B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.     

Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin

Ferroportin 1 (FPN1) is transmembrane protein involved in iron homeostasis. In the duodenum, FPN1 localizes to the basolateral surface of enterocytes where it appears to export iron out of the cell and into the portal circulation. FPN1 is also abundantly expressed in reticuloendothelial macrophages of the liver, spleen, and bone marrow, suggesting that this protein serves as an iron exporter in cells that recycle iron from senescent red blood cells. To directly test the hypothesis that FPN1 functions in the export of iron after erythrophagocytosis, FPN1 was stably expressed in J774 mouse macrophages by using retroviral transduction, and release of 59Fe after phagocytosis of 59Fe-labeled rat erythrocytes was measured. J774 cells overexpressing FPN1 released 70% more 59Fe after erythrophagocytosis than control cells, consistent with a role in the recycling of iron from senescent red cells. Treatment of cells with the peptide hormone hepcidin, a systemic regulator of iron metabolism, dramatically decreased FPN1 protein levels and significantly reduced the efflux of 59Fe after erythrophagocytosis. Subsequent fractionation of the total released 59Fe into heme and nonheme compounds revealed that hepcidin treatment reduced the release of nonheme 59Fe by 50% and 25% from control and FPN1-overexpressing cells, respectively, but did not diminish efflux of 59Fe-heme. We conclude that FPN1 is directly involved in the export of iron during erythrocyte-iron recycling by macrophages.     

Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.     

Distinct XPPX sequence motifs induce ribosome stalling,which is rescued by the translation elongation factor EF-P

Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.Protein synthesis in the cell occurs on macromolecular machines called ribosomes. The ribosome synthesizes polypeptide chains by providing a platform upon which peptide-bond formation can occur between a peptidyl-tRNA located at the ribosomal P-site and an aminoacyl-tRNA in the A-site. However, the ribosome cannot form peptide bonds between all amino acids with the same efficiency; this is exemplified by the amino acid proline, which has an imino group instead of a primary amino group in other amino acids. Proline has been shown to be a particularly poor substrate for peptide-bond formation, both as a donor in the P-site and as an acceptor in the A-site (1–4). In fact, ribosomes stall when attempting to incorporate three or more consecutive proline residues (PPP) into the polypeptide chain (5–7). In this case, ribosome stalling results from the slow rate of peptide-bond formation between the peptidyl-Pro-Pro-tRNA located in the P-site and the Pro-tRNA in the A-site (6). In bacteria, the translational arrest is relieved by the translation elongation factor P (EF-P), which binds to the stalled ribosomes and stimulates peptide bond formation (5, 6). In Escherichia coli, EF-P is posttranslationally modified by YjeA, YjeK, and YfcM (EpmA, EpmB, and EpmC) (8–10) and the resulting lysinylation modification has been shown to be critical for the rescue activity of EF-P in vivo and in vitro (5, 6). EF-P homologs exist in all archaea and eukaryotes, termed aIF-5A and eIF-5A, respectively (11). Yeast eIF-5A has recently been shown to also rescue translational stalling at polyproline-stretches (12). Like EF-P, a/eIF-5A is also posttranslationally modified, but via hypusinylation rather than lysinylation (11). In addition to PPP, PPG triplets also induce ribosome stalling in bacteria, which is rescued by EF-P (6). Moreover, a recent proteomics study identified APP, YIRYIR, and GSCGPG motifs as conferring EF-P dependent translation (13). Ribosome stalling has also been observed at PPA, PPD, PPE, PPN, PPW, APP, and WPP triplets during in vitro translation (7), however it was not shown whether EF-P can relieve the translational arrest at these triplets. Ribosome profiling data indicate ribosome accumulation at PPP, PPG as well as PPD and PPE triplets, despite the profiling being performed with wild-type cells containing EF-P or eIF-5A (7, 14, 15).To address the role of EF-P during translation in E. coli in vivo, we used SILAC (stable isotope labeling by amino acids in cell culture) coupled with high-resolution mass spectrometry (MS) to monitor the changes in expression levels of proteins in E. coli strains lacking either the efp gene, or one of the genes (yjeK, yjeA, yfcM) encoding the modification enzymes, relative to the parental wild-type E. coli strain. We found that in the absence of EF-P, YjeA, or YjeK, the majority of PPP-containing proteins are strongly down-regulated, whereas only specific subsets of XPP- and PPX-containing proteins are down-regulated. A systematic analysis of each of the 39 XPP/PPX combinations (where X means any amino acid) reveals the hierarchy of EF-P dependence. Moreover, we show in vitro and in vivo that the combinations of strong XPP with strong PPX motifs lead to XPPX quadruplets with the strongest effects, which are nevertheless efficiently relieved by EF-P. Collectively, our findings broaden the substrate range for EF-P activity from ∼100 PPP-containing proteins in E. coli to encompass the >1,300 additional XPPX-containing proteins. Eukaryotic proteomes, such as that of Homo sapiens contain >7,000 PPP-containing proteins and >15,000 XPPX-containing proteins that are all potential substrates for eIF-5A.     

