Comparative Hepatic and Intestinal Metabolism and Pharmacodynamics of Statins.

This study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and their metabolites were investigated using human liver microsomes (HLMs), human intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism [intrinsic clearance (CLint) values of 3700 and 7400 µl/min per milligram], whereas the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CLint 20-840 µl/min per milligram). The acids had CLint values in the range <0.1-80 µl/min per milligram. In HIMs, only atorvastatin lactone and simvastatin (lactone) exhibited notable metabolism, with CLint values corresponding to 20% of those observed in HLMs. CYP3A4/5 and CYP2C9 were the main statin-metabolizing enzymes. The majority of the acids inhibited HMG-CoA reductase, with 50% inhibitory concentrations of 4-20 nM. The present comparison of the metabolism and pharmacodynamics of the various statins using identical methods provides a strong basis for further application, e.g., comparative systems pharmacology modeling. SIGNIFICANCE STATEMENT: The present comparison of the in vitro metabolic and pharmacodynamic properties of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin and their metabolites using unified methodology provides a strong basis for further application. Together with in vitro drug transporter and clinical data, the present findings are applicable for use in comparative systems pharmacology modeling to predict the pharmacokinetics and pharmacological effects of statins at different dosages.

Preparation of crude rough microsomes from tissue culture cells.

There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

Prostasomes are mediators of intercellular communication: from basic research to clinical implications.

Prostasomes are nanosized microvesicles secreted by acinar epithelial cells of the prostate gland. Furthermore, they are intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies of late endosomal origin. Prostasomes are thought to play an important role in intercellular communication by direct interaction primarily between the immobile acinar cells of the prostate gland and the mobile spermatozoa. Prostasomes transfer not only membrane components but also genetic material to spermatozoa. They are rich in various transferable bioactive molecules (e.g., receptors and enzymes) that promote the fertilizing ability of spermatozoa. In this review, the pleiotropic biological effects of prostasomes that are relevant for successful fertilization will be discussed. The ability to synthesize and export prostasomes to the extracellular space is observed not only in normal prostate epithelial cells but also in malignant prostate cells. Release of prostasomes by prostate cancer cells suggests a role in malignant cell growth and proliferation. These findings may provide new therapeutic and diagnostic strategies.

Discovery of PF-184563, a potent and selective V1a antagonist for the treatment of dysmenorrhoea. The influence of compound flexibility on microsomal stability.

The V1a receptor has emerged as an attractive target for a range of indications including Raynaud's disease and dysmenorrhoea. As part of an effort to discover a new class of orally active V1a antagonist, we optimised a highly lipophilic, metabolically unstable lead into a range of potent, selective and metabolically stable V1a antagonists. In this communication, we demonstrate the series-dependent effect of limiting the number of rotatable bonds in order to decrease Cytochrome P450-mediated metabolism. This effort culminated in the discovery of PF-184563, a novel, selective V1a antagonist with excellent in vitro and in vivo properties.

New lipid modulating drugs: the role of microsomal transport protein inhibitors.

Microsomal triglyceride transfer protein (MTP) is involved in the synthesis of very low density lipoprotein in the liver. Its deficiency results in abetalipoproteinemia. MTP inhibitors target the assembly and secretion of apolipoprotein B-containing lipoproteins. These agents may potentially play a role, alone or in combination, in the treatment of hypercholesterolemia or hypertriglyceridaemia. Clinical applications of MTP inhibitors initially focused primarily on high-dose monotherapy in order to produce substantial reductions in LDL-cholesterol levels but these proved to induce significant hepatic steatosis and transaminase elevations. However, likely orphan indications for MTP inhibitors, where a different risk-benefit profile applies, include patients with homozygous familial hypercholesterolemia where statins often show a low response. Development of MTP inhibitors has continued to enter clinical trials at lower doses or in formulations aimed at utilizing their efficacy while avoiding their side effects. These have shown promising results in reducing cholesterol, triglycerides and apolipoprotein B with a far lower incidence of, often, transient side-effects. The clinical efficacy and safety of MTP inhibition in patients with hyperlipidaemia remains to be fully determined and to be proven in both surrogate and clinical endpoint trials but there may be a role for these agents in orphan indications for rarer severe hyperlipidaemias.

