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Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3'-phosphoadenosine 5'-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3',5'-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions. Disruption of one allele of the Sult2a8 gene in male HT mice resulted in losing ~ 50% of the 7α-OH sulfonating activity compared to wild-type (WT) control, but no significant change in female HT mice. Under the fed condition comparing the levels of hepatic and biliary bile acids as well as plasma/serum energy metabolites, HT mice displayed a profile similar to that of WT mice, suggesting that the Sult2a8-haplodeficient mice conducted normal energy metabolism. However, after 48-h fasting, a significant decrease in plasma cholesterol level was found in male HT mice but without any significant reduction in female HT mice. Of interest, in male Sult2a8-haplodeficient mice, an increase of the hepatic taurine-conjugated cholic acid level was noted but no noticeable change in other tested bile acids after fasting. Taken together, SULT2A8 is a male-specific and key hepatic SULT in metabolizing 7α-OH primary bile acids. During energy deprivation, SULT2A8 is required to maintain the bile acid and cholesterol metabolism, suggesting SULT is a potential therapeutic target for controlling metabolic diseases.

Previously our lab has created a mouse ovarian xenograft model of copy number variation (CNV)-mediated G protein-coupled receptor (GPCR) MAS-driven tumorigenesis, and RNA profiling identified a putative chemokine tumor-induced factor (Tif). Sequence analysis and chemotactic study suggested that Tif was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316-1327]. In the present study, we report the molecular and functional characterization of the Tif gene. Genomic study of CHO-K1 cells indicated that Tif gene consisted of 4 exons, characterized with an antisense B1 element which is embedded in the fourth exon. Two Tif transcripts were identified which shared identical sequences except that a string of 71-nt derived from the antisense B1 element was deficient in the shorter transcript. Of interests, B1-like RNA ladder was detected in xenografts. Functional studies showed that TIF induced chemotaxis and neovessel formation. Pharmacological studies suggested that TIF activated Gi-coupled CXCR2 and induced both calcium mobilization and ERK1/2 phosphorylation, and suppressed forskolin-stimulated cAMP accumulation. In addition, secreted matured TIF functioned as an autocrine factor and promoted anchorage-independent growth. Unexpectedly, TIF delayed the onset of tumor formation, possibly via suppressing proliferation of stromal fibroblasts. However, TIF did not exert any inhibitory effect on tumor growth. Potentially, TIF could be used for preventing cancer relapse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression during energy deprivation. Recent evidence has also suggested that PPARα is involved in bile acid metabolism, but how PPARα modulates the homeostasis of bile acids during fasting is still not clear. In a mechanistic study aiming to dissect the spectrum of PPARα target genes involved in metabolic response to fasting, we identified a novel mouse gene (herein named mL-STL for mouse liver-sulfotransferase-like) that shared extensive homology with the Sult2a subfamily of a superfamily of cytosolic sulfotransferases, implying its potential function in sulfonation. The mL-STL gene expressed predominantly in liver in fed state, but PPARα was required to sustain its expression during fasting, suggesting a critical role of PPARα in regulating the mL-STL-mediated sulfonation during fasting. Functional studies using recombinant His-tagged mL-STL protein revealed its narrow sulfonating activities toward 7α-hydroxyl primary bile acids, including cholic acid, chenodeoxycholic acid, and α-muricholic acid, and thus suggesting that mL-STL may be the major hepatic bile acid sulfonating enzyme in mice. Together, these studies identified a novel PPARα-dependent gene and uncovered a new role of PPARα as being an essential regulator in bile acid biotransformation via sulfonation during fasting., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel "surrogate target cells" for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these "surrogate target cells" proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl- secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such "cytokine networks" play an important role in sustaining the airway inflammatory disease.