Action control is mediated by prefrontal BDNF and glucocorticoid receptor binding

Stressor exposure biases decision-making strategies from those based on the relationship between actions and their consequences to others restricted by stimulus-response associations. Chronic stressor exposure also desensitizes glucocorticoid receptors (GR) and diminishes motivation to acquire food reinforcement, although causal relationships are largely not established. We show that a history of chronic exposure to the GR ligand corticosterone or acute posttraining GR blockade with RU38486 makes rodents less able to perform actions based on their consequences. Thus, optimal GR binding is necessary for the consolidation of new response-outcome learning. In contrast, medial prefrontal (but not striatal) BDNF can account for stress-related amotivation, in that selective medial prefrontal cortical Bdnf knockdown decreases break-point ratios in a progressive-ratio task. Knockdown also increases vulnerability to RU38486. Despite the role of BDNF in dendritic spine reorganization, deep-layer spine remodeling does not obviously parallel progressive-ratio response patterns, but treatment with the Na⁺-channel inhibitor riluzole reverses corticosteroid-induced motivational deficits and restores prefrontal BDNF expression after corticosterone. We argue that when prefrontal neurotrophin systems are compromised, and GR-mediated hypothalamic-pituitary-adrenal axis feedback is desensitized (as in the case of chronic stress hormone exposure), amotivation and inflexible maladaptive response strategies that contribute to stress-related mood disorders result.     

Visual detection is gated by attending for action: evidence from hemispatial neglect

We report observations in patients with visual extinction demonstrating that detection of visual events is gated by attention at the level of processing at which a stimulus is selected for action. In one experiment, three patients reported the identity of numerical words and digits presented either in the ipsilesional field, the contralesional field, or both fields. On the critical bilateral trials, extinction was greater when the competing items shared the same meaning and response, regardless of whether the items were visually different (e.g., ONE + 1), or identical (e.g., 1 + 1). A fourth patient was tested in a second experiment in which the competing items on bilateral trials were either different (e.g., ONE + TWO), identical (e.g., ONE + ONE) or homophones that were visually and semantically different but shared the same response (e.g., ONE + WON). Homophones and identical items caused similar extinction with less extinction occurring on different item trials.     

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18⁺ cells but failed to further differentiate into K14⁺ cells (epidermis/limbus) or K3/K12⁺ cells (corneal epithelium). APR-246 (PRIMA-1^MET), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.     

Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin

Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson's disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. Here we show that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, we find that, in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila.     

An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele.

If deprived of wild-type p53 function, the body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.     

Calcitriol-induced apoptosis in LNCaP cells is blocked by overexpression of Bcl-2

While the role of vitamin D in bone and mineral metabolism has been investigated extensively, the role of the vitamin D receptor in other tissues is less well understood. 1,25-Dihydroxyvitamin D3 (calcitriol) can act as a differentiating agent in normal tissues and can inhibit the growth of many cancer cell lines including LNCaP prostate cancer cells. We have shown previously that calcitriol causes LNCaP cell accumulation in the G0/G1 phase of the cell cycle. In this study, we demonstrate that calcitriol also induces apoptosis of LNCaP cells. The calcitriol-induced apoptosis is accompanied by a down-regulation of Bcl-2 and Bcl-X(L) proteins, both of which protect cells from undergoing apoptosis. Other proteins important in apoptotic control, Bax, Mcl-1, and Bcl-X(S), are unaffected by calcitriol treatment. We find that overexpression of Bcl-2 blocks calcitriol-induced apoptosis and reduces, but does not eliminate, calcitriol-induced growth inhibition. We conclude that both regulation of cell cycle and the apoptotic pathway are involved in calcitriol action in prostate cancer cells.     

Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin

Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation.Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family of secreted factors, is known to play important regulatory roles in neuronal survival and differentiation, synaptic development and plasticity, as well as many cognitive functions (1, 2). The findings that the synthesis and secretion of neurotrophins are regulated by neuronal activity prompted the suggestion that neurotrophins may regulate activity-dependent neural plasticity in the brain (3). Indeed, there is now substantial evidence indicating that activity-induced BDNF secretion at glutamatergic synapses is essential for long-term potentiation (LTP) (4), a cellular substrate for the learning and memory functions of neural circuits.The BDNF protein is first synthesized in the endoplasmic reticulum as a precursor protein, prepro-BDNF, which is then converted to pro-BDNF by removal of the signal peptide and further cleaved to generate the mature BDNF (5). Immunostaining and electron microscope studies using specific antibodies to the pro- and mature form of BDNF showed that pro-BDNF is colocalized with mature BDNF in secretory granules in presynaptic axon terminals (6), suggesting that the cleavage may occur in the secretory granule. However, under some experimental conditions, the processing of pro-BDNF into mature BDNF may occur extracellularly (7, 8). The secretory granule containing BDNF and pro-BDNF could undergo exocytosis upon neuronal excitation, as readily demonstrated in cell cultures using ELISA or fluorescent protein-tagged BDNF expressed in the neuron (9, 10). Besides secretory granules, neurotrophins within neuronal cytoplasm could also reside in endosomal compartments, resulting from endocytic uptake of extracellular neurotrophins secreted by the neuron itself or other nearby cells. Initially discovered as factors derived by target tissues, neurotrophins exert their actions via binding to neuronal surface receptors, including tropomyosin related kinase B (TrkB) and pan-neurotrophin receptor p75 (11). Neurotrophin binding to its receptor leads to cytoplasmic signaling as well as internalization of the neurotrophin-receptor complexes. These endocytosed neurotrophin-receptor complexes remain active in the form of “signaling endosomes” that could be transported over long distances within neuronal cytoplasm to exert its regulatory functions within the neuron (12–15). In this study, we have examined the possibility that these endosomes may undergo activity-dependent exocytosis at postsynaptic dendrites, thus providing an additional source of synaptic BDNF.To mark endosomes containing BDNF via the endocytic pathway, it is necessary to monitor BDNF trafficking in neurons. Although YFP-tagged BDNF has been used to study internalization of exogenous BDNF (16), such fluorescent protein-labeled BDNF was not suitable for real-time tracking of BDNF-containing endosomes at a high spatiotemporal resolution. In this study, we used BDNF linked to quantum dots (QDs), which are fluorescent nanoparticles with excellent photostability (17) and could be tracked in live cells with high signal-to-noise ratio and over unprecedented duration. This method has been used to examine endocytic recycling of synaptic vesicles (18) and axonal transport of endosomes containing neurotrophins (19, 20). In this study, we used time-lapse imaging of BDNF-QDs within cultured hippocampal neurons to monitor intracellular transport and localization of these endosomes. Furthermore, the sudden disappearance of cytoplasmic QD fluorescence in a solution containing fluorescence quencher was used to indicate the exocytosis of QD-containing endosomes. Previous studies have shown that extracellular false transmitters, soluble fluorescent markers, and membrane-bound fluorescent lipid dyes could be loaded into endosomes, which undergo exocytosis upon membrane depolarization (21–25). However, whether endosomes formed by receptor-mediated endocytosis is similarly regulated by activity remains unclear. Furthermore, the Ca²⁺-dependence and the kinetics of exocytosis of different endosomal vesicle populations may be differentially regulated by distinct vesicle-associated proteins.In the present study, we have explored the role of synaptotagmin (Syt) and complexin (Cpx) in regulating activity-induced exocytosis of BDNF-containing endosomes. As a universal cofactor in all Ca²⁺-triggered vesicular fusion reactions that have been examined (26), Cpx is known to serve both activating and clamping functions for vesicular exocytosis, by interacting with the Ca²⁺ sensor Syt and the assembled SNARE complexes at the plasma membrane (27). Various isoforms of Syt play distinct regulatory roles in various types of neurosecretion, presumably via their differential Ca²⁺ sensitivity. By manipulating the expression of various Syt and Cpx isoforms in cultured hippocampal neurons, we found that Syt6 and Cpx1/2 play essential regulatory roles in activity-dependent exocytosis of BDNF-containing endosomes. These results support the notion that BDNF-containing endosomes may serve as a source of extracellular BDNF for activity-dependent synaptic modulation and that Syt6 specifically regulates the exocytosis of BDNF-containing endosomes.     