An octahydro-cyclopenta[c]pyrrole series of inhibitors of the type 1 glycine transporter.

We describe a novel series of inhibitors of the type 1 glycine transporter (GlyT1) as an approach to relieving the glutamatergic deficit that is thought to underlie schizophrenia. Synthesis and SAR follow-up of a series of octahydro-cyclopenta[c]pyrrole derivatives afforded potent in vitro inhibition of GlyT1 as well as in vivo activity in elevating CSF glycine. We also found that a 3-O(c-pentyl), 4-F substituent may serve as a surrogate for the widely used 3-trifluoromethoxy group, suggesting its application as an isostere for future medicinal chemistry studies.

Differential ligand binding and agonist-induced regulation characteristics of the two rainbow trout GH receptors, Ghr1 and Ghr2, in transfected cells.

Previously, we isolated and characterized two distinct GH receptor (GHR)-encoding mRNAs, ghr1 and ghr2, from rainbow trout. In this study, Chinese hamster ovary-K1 cells were individually transfected with plasmids that contained cDNAs encoding rainbow trout ghr1 or ghr2. High affinity binding of (125)I-salmonid GH (sGH) by the expressed receptors was saturable, displaceable, and ligand selective. Whole-cell binding analysis revealed a single class of binding site; for Ghr1 K(d)=8 nM, for Ghr2 K(d)=17 nM. While salmonid prolactin (sPrl) displaced (125)I-sGH from both Ghr1 and Ghr2, the affinity of either receptor subtype for sPrl was substantially less than for sGH; salmonid somatolactin, another member of the GH-PRL family, did not displace labeled sGH except at pharmacological concentrations. (125)I-sGH was internalized by Ghr1- and Ghr2-expressing cells in a time-dependent manner; the maximum internalization reached was 71% for Ghr1 and 55% for Ghr2. Long-term exposure (24 h) of transfected cells to sGH up-regulated surface expression of both Ghr1 and Ghr2; however, sGH induced surface expression of Ghr1 to a greater extent than that of Ghr2. These results indicate that rainbow trout ghrs display both overlapping and distinct characteristics that may be important for ligand selection and differential action in target organs.

Peroxidación lipídica de microsomas obtenidos del hígado y riñón de rata: efecto del isómero CIS- 9, TRANS-11 del ácido linoléico / Lipid peroxidation of microsomes obtained from rat liver and kidney: effect of CIS-9, TRANS-11_ conjugated linoleic acid isomer

La composición de ácidos grasos, la quimioluminescencia y el índice de peroxidabilidad de microsomas obtenidos de hígado y riñón de rata fueron estudiados después de la administración oral del isómero ácido linoleico conjugado-9-cis, 11-trans (c9-t11-ALC). Mediante la incubación de microsomas en un sistema ascorbato-Fe++ (120 minutos en 37°C), se observó que las cuentas totales por minuto/mg de proteína, originadas por la quimioluminiscencia, eran más bajas en microsomas de hígado y riñón pertenecientes al grupo c9-t11-ALC que en los microsomas obtenidos de grupo control. El efecto de c9-t11-ALC sobre la composición de ácidos grasos poli-insaturados de microsomas hepáticos nativos fue evidenciado por una disminución estadísticamente significativa p<0.007 del ácido linoleico C18:2 n6. Cuando los microsomas peroxidados obtenidos de hígado y riñón, de ambos grupos (control y c9-t11-ALC), fueron comparados con sus respectivos nativos, se observó que C18:2 n6, C20: 4 n6 disminuyeron en todas las membranas microsomales usadas en este trabajo, mientras que en los microsomas obtenidos de hígado del grupo c9-t11-ALC y grupo control también disminuyó C22: 6n3. Por consiguiente, el índice del peroxidabilidad – parámetro basado en el índice máximo de oxidación de ácidos grasos – mostró cambios significativos en microsomas de hígado y riñón. Estos cambios fueron menos pronunciados en las membranas microsomales obtenidas de las ratas que recibieron c9-t11-ALC por vía oral. Nuestros resultados confirmarían y ampliarían observaciones anteriores que indicaron que ALC podría actuar como antioxidante, protegiendo las membranas contra efectos dañinos (AU)