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Background. Exercise has been suggested to be a viable treatment for depression. This study investigates the effect of supervised aerobic exercise training on depressive symptoms and physical performance among Chinese patients with mild to moderate depression in early in-patient phase. Methods. A randomized repeated measure and assessor-blinded study design was used. Subjects in aerobic exercise group received 30 minutes of aerobic training, five days a week for 3 weeks. Depressive symptoms (MADRS and C-BDI) and domains in physical performance were assessed at baseline and program end. Results. Subjects in aerobic exercise group showed a more significant reduction in depressive scores (MADRS) as compared to control (between-group mean difference = 10.08 ± 9.41; P = 0.026) after 3 weeks training. The exercise group also demonstrated a significant improvement in flexibility (between-group mean difference = 4.4 ± 6.13; P = 0.02). Limitations. There was lack of longitudinal followup to examine the long-term effect of aerobic exercise on patients with depression. Conclusions. Aerobic exercise in addition to pharmacological intervention can have a synergistic effect in reducing depressive symptoms and increasing flexibility among Chinese population with mild to moderate depression. Early introduction of exercise training in in-patient phase can help to bridge the gap of therapeutic latency of antidepressants during its nonresponse period.

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

DHFR-deficient Chinese hamster ovary (CHO DHFR(-)) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR(-) cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-beta-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400V, 375microF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR(-) cells by electroporation.

Overexpressions of G protein-coupled receptor (GPCR) with elevated downstream signaling events have been reported in various tumors. However, the cellular mechanism that GPCR overexpression leads to tumor formation is largely unknown. The orphan GPCR mas was originally isolated from a human epidermoid carcinoma. In vivo studies of mas-overexpressing cells suggested that xenograft tumor formation was positively correlated with the levels of mas expression. Histochemical analysis indicated that xenograft tumor consisted of mas-transfected and stromal cells. Biochemical analyses revealed that cells overexpressing mas exhibited significantly increased anchorage-independent growth, whereas there was no significant difference in cell proliferation in comparison with empty vector-transfected control cells. Expression profiling using mRNA differential display and Northern analysis indicated an elevated expression of GRO and a novel CXC chemokines, tumor-induced factor (TIF), in mas-transfected cells and xenograft tumor. Bacterially expressed recombinant TIF was found to act as a neutrophil chemoattractant in a chemotactic assay. These results suggest that mas overexpression enables anchorage-independent growth of transformed cells, and interplays of secreted chemokines with stromal cells modulate xenograft tumor formation. Importantly, a novel CXC chemokine, TIF, was identified in the xenograft tumor tissues., (2009 UICC)

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been known to play a pivotal role in maintaining the energy balance during fasting; however, the battery of PPARalpha target genes involved in this metabolic response is still not fully characterized. Here, we report the identification and characterization of Ppsig (for PPARalpha-regulated and starvation-induced gene) with unknown biological function from mouse liver. Multiple Ppsig cDNAs which differed in the 3'-untranslated regions were identified. The open reading frame of Ppsig cDNA is 1830 bp which encodes a protein of 67.33 kDa. Ppsig contains 11 exons spanning at least 10 kb. Although the exact biological function of Ppsig is still not known, we found that Ppsig mRNA transcript was dramatically up-regulated during 72 h fasting and following treatment with a potent PPARalpha agonist, in a tissue-specific and PPARalpha-dependent manner. A functional peroxisome proliferator-response element was found in the intron 1 of Ppsig, thus confirming that Ppsig is a novel direct mouse PPARalpha target gene. This finding might help in elucidating the transcriptional regulatory mechanism of Ppsig in the cellular response to fasting.

The functional activity of G protein-coupled receptors can be modified by their ability to form oligomeric complexes with G protein-coupled receptors from other receptor families. Emerging evidence suggests that the appetite-regulating hormone ghrelin is a directly acting vasodilator peptide with anti-inflammatory activity, therefore, we have examined the ability of ghrelin receptors to oligomerize with members of the prostanoid receptor family which are also involved in modulating vascular activity and inflammatory responses. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the ability of ghrelin receptors to hetero-oligomerize with prostaglandin E2 receptor subtype EP3-I, prostacyclin receptors, and thromboxane A2 (TPalpha) receptors, when transiently over-expressed in human embryonic kidney 293 cells. These results suggest that hetero-oligomeric interactions between ghrelin receptors and prostanoid receptors are likely to be of biological relevance. Co-transfection of cells with ghrelin receptor and prostanoid receptors significantly decreased ghrelin receptor expression and attenuated its constitutive activation of phospholipase C without changing its affinity for ghrelin. We also observed an increase in the proportion of ghrelin receptors localized intracellularly in the presence of prostanoid receptors. Taken together, these results suggest that the increased expression of prostanoid receptors in conditions of vascular inflammation, such as in atherosclerotic plaques, could influence those cellular responses dependent on the constitutive activation of ghrelin receptors.

