinuclear fashion, cRILP moves to the cell periphery. Both knockdown of RILP and expression of cRILP reproduced the HCV-induced trafficking defect, and restoring full-length RILP reversed the trafficking effects of virus. For the first 3 d after electroporation of HCV RNA, intracellular virus predominates over secreted virus, but the quantity of intracellular virus then rapidly declines as secreted virus dominates. The transition from the intracellular-predominant to the secretion-predominant phenotype corresponds to the time course of cRILP generation. Expressing cRILP directly prevents intracellular virus accumulation at early times without affecting net virus production. The ability of cRILP to promote virus secretion could be prevented by a kinesin inhibitor. HCV thus modifies cellular trafficking by cleaving RILP, which serves to redirect Rab7-containing vesicles to a kinesin-dependent trafficking mode promoting virion secretion. Cleavage of a Rab adaptor protein is thus a mechanism by which viruses modify trafficking patterns of infected cells.

Caspase-1 regulates cellular trafficking via cleavage of the Rab7 adaptor protein RILP.

Intracellular trafficking is a tightly regulated cellular process, mediated in part by Rab GTPases and their corresponding effector proteins. Viruses have evolved mechanisms to hijack these processes to promote their lifecycles. Here we describe a mechanism by which cleavage of the Rab7 adaptor protein, RILP (Rab interacting lysosomal protein) is induced by viral infection. We report that RILP is directly cleaved by caspase-1 and we have identified a novel caspase-1 recognition site at aspartic acid 75 within the RILP sequence. Alanine substitution at D75 blocks caspase-1-mediated RILP cleavage. Full-length RILP localizes in a tight vesicular structure near the perinuclear region while the cleaved form of RILP re-distributes throughout the cytoplasm. However, cleavage alone was insufficient to re-localize RILP to the cellular periphery and re-localization required specific phosphorylation events near the caspase-1 recognition site. The combination of cleavage and phosphorylation were both needed for release from the dynein component p150Glued and redistribution of CD63+ve intracellular vesicles.

TRIM25 dictates selective miRNA loading into extracellular vesicles during inflammation.

Extracellular vesicles (EVs) such as exosomes are loaded with specific biomolecules in order to perform cell-to-cell communication. Understanding the mechanism of selective cargo loading is important to better understand the physiological and pathological function of EVs. Here we describe a novel target of the E3 ligase TRIM25 and show that inflammation-mediated EV loading of the RNA binding protein FMR1 and its associated microRNA, miR-155, is promoted by TRIM25-mediated K63-ubiquitination of FMR1. This ubiquitination promotes an interaction between FMR1 and the EV loading machinery via the cleavage of the trafficking adaptor protein RILP. These interactions are lost when TRIM25 is knocked down. Loss of TRIM25 also prevents the loading of both FMR1 and miR-155. These findings suggest that inflammation-mediated loading of FMR1 and its associated microRNAs into the EV are dependent on K63-ubiquitination by TRIM25 and provide novel insights and tools to manipulate EV biogenesis for therapeutic benefit.

Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+)) conductance in vesicles and was able to rapidly equilibrate H(+) gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H(+) channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H(+) permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process.

Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel.

UNLABELLED: The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. CONCLUSION: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.

Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.

Fas receptor is a member of the tumor necrosis factor-alpha family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum-mediated calcium release in a mechanism dependent on phospholipase C-gamma1 (PLC-gamma1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-gamma1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-gamma1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.

The RNA binding protein FMR1 controls selective exosomal miRNA cargo loading during inflammation.

Cells respond to inflammatory disease states by releasing exosomes containing highly specific protein and RNA cargos, but how inflammation alters cargo specificity and secretion of exosomes is unknown. We show that increases in exosome secretion induced by either viral infection or LPS/ATP exposure result from inflammasome activation and subsequent caspase-1-dependent cleavage of the trafficking adaptor protein RILP. This cleaved form of RILP promotes the movement of multivesicular bodies toward the cell periphery and induces selective exosomal miRNA cargo loading. We have identified a common short sequence motif present in miRNAs that are selectively loaded into exosomes after RILP cleavage. This motif binds the RNA binding protein FMR1 and directs miRNA loading into exosomes via interaction with components of the ESCRT (endosomal sorting complex required for transport) pathway. These results indicate that inflammasome-mediated RILP cleavage, and sequence-specific interactions between miRNAs and FMR1, play a significant role in exosome cargo loading and enhanced secretion during cellular inflammatory responses.

SIRT7 regulates hepatocellular carcinoma response to therapy by altering the p53-dependent cell death pathway.