Endocrine effects of relaxin overexpression in mice

Relaxin is a small peptide hormone with a variety of biological functions. To investigate the systemic endocrine effects of relaxin, we produced mice with transgenic overexpression of the Rln1 gene, Tg(Rln1), driven by rat insulin 2 promoter. The expression of relaxin was detected in the pancreas of the transgenic animals. An analysis of the sera from the transgenic animals revealed at least 20-fold elevation of the level of bioactive relaxin. Transgenic animals had normal viability and fertility in both sexes. Transgenic overexpression of Rln1 did not rescue the undescended testis phenotype in Insl3-deficient males, suggesting that in vivo relaxin does not interact with the insulin-like 3 factor receptor, leucine-rich repeats-containing G protein-coupled receptor 8, Lgr8. Phenotypically, the excess of relaxin resulted in hypertrophic nipple development in virgin female mice. Deletion of the relaxin receptor, leucine-rich repeats-containing G protein-coupled receptor 7, Lgr7, in Tg(Rln1) animals abrogated the development of enlarged nipples in females, indicating that relaxin exerts its effect through Lgr7 alone. The levels of previously defined targets of relaxin signaling, such as matrix metalloproteinases 2 and 9, vascular endothelial growth factor, or nitric oxide, were similar in the sera of the transgenic and wild-type mice. However, the total plasma protein concentration in male Tg(Rln1) mice was lower than that in control animals. The livers of male Tg(Rln1) mice exhibited significantly higher hydroxyproline content, indicative of increased collagen deposition. Our results indicate that relaxin overexpression causes gender-specific changes in liver collagen metabolism.     

Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality

Erythropoietin (Epo) and its receptor (EpoR) are indispensable to erythropoiesis. Although roles besides angiogenesis, such as neuroprotection and heart development, have been reported for the Epo-EpoR system, the precise contribution of Epo-EpoR to these nonhematopoietic tissues requires clarification. Exploiting a GATA-1 minigene cassette with hematopoietic regulatory domains, we established 2 lines of transgene-rescued EpoR-null mutant mice expressing EpoR exclusively in the hematopoietic lineage. Surprisingly, despite the lack of EpoR expression in nonhematopoietic tissues, these mice develop normally and are fertile. As such, we could exploit them for analyzing the roles of the Epo-EpoR system in adult hematopoiesis and in nonhematopoietic tissues. These rescued lines showed a differential level of EpoR expression in erythroid cells; one expressed approximately 40%, and the other expressed 120% of the wild-type EpoR level. A colony formation assay showed that erythroid progenitors in the 2 mutant lines exhibit distinct sensitivity to Epo. The circulating Epo level was much higher in the transgenic line with a lower EpoR expression. In response to induced anemia, the plasma Epo concentrations increased in both lines. Notably, the timing of the peak of plasma Epo concentration was delayed in both lines of rescued mice compared with wild type, suggesting that, in wild-type mice, nonhematopoietic EpoR contributes to the regulation of plasma Epo concentration. We thus conclude that nonhematopoietic expression of EpoR is dispensable to normal mouse development and that the expression level of EpoR regulates erythropoiesis by controlling the sensitivity of erythroid progenitors to Epo.     

Aldosterone secretion by the rat adrenal cortex is stimulated by the activation of protease-activated receptor 1

Stimulation of aldosterone by a serine protease, trypsin, was first reported in 1982, although the mechanism of this effect was unclear. Recently, a family of protease-activated receptors (PARs) has been described and four members of the family characterised and cloned, including the previously recognised thrombin receptor. This study investigated whether PARs mediate the action of trypsin on aldosterone secretion. Using intact rat adrenal capsular tissue, thrombin was found to increase aldosterone secretion, and the effects of trypsin on aldosterone secretion were confirmed. Both trypsin and thrombin were shown to activate phospholipase C, as measured by an increase in inositol triphosphate turnover by adrenal capsular tissue. It was also shown that U73122, a phospholipase C inhibitor, attenuated the aldosterone response to trypsin. These effects were consistent with the activation of a PAR. Northern blot analysis revealed the presence of mRNA encoding PAR-1, but not PARs-2, -3 or -4 in the adrenal capsule/zona glomerulosa. Messenger RNA encoding PAR-1 was increased by dietary sodium depletion, consistent with previous reports of an increased response to trypsin after sodium depletion. These data suggest that the actions of trypsin on aldosterone secretion are mediated by PAR-1.