Comparison of ELISA and HPLC-MS methods for the determination of exenatide in biological and biotechnology-based formulation matrices / 药物分析学报

The development of biotechnology-based active pharmaceutical ingredients, such as GLP-1 analogs, brought changes in type 2 diabetes treatment options. For better therapeutic efficiency, these active pharmaceutical ingredients require appropriate administration, without the development of adverse effects or toxicity. Therefore, it is required to develop several quantification methods for GLP-1 analogs products, in order to achieve the therapeutic goals, among which ELISA and HPLC arise. These methods are developed, optimized and validated in order to determine GLP-1 analogs, not only in final formu-lation of the active pharmaceutical ingredient, but also during preclinical and clinical trials assessment. This review highlights the role of ELISA and HPLC methods that have been used during the assessment for GLP-1 analogs, especially for exenatide.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

Papillary and muscle invasive bladder tumors with distinct genomic stability profiles have different DNA repair fidelity and KU DNA-binding activities.

Low-grade noninvasive papillary bladder tumors are genetically stable whereas muscle invasive bladder tumors display high levels of chromosomal aberrations. As cells deficient for nonhomologous end-joining (NHEJ) pathway components display increased genomic instability, we sought to determine the NHEJ repair characteristics of bladder tumors and correlate this with tumor stage and grade. A panel of 13 human bladder tumors of defined stage and grade were investigated for chromosomal aberrations by comparative genomic hybridization and for NHEJ repair fidelity and function. Repair assays were conducted with extracts made directly from bladder tumor specimens to avoid culture-induced phenotypic alterations and selection bias as only a minority of bladder tumors grow in culture. Four noninvasive bladder tumors (pTaG2), which were genetically stable, repaired a partially incompatible double-strand break (DSB) by NHEJ-dependent annealing of termini and fill-in of overhangs with minimal loss of nucleotides. In contrast, four muscle invasive bladder cancers (pT2-3G3), which displayed gross chromosomal rearrangements, repaired DSBs in an error-prone manner involving extensive resection and microhomology association. Four minimally invasive bladder cancers (pT1G3) had characteristics of both repair types. Error-prone repair in bladder tumors correlated with reduced KU DNA-binding and loss of TP53 function. In conclusion, there were distinct differences in DSB repair between noninvasive papillary tumors and higher stage/grade invasive cancers. End-joining fidelity correlated with stage and was increasingly error-prone as tumors became more invasive and KU binding activity reduced; these changes may underlie the different genomic profiles of these tumors.

The prevalence of newly diagnosed hyperlipidaemia in men with erectile dysfunction.

OBJECTIVE: To determine the prevalence of newly diagnosed hypercholesterolaemia and hypertriglyceridaemia in patients presenting to an andrology clinic with erectile dysfunction (ED), and to assess the relationship between