BACKGROUND: Optimal therapeutic strategies for hepatocellular carcinoma (HCC) patients are still challenging due to the high recurrence rate after surgical resection and chemotherapy resistance. Growing evidence shows that genetic and epigenetic alterations are involved in HCC progression and resistance to therapy, however the molecular mechanisms underlying resistance to therapy have not been fully understood. METHODS: Expression of SIRT7 in 17 paired paraffin-embedded HCC tissues and adjacent nontumoral liver tissues was examined by immunohistochemistry and Western blot. The mRNA expression of SIRT7 in 20 paired frozen HCC tissues and adjacent nontumoral liver tissues was analyzed by quantitative RT-PCR. The biologic consequences of overexpression and knockdown of SIRT7 in HCC therapy sensitivity were studied in vitro and in vivo. Interaction between SIRT7 and p53 were studied in HCC cell lines. RESULTS: SIRT7 expression was frequently upregulated in clinical HCC samples, and its expression was highly associated with TACE-resistance and poor survival (P = 0.008.) Depletion of SIRT7 from multiple liver cancer cell lines significantly increased doxorubicin toxicity while overexpression of SIRT7 largely abolished doxorubicin induced apoptosis. At the molecular level, we observed that SIRT7 interacts with and induces deacetylation of p53 at lysines 320 and 373. Deacetylated p53 showed significantly less affinity for the NOXA promoter and its transcription. In mouse xenografts, SIRT7 suppression increased doxorubicin induced p53 activation, inhibited tumor growth and induced apoptosis. CONCLUSION: The newly identified SIRT7-p53-NOXA axis partially illustrates the molecular mechanism of HCC resistance to therapy and represents a novel potential therapeutic target for HCC treatment.

Xenoestrogens are potent activators of nongenomic estrogenic responses.

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E(2)) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-alpha on pituitary cells, and can provoke Ca(2+) influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E(2). It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.

Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes and prolactin release in GH3/B6 pituitary tumor cells.

Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 versus several XEs representing organochlorine pesticides (dieldrin, endosulfan, o',p'-dichlorodiphenylethylene), plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-alpha. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca2+ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca2+ elevation. Removing Ca2+ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca2+ changes, as did 10 microM nifedipine. This suggests that XEs mediate their actions via voltage-dependent L-type Ca2+ channels in the plasma membrane. None of the Ca2+ fluxes came from intracellular Ca2+ stores. E2 and each XE also caused unique time- and concentration-dependent patterns of prolactin (PRL) secretion that were largely complete within 3 min of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca2+ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular CCa2+ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies that could explain their abilities to disrupt endocrine functions.

Signaling from the membrane via membrane estrogen receptor-alpha: estrogens, xenoestrogens, and phytoestrogens.

Estrogen mimetics in the environment and in foods can have important consequences for endocrine functions. When previously examined for action via genomic steroid signaling mechanisms, most of these compounds were found to be very weak agonists. We have instead tested their actions via several membrane-initiated signaling mechanisms in GH3/B6 pituitary tumor cells extensively selected for high (responsive) or low (nonresponsive) expression of the membrane version of estrogen receptor-alpha (mERalpha). We found many estrogen mimetic compounds to be potently active in our quantitative extracellular-regulated kinase (ERK) activation assays, to increase cellular Ca++ levels, and to cause rapid prolactin release. However, these compounds may activate one or both mechanisms with different potencies. For instance, some compounds activate ERKs in both pM and nM concentration ranges, while others are only active at nM and higher concentrations. Compounds also show great differences in their temporal activation patterns. While estradiol causes a bimodal time-dependent ERK activation (peaking at both 3 and 30 min), most estrogen mimetics cause either an early phase activation, a late phase activation, or an early sustained activation. One xenoestrogen known to be a relatively potent activator of estrogen response element-mediated actions (bisphenol A) is inactive as an ERK activator, and only a modest inducer of Ca++ levels and prolactin release. Many different signaling machineries culminate in ERK activation, and xenoestrogens differentially affect various pathways. Clearly individual xenoestrogens must be individually investigated for their differing abilities to activate distinct membrane-initiated signal cascades that lead to a variety of cellular functions.

Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I.

UNLABELLED: Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERß and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, ß, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/ß antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. CONCLUSIONS: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.

Dissecting the dynamic turnover of GFP-LC3 in the autolysosome.

Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.

Mechanisms of membrane estrogen receptor-alpha-mediated rapid stimulation of Ca2+ levels and prolactin release in a pituitary cell line.

The role of membrane estrogen receptor-alpha (mERalpha) in rapid nongenomic responses to 17beta-estradiol (E(2)) was tested in sublines of GH3/B6 rat prolactinoma cells selected for high (GH3/B6/F10) and low (GH3/B6/D9) mERalpha expression. E(2) elicited rapid, concentration-dependent intracellular Ca(2+) concentration ([Ca(2+)](i)) increases in the F10 subline. Lack of inhibition by thapsigargin depletion of intracellular Ca(2+) pools, together with abrogation of the response in Ca(2+)-free medium, suggested an extracellular source of Ca(2+) for this response. The participation of voltage-dependent channels in the E(2)-induced [Ca(2+)](i) increase was confirmed by the specific L-type Ca(2+) channel inhibitor nifedipine. For comparison, the D9 mERalpha-depleted subline was insensitive to steroid action via this signaling mechanism. [Ca(2+)](i) elevation was correlated with prolactin (PRL) release in the F10 cell line in as little as 3 min. E(2) caused a much higher PRL release than KCl treatment (which caused maximal Ca(2+) elevation), suggesting that secretion was also controlled by additional mechanisms. Participation of mERalpha in these effects was confirmed by the ability of E(2)-peroxidase (a cell-impermeable analog of E(2)) to cause these responses, blockage of the responses with the ER antagonist ICI 182 780, and the inability of the E(2) stereoisomer 17alpha-E(2) to elicit a response. Thus rapid exocytosis of PRL is regulated in these cells by mERalpha signaling to specific Ca(2+) channels utilizing extracellular Ca(2+) sources and additional signaling mechanisms.