Angiotensin and endothelin are vasoactive peptides with neuromodulatory effect, however their interactions in facilitating neurotransmission are largely unknown. In the present study, effort was made to examine how endothelin 1 modulates angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens. Both peptides facilitated field-stimulated muscle contraction in a concentration-dependent manner with Kd values of 16.97+/-6.47 and 2.46+/-0.83 nM for angiotensin II and endothelin 1, respectively. Hill plot analysis gave Hill constants of 0.91+/-0.15 and 0.97+/-0.26 for angiotensin II and endothelin 1, respectively. Correlation analysis indicated that the extent of potentiation by angiotensin II, but not endothelin 1, was proportional to the basal field-stimulated muscle contraction. In the presence of low concentrations of endothelin 1 (< or = 3 nM), angiotensin II-potentiated field-stimulated contraction was further enhanced by endothelin. However, in the presence of high concentrations of endothelin 1 (> or = 10 nM), a much increased basal field-stimulated contraction was observed, and the addition of angiotensin II did not elicit any further enhancement in the contractile response. Intriguingly, after prolonged exposure of prostatic rat vas deferens to a high concentration of endothelin 1, the addition of angiotensin II induced a refractory response to field-stimulation. Taken together, our result indicated that endothelin 1 augmented angiotensin II-facilitated purinergic neurotransmission in prostatic rat vas deferens at low concentrations, but inhibited gradually at high concentrations.

Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl(4)-induced liver injury at 24h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1(+/+) and Cyp2e1(-/-) mice after treating with either corn oil or CCl(4) (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1(+/+) mice treated with CCl(4) for 24 and 48 h, while the Cyp2e1(+/+) mice treated with corn oil and Cyp2e1(-/-) mice treated with either corn oil or CCl(4) showed normal reddish brown colored liver. Ballooned hepatocytes with multiple vacuoles in their cytoplasm were observed in the livers of Cyp2e1(+/+) mice 24 and 48 h after treating with CCl(4). The levels of serum alanine aminotransferase and aspartate aminotransferase, markers for liver injury, were significantly higher at 12h, peaked at 24h and gradually decreased at 48 h after CCl(4) intoxication. In contrast, this kind of damage was not apparent in the Cyp2e1(-/-) mice treated with CCl(4). Altered expressions of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigenesis, structural organization, regeneration and inflammatory response were identified, and the time-dependent changes in expression of these genes were varied. Overall, the present study provides insights into the mechanism of CCl(4)-induced hepatotoxicity in animal models.

In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca(2+) influx was resolved into capacitative Ca(2+) entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 microM) and a phospholipase C (PLC) inhibitor (U73122; 10 microM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.

Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.

The universal HIV antibody testing programme was implemented in Hong Kong in September 2001. A survey on acceptance of the test was conducted in the territory's maternal and child health centres in a two-month period. The response rate was 98.2% and 2,669 valid questionnaires were analysed. Seventy per cent (n=1,825) of the respondents indicated their acceptance of the test. A significant association was noted between clients' acceptance and access to HIV information (adjusted odds ratio (OR)=10.45, 95% confidence interval (CI)=6.33-17.26) by means of posters, pamphlets, videos and group talks. Perceived benefits and health care workers' recommendation were the main reported reasons for acceptance, whereas no or low perceived susceptibility was the main reason for refusal. Acceptance was also positively correlated with level of education (adjusted OR=3.99, 95% CI=2.15-7.43) and HIV knowledge (adjusted OR=3.61, 95% CI=2.19-5.93). A high uptake rate (99.6%) reflects that most had the test done eventually despite some initial uncertainty. It is concluded that access to appropriate HIV information was important to maximize the acceptance of the programme.