The polyunsaturated fatty acid composition, chemiluminescence and peroxidizability index of microsomes obtained from rat liver and kidney were studied after oral administration of cis-9, trans-11-conjugated linoleic acid isomer (c9-t11-CLA). After incubation of microsomes in an ascorbate Fe++ system (120 min at 37°C) it was observed that the total counts per min/mg protein originated from light emission: chemiluminescence was lower in liver and kidney microsomes in the c9-t11-CLA group than in the microsomes obtained from control group. The effect of c9-t11-CLA on the polyunsaturated fatty acid composition of native liver microsomes was evidenced by a statistically significant p<0.007 decrease of linoleic acid C18:2 n6. When peroxidized microsomes obtained from liver and kidney of both groups (control and c9-t11-CLA) were compared with respective natives, it was observed that C18:2 n6, C20:4 n6 decreased in all membranes used in this work, whereas in microsomes obtained from liver c9-t11-CLA and control groups C22:6 n3 also decreased. As a consequence, the peroxidizability index – a parameter based on the maximal rate of oxidation of fatty acids – showed significant changes in liver and kidney microsomes. These changes were less pronounced in membranes derived from rats receiving c9-t11-CLA per os. Our results would confirm and extend previous observations which indicated that CLA could act as an antioxidant, protecting membranes from deleterious effects (AU)

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle.

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3.

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.

Relationship between the expression of hepatic but not testicular 3beta-hydroxysteroid dehydrogenase with androstenone deposition in pig adipose tissue.

This study investigated the relationship between expression of hepatic and testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and accumulation of androstenone in adipose tissue because of its relation to boar taint. The experiments were performed on 13 Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%), pigs, which differed in the level of backfat androstenone. Our previous work showed that the major product of the hepatic androstenone metabolism is 3beta-androstenol. In this study, the formation of 3beta-androstenol was inhibited by the specific 3beta-HSD inhibitor trilostane. These results are the first direct confirmation that 3beta-HSD is the enzyme responsible for androstenone metabolism in the pig. The expression of the hepatic but not testicular 3beta-HSD protein showed a negative relationship with the level of backfat androstenone (r2 = 0.64; P < 0.001) and was accompanied by a reduced rate of the hepatic androstenone clearance. Low expression of 3beta-HSD protein in the liver of high androstenone pigs was also accompanied by a reduced level of 3beta-HSD mRNA (P < 0.001), which suggests a defective regulation of the hepatic 3beta-HSD expression at the level of transcription. In contrast, expression of the testicular 3beta-HSD protein did not differ between animals with high and low androstenone levels (P > 0.05) and was lower compared with the hepatic 3beta-HSD expression. Cloning and sequencing of the 3beta-HSD coding regions established that the hepatic and testicular 3beta-HSD cDNA have identical sequences, which were 98% similar to the human 3beta-HSD isoform I. It is suggested that expression of a single 3beta-HSD gene is regulated by different mechanisms in pig liver and testis. The liver-specific regulation of 3beta-HSD expression contributes to the low rate of hepatic androstenone metabolism and therefore can be considered as one of the factors regulating deposition of androstenone in pig adipose tissue and subsequent development of boar taint.

Experimental methods to study human transplacental exposure to genotoxic agents.

Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta. The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture. Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.