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARalpha-null (KO) mice and their wild-type (WT) counterparts were investigated. Our results indicated that mice deficient in PPARalpha displayed hepatomegaly and hypoketonemia following 72 h starvation. Histochemical analyses revealed that severe fatty infiltration was observed in the livers of KO mice under fasted conditions. Furthermore, 72 h fasting resulted in a 2.8-fold higher accumulation of hepatic TG in KO mice than in WT mice fasted for the same length of time. Surprisingly, the total hepatic PL contents in fasted KO mice decreased by 45%, but no significant change in hepatic PL content was observed in WT mice following starvation. Gas chromatographic analysis indicated that KO mice were deprived of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids during fasting. Taken together, these results show that PPARalpha plays an important role in regulation of fatty acid metabolism as well as phospholipid homeostasis during energy deprivation.

Earlier studies indicate that binding sites of type II angiotensin (AT2) receptors are detected all over the pancreas, as well as in the pancreatic exocrine cell line AR4-2J. However, lack of corresponding functional AT2 receptor responses can be detected in the exocrine pancreas. The aim of present study is to determine the protein expression of AT2 receptors in the pancreas by probing with an AT2 receptor-specific antibody, and to examine the role of AT2 receptors in the regulation of pancreatic endocrine hormone release. In Western protein analysis of adult rat tissues, expression of AT2 receptor-immunoreactive bands of 56, 68, and 78 kDa was detected in the adrenal, kidney, liver, salivary glands, and pancreas. In adult rat pancreas, strong immunoreactivity was detected on cells that were located at the outer region of Langerhans islets. Immunohistochemical studies indicated that AT2 receptors colocalized with somatostatin-producing cells in the endocrine pancreas. Consistent with the findings in adult pancreas, abundant expression of AT2 receptors was also detected in immortalized rat pancreatic endocrinal cells lines RIN-m and RIN-14B. To examine the role of AT2 receptors on somatostatin secretion in the pancreas, angiotensin-stimulated somatostatin release from pancreatic RIN-14B cells was studied by an enzyme immunoassay in the absence or presence of various subtype-selective angiotensin analogues. There was a basal release of somatostatin from RIN-14B cells at a rate of 8.72 +/- 4.21 ng/10(6) cells (n = 7). Angiotensin II (1 nM-10 microM) stimulated a biphasic somatostatin release in a dose-dependent manner with an apparent EC50 value of 49.3 +/- 25.9 nM (n = 5), and reached maximal release at 1 microM angiotensin II (982 +/- 147.34% over basal secretion; n = 5). Moreover, the AT2 receptor-selective angiotensin analogue, CGP42112, was 1000 times more potent than the AT1 receptor-selective angiotensin analogue, losartan, in inhibiting angiotensin II-stimulated somatostatin release. These results suggest that angiotensin may modulate pancreatic hormone release via regulation of somatostatin secretion.

In conclusion, we have applied the fluorescent differential method and the PPAR alpha-null mouse model for the rapid isolation of expression tags of PPAR alpha target genes that are involved in the action of peroxisome proliferators and in the regulation of lipid homeostasis under energy deprivation. Identification of a wide spectrum of PPAR alpha target genes will provide new insights into the diverse cellular pathways regulated by these receptor, and this information will be critical for understanding the complicated biological interactions among members of the PPAR alpha target genes. With the recent technological advancement, a newer method, such as DNA microarray, has emerged in the identification of differential gene expressions. This new DNA microarray method, in conjunction with the differential display method, is the first important step toward understanding the molecular mechanisms of gene interactions in any biological systems and can speed up the search for differential gene expressions.