Dual role of beta-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2.

Results from some intervention trials indicated that supplemental beta-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100-150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of beta-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying beta-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6-25 microg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), beta-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50-100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of alpha-tocopherol (25 microM) prevented the prooxidant effects of the carotenoid. beta-Carotene auto-oxidation, measured as formation of 5,6-epoxy-beta,beta-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

Effects of drugs with muscle-related side effects and affinity for calsequestrin on the calcium regulatory function of sarcoplasmic reticulum microsomes.

The tight regulation of Ca2+ release to and clearance from the cytosol is essential for normal excitation-contraction coupling in both skeletal and cardiac muscles. Calsequestrin (CSQ) is one of the major components in the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Previously, we showed that several pharmaceutical drugs, such as phenothiazine derivatives, tricyclic antidepressants, anthracycline derivatives, and other hydrophobic compounds bind CSQ with K(d) values in the micromolar range and significantly reduce the Ca2+ binding capacity of cardiac CSQ (Mol Pharmacol 67:97-104, 2005). Because of its key role in Ca2+ regulation, this interference with CSQ function could well produce adverse physiological consequences and potentially be linked to the known muscle-related side effects of these drugs. To further understand the molecular mechanism of undesirable drug effects or adverse drug reactions among those compounds, we examined their effect on the SR microsome. The results clearly showed that these compounds affect Ca2+ release and reduce the total Ca2+ content of the purified SR microsomes, matching well with our previous results with purified recombinant CSQ. Liquid chromatography-mass spectrometry/mass spectrometry showed that the antipsychotic drug trifluoperazine penetrates well into the SR microsome as expected from the reported and calculated log S (aqueous solubility) and log P (partition coefficient) values among the phenothiazine derivatives. We therefore propose that a certain portion of the muscle-related (both cardiac and skeletal) complications of these drugs is caused by the altered Ca2+ regulation of the SR mediated by their adverse interaction with CSQ.

Extracts obtained from predegenerated nerves improve functional recovery after sciatic nerve transection.

Gap injuries of peripheral nerves, resulting from trauma or neurosurgical procedures, presage badly, for the presence of the distal stump of the nerve seems to be indispensable for regeneration. The standard grafting method requires a lesion of a healthy nerve, and therefore various substitutional materials are under consideration. The aim of the present work was to examine the recovery of rat sciatic nerves after supplying 10-mm-long gaps with an autologous connective-tissue chambers filled with fibrin only or fibrin and various neuroactive substances (brain-derived neurotrophic factor (BDNF), extracts from predegenerated or non-predegenerated nerves). The nerves were allowed to regenerate for 16 weeks. Recovery was measured functionally using the sciatic functional index, and by comparing the weight ratios of calf muscles. The histologic features of regeneration were assessed by counting the number of acetylcholinesterase-positive nerve fibers present inside implanted chambers. We found that chambers filled with fibrin and predegenerated peripheral nerve extracts or BDNF supported functional nerve regeneration much more strongly than chambers filled with fibrin only or fibrin and non-predegenerated peripheral nerve extracts. We conclude that autologous connective-tissue chambers filled with fibrin and predegenerated peripheral nerve extracts or BDNF seem to be a promising tool in peripheral nerve gap injury treatment, with likely clinical implications.

Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.

Alpha-synuclein (alpha-syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of alpha-syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, alpha-syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized alpha-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular alpha-syn is present in the lumen of vesicles. Importantly, the intravesicular alpha-syn is more prone to aggregation than the cytosolic protein, and aggregated forms of alpha-syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated alpha-syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of alpha-syn and might also contribute to pathological function of this protein.

Entamoeba histolytica: biochemical and molecular insights into the activities within microsomal fractions.

One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.

Prolactin receptor knockdown in the rat paraventricular nucleus by a morpholino-antisense oligonucleotide causes hypocalcemia and stress gastric erosion.