The nuclear rRNA gene of Ophiostoma piliferum was analyzed to understand its phylogenetic relationships to other sapstain fungi. Phylograms based on nucleotide sequences of the rRNA gene showed that the relationships between O. piliferum and other Ophiostoma species varied depending on the regions of the rRNA gene analyzed. Intraspecies variation in O. piliferum was found in the internal transcribed spacer regions, and the variation was related to the geographic origin of O. piliferum strains. A useful molecular marker for differentiating O. piliferum from other sapstain Ophiostoma species was generated by the HaeIII restriction fragment length polymorphism of the 26S rRNA gene.

Ilex latifolia is widely used as an ingredient to prepare traditional beverage drinks in southern China. In fact, various Ilex species have been used in Chinese folk medicine to treat coronary heart diseases. The mode of action is believed to be mediated by their coronary vasodilative effects. In this study, the water extract of the leaves of Ilex latifolia (IK-TP) was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. IK-TP was found to inhibit Na+/K+-ATPase activities in rat heart sarcolemma, rat brain microsomes and a purified enzyme from porcine cerebral cortex. IK-TP also inhibited Ca2+-dependent ATPase at a similar dose. Following exposure of the isolated rat heart to IK-TP at a dose that produces pronounced cardiac effects, inhibition of Na+/K+-ATPase activity can be readily detected in the heart. This study suggests the presence of ATPase inhibitory compounds in Ilex latifolia with specificities different from that of ouabain.

All the components of the renin-angiotensin system (RAS), including the AT(1)receptor, have previously been shown to be present in the human term placenta. However, the presence of the RAS components has not been fully investigated in the human placenta throughout pregnancy. The aim of this study was to examine the localization of the angiotensin receptors AT(1)and AT(2)using immunocytochemistry and the expression of prorenin, angiotensinogen and the AT(1)and AT(2)receptor mRNA using RT-PCR in the human placenta in the first, second and third trimesters of pregnancy. Localization of the AT(1)receptor was shown throughout gestation in the syncytiotrophoblast, cytotrophoblast, Hofbauer cells and the fetal vascular endothelium. Expression of mRNA for prorenin, angiotensinogen and the AT(1)receptor was shown in the placenta throughout gestation. However, localization or mRNA expression of the AT(2)receptor was not detected in any of the placental samples studied. These results clearly show the expression of a majority of the components of the RAS in the placenta from early gestation onwards. Therefore, these results suggest that the RAS may have a role in the human placenta throughout gestation., (Copyright 1999 W.B. Saunders Company Ltd.)

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4%), 2-repeat allele (21.4%), and 7-repeat allele (3.8%) which accounted for over 97% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6%), followed by 4/2 (38.6), 4/7 (7.6%), and 2/2 (3.0%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

Functional angiotensin receptors were characterized in the rat pancreatic acinar cell line AR4-2J. Angiotensin II stimulated a dose-dependent release of amylase and production of inositol phosphates. Results of high-performance liquid chromatography separation of inositol phosphates indicated that angiotensin stimulated the rapid accumulation of inositol 1,3,4-trisphosphate. Angiotensin II and angiotensin III were at least an order of magnitude more potent than angiotensin I in the stimulation of amylase release. The angiotensin II-stimulated amylase release was blocked by losartan, a selective AT1 angiotensin antagonist. The selective AT2 angiotensin receptor ligands CGP42112 did not alter angiotensin II-stimulated amylase released. However, CGP42112 stimulated amylase release at micromolar concentrations with a potency similar to angiotensin I. Analysis of mRNA expression by reverse transcription polymerase chain reaction suggested that AT1A was the predominant type-I angiotensin receptor expressed in the AR4-2J cells.

A polyclonal antibody has been prepared against a synthetic peptide derived from the C-terminal tail of the cloned rat AT2 angiotensin receptor, corresponding to amino acid residue 341-351. The antibody was of high titer and displayed monospecific activity toward the synthetic peptide in the ELISA assay. Western blot analysis indicated that the antiserum recognised only a single protein band with a mean apparent molecular mass of 75.4 kDa in the rat adrenals. Immunohistochemical studies with affinity purified antibody localised immunoreactive AT2 angiotensin receptor in medulla cells of the adrenals. Immunoreactivity was also observed in pyramidal tract, but no specific immunoreactivity can be detected in regions of rat brain that are known to express AT2 angiotensin receptors, including inferior olive, locus coeruleus and cerebellum.

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 microM) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat.

The present study explored regulation of anion secretion across cystic fibrosis pancreatic ductal epithelium by extracellular ATP with the short-circuit current (Isc) technique. CFPAC-1 cells grown on Millipore filters formed polarized monolayers with junctional complexes as revealed by light and electron microscopy. The cultured monolayers exhibited an increase in Isc in response to apical application of ATP in a concentration-dependent manner (concentration eliciting 50% of maximal response = 3 microM). Replacement of Cl- in the bathing solution or treatment of the cells with a Cl- channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), markedly reduced Isc, indicating that a substantial portion of ATP-activated Isc was Cl- dependent. The effects of different adenosine nucleosides and/or nucleotides on Isc were also studied to identify the type of purinoceptors involved. The order of potency, ATP = UTP > ADP > adenosine, was consistent with that for P2 purinoceptors. Reactive blue 2 (100 microM), a P2 antagonist, was found to inhibit 86% of ATP-induced Isc. ATP-induced Isc was also inhibited by pretreatment of the cells with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (50 microM). Confocal microscopic study also demonstrated a rise in intracellular Ca2+ with stimulation by extracellular ATP, indicating a role of intracellular Ca2+ in mediating the ATP response. ATP-induced Isc was observed in monolayers whose basolateral membranes had been permeabilized by nystatin, which was also sensitive to apical addition of DIDS, suggesting that Isc was mediated by apical Cl- channels. The results of the present study demonstrate the presence of a purinergic regulatory mechanism involving P2U receptor and Ca2+ mobilization in pancreatic duct anion secretion.

1. Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation. 2. Angiotensin-potentiated contractile response to field stimulation was concentration-dependent, and the order of potency was AII > AIII approximately AI. The EC50 of AII was 8.11 +/- 2.79 nM. 3. AII did not modify the contractile response of exogenous noradrenaline (NA) on non-stimulated prostatic vas deferens. Furthermore, the concentration-response curve for AII-potentiated contractile responses to field stimulation in reserpine-treated rats did not significantly differ from the control group. 4. Desensitization of purinoceptors with 30 microM alpha, beta-methylene-ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII. 5. The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75. 6. In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.

Angiotensin had a dual action on the epididymal half of rat vas deferens. It potentiated electrical stimulated contraction and exerted a direct contractile effect on the muscle. The potentiation of electrically stimulated response may be mediated by presynaptic facilitation of neurotransmitter release. Muscular contractile response to angiotensin is concentration dependent. Angiotensin II was found to be much more potent than angiotensin III, and the order of potencies was angiotensin II > angiotensin I > angiotensin III. The presence of a mixture of protease inhibitors (10 microM chymostatin, 50 microM bacitracin, 10 microM leupeptin and 10 microM pepstatin) did not alter the contractile activity of angiotensin II. In contrast, angiotensin I (10 nM)-induced contraction was significantly reduced in the presence of ACE inhibitor SQ 20881 (500 nM). The angiotensin II induced contraction was not reduced by CGP 42112, a specific AT2 receptor antagonist, but was significantly inhibited by losartan, a specific AT1 receptor antagonist. Losartan shifted the dose-response curve of angiotensin II to the right with a pA2 value of 8.68. In addition, p-aminophenylalanine6 angiotensin II, which is proposed as an AT2 receptor agonist, did not induce contraction. It is concluded that the AT1 receptor predominantly mediates angiotensin-induced contraction in epididymal rat vas deferens.

Calretinin is a member of the EF-hand calcium-binding protein family, with a high similarity with calbindin D28k. The chick calretinin cDNA sequence was reconstructed in a M13 vector and transferred into an expression plasmid derived from the pET series. The calretinin gene was expressed in Escherichia coli and produced immunoreactive calretinin of the expected size. Bacterially expressed calretinin was purified with successive ammonium-sulfate precipitation, DEAE chromatography, hydroxyapatite chromatography, Sephadex G-75 chromatography and Mono-Q chromatography. Normally, 1.0-1.5 mg calretinin was obtained from 1 l bacterial culture with a protein recovery of 0.5-1.5%. Calbindin D28k was purified similarly from bacteria using an expression plasmid provided by W. Hunziker. Calcium-binding activity of purified proteins was measured by equilibrium dialysis in calcium/EGTA mixtures with 45Ca as tracer. Both calretinin and calbindin D28k bound 3-4 Ca2+/molecule (calretinin, 4.0 +/- 0.5; calbindin D28k, 3.5 +/- 0.4), implying that at least one of the canonical EF-hand domains does not bind calcium. The Kd was 0.3-0.5 microM with little difference between the values for the two proteins.

The human mas oncogene, which renders transfected NIH/3T3 cells tumorigenic, was identified as a subtype of angiotensin receptor by transient expression in Xenopus oocytes and stable expression in the mammalian neuronal cell line, NG115-401L. The mas receptor preferentially recognizes angiotensin III, and is expressed at high levels in brain. The mas/angiotensin receptor functions through the breakdown of inositol lipids and can drive DNA synthesis, unlike another inositol-linked peptide receptor, that for bradykinin. Comparative analysis of several early biochemical events elicited by either angiotensin or bradykinin stimulation of mas-transfected cells has not indicated a specific difference correlated with mitogenic activity. In particular, the inositol lipid kinase, phosphatidylinositol-3-kinase, thought to be involved in the mitogenic mechanism of platelet-derived growth factor receptors, is unaffected by activation of mas. These results have shown that a proto-oncogene encodes a neural peptide receptor, indicating that peptide receptors may be involved in differentiation and proliferation processes, as are other identified proto-oncogenes.

1. The effect of neonatal monosodium-L-glutamate (MSG) treatment on lipolysis in rat epididymal adipose tissue was studied. A reduction in the basal lipolysis was observed in the MSG-treated rats. 2. This was accompanied by a decrease lipolytic response to isoprenaline, adrenocorticotropic hormone, forskolin, isobutylmethylxanthine and dibutyryl-cAMP. 3. The addition of adenosine deaminase, which inactivates endogenous adenosine in the medium, did not normalize the basal and the hormone stimulated lipolytic responses. 4. The maximal lipolysis stimulated by adenosine deaminase or 8-(p-sulfophenyl)-theophylline (8-SPT), an adenosine antagonist, was significantly lower in the MSG-treated rats. 5. Moreover, there was no change in the sensitivity of adenosine receptors to its antagonist as reflected by the similar potency of 8-SPT in eliciting the lipolytic response in both the control and MSG-treated rats. 6. In conclusion, neonatal MSG treatment in rats induced a general reduction of lipolytic response in the epididymal adipocytes which cannot be explained by an enhancement of the adenosine inhibitory system.

The effect of chronic caffeine treatment on lipolysis in rat epididymal adipose tissue was studied. There was a decrease in body weight, epididymal fat pad weight and mean adipocyte diameter in caffeine-treated rats when compared with control rats. No difference in adipocyte triglyceride content or mean adipocyte weight between control and caffeine-treated rats was observed. Lipolysis in adipocytes induced by adenosine deaminase (1 U/ml) decreased by 35% in caffeine-treated rats. This was accompanied by a 2.5-fold increase in the anti-lipolytic potency of 2-chloroadenosine and an increase of adipocyte adenosine A1 receptor number.