Under acute stress conditions in the rat, there is rapid and transient increase in circulating prolactin (PRL). This leads to an elevated expression of the long form of PRLR (PRLR-L) first in the hypothalamus and the choroid plexus. This increase in PRL is involved in the inhibition of stress-induced hypocalcemia and gastric erosion. In this study we used rat PRL and a PRLR morpholino-antisense oligonucleotide to elucidate the mechanism by which hypothalamic PRLR mediates the inhibition of restraint stress in water (RSW)-induced hypocalcemia and gastric erosion. We found that this effect is largely mediated by PRLRs in the paraventricular nucleus (PVN), medial preoptic nucleus, and ventromedial hypothalamus. We also show that when measured after 7 h of RSW, microinjection of the PRLR antisense oligonucleotide into these areas down-regulates RSW-enhanced expression of PRLR-L protein in the PVN and increases the plasma PRL level, but does not affect plasma levels of another hormone, GH. Furthermore, our experiments demonstrated that under nonstress conditions, knockdown of the PRLR in the PVN significantly lowers circulating Ca2+ levels, but does not affect gastric erosion. These results suggest that PRL acting on the PRLR-L in the PVN is one of the critical pathways for regulating circulating Ca2+ levels under both acute stress and nonstress conditions.

Microsomal triglyceride transfer protein gene expression and ApoB secretion are inhibited by bitter melon in HepG2 cells.

Momordica charantia or bitter melon is traditionally used as an antidiabetic agent in Asia, Africa, and South America. Recent studies indicate that bitter melon can also lower plasma lipids and VLDL in diabetic animal models as well as animals fed a high-fat diet, suggesting an effect on lipoprotein metabolism. The aim of this study was to delineate the cellular and molecular mechanisms involved in the lipid-lowering properties of bitter melon and regulation of apolipoprotein B (apoB). Human hepatoma cells, HepG2, treated with bitter melon juice (BMJ) for 24 h reduced apoB secretion with and without the addition of lipids (P < 0.05). However, BMJ did not increase apoB secretion in cells treated with N-acetyl-leucyl-leucyl-norleucinal, indicating a lack of effect on the proteasomal degradation pathway. BMJ reduced the secretion of new triglycerides (P < 0.05) and decreased microsomal triglyceride transfer protein (MTP) mRNA expression, suggesting that lipid bioavailability and lipidation of lipoprotein assembly are likely involved in decreased apoB secretion. Interestingly, BMJ increased the nuclear translocation of the mature form of sterol regulatory element-binding protein-1c (SREBP-1c, P < 0.05), involved in MTP secretion. Our data suggest that BMJ is a potent inhibitor of apoB secretion and TG synthesis and secretion that may be involved in the plasma lipid- and VLDL-lowering effects observed in animal studies.

Triadin overexpression stimulates excitation-contraction coupling and increases predisposition to cellular arrhythmia in cardiac myocytes.

Triadin 1 (TRD) is an integral membrane protein that associates with the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin to form a macromolecular Ca signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). To define the functional role of TRD, we examined the effects of adenoviral-mediated overexpression of the wild-type protein (TRD(WT)) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRD(Del.200-224)) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRD(WT) reduced the amplitude of I(Ca)- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small I(Ca) at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRD(WT) myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRD(WT) myocytes. Periodic electrical stimulation of TRD(WT) myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRD(Del.200-224) failed to produce any of the effects of the wild-type protein. The lipid bilayer technique was used to record the activity of single RyR2 channels using microsome samples obtained from control, TRD(WT) and TRD(Del.200-224) myocytes. Elevation of TRD(WT) levels increased the open probability of RyR2 channels, whereas expression of the mutant protein did not affect RyR2 activity. We conclude that TRD enhances cardiac excitation-contraction coupling by directly stimulating the RyR2. Interaction of TRD with RyR2 may involve amino acids 200 to 224 in C-terminal domain of TRD.