Carbachol (0.1-300 mumol/L) potentiated contractions to field stimulation (0.1 Hz, 1 ms, supramaximal V) in the rat epididymal and prostatic vas deferens. Desensitization of P2-purinoceptors by exposure to alpha,beta-methylene ATP (30 mumol/L) markedly reduced (greater than 80%) the potentiating effect of carbachol in the prostatic vas deferens but only moderately reduced (about 20%) the maximal stimulated response to carbachol in the epididymal segment. The presence of prazosin (10 mumol/L) and yohimbine (10 mumol/L), being selective alpha 1- and alpha 2-adrenoceptor antagonists, did not modify the attenuation of carbachol potentiation caused by alpha,beta-methylene ATP treatment. At 0.1 mmol/L, alpha,beta-methylene ATP had no significant effect on the binding of [3H]QNB to muscarinic cholinergic receptors. It is concluded that carbachol may potentiate the contractions to field stimulation in the prostatic vas deferens via an enhancement of purinergic neurotransmission. The molecular mechanism of carbachol potentiation in the epididymal vas deferens remains to be established.

We have demonstrated a high affinity specific binding of 125I-Bolton Hunter conjugate of substance P (125I-BHSP) to rat retina membranes. The binding was saturable and monophasic with a Kd of 0.2 nM and a Bmax of 290 fmol/mg protein. The rank order of potency of tachykinins and related analogues in inhibiting 125I-BHSP binding conformed to that of the NK-1 (identical to SP-P) tachykinin receptor, with SP greater than NKA greater than or equal to KAS greater than or equal to NKB and [Glp6, L-Pro9]SP(6-11) being 60 times more active than [Glp6, D-Pro9]SP(6-11). After neonatal monosodium glutamate (MSG) treatment, there was a marked reduction in the number but not binding affinity of 125I-BHSP binding sites in the retina. The same treatment had no effect on either the number or binding affinities of NK-1 sites in the salivary gland.

The inhibitory effects of adenosine as well as its related analogues on the contractile response of the rat vas deferens to field stimulation were compared in the absence and in the presence of nitrobenzylthioguanosine (NBTGR), a potent adenosine uptake inhibitor. In the presence of NBTGR, the order of potency was N6-cyclohexyladenosine (CHA) greater than or equal to L-N6-phenylisopropyladenosine (L-PIA) greater than 2-chloroadenosine greater than D-N6-phenylisopropyladenosine (D-PIA) greater than or equal to adenosine greater than 2'-deoxyadenosine. The inhibitory effect of adenosine but not that of clonidine, beta-endorphin and somatostatin was blocked by 1,3-diethyl-8-phenylxanthine (DPX, pA2 = 7.2), a potent P1-purinergic antagonist. The results suggest that adenosine inhibited the electrically evoked contractions of the rat vas deferens via the activation of the A1 subtype of P1-purinergic receptors.

Two dihydropyridine compounds, Bay K8644 (a calcium entry activator) and nifedipine (a calcium entry blocker), were found to inhibit the binding of [3H]phenylisopropyladenosine ([3H]PIA) to A1 adenosine receptors in rat cerebral cortex membranes with comparable potencies (IC50 10-30 microM). Scatchard analyses indicated that both Bay K8644 and nifedipine inhibited the binding of [3H]PIA by increasing the KD but without significant effect on the Bmax. When tested at 100 microM, neither Bay K8644 nor nifedipine showed a significant effect on [3H]-p-aminoclonidine ([3H]PAC; alpha 2-adrenergic receptor), [3H]dihydroalprenolol ([3H]DHA; beta-adrenergic receptor), [3H]spiperone (dopamine receptor), and [3H]nitrobenzylthioinosine [( 3H]NBMPR; nucleoside transporter) binding. In the presence of 10 mM Mg2+, the ability of 2-chloroadenosine (2-Cl-Ad, an A1 adenosine receptor agonist) to displace [3H]PIA binding was increased. Conversely, the potencies of 1,3-diethyl-8-phenylxanthine (DPX; an A1 receptor antagonist), Bay K8644 and nifedipine in inhibiting [3H]PIA binding were unchanged. It is suggested that both Bay K8644 and nifedipine may act as antagonists of adenosine A1 receptors, in addition to their well-known effects on calcium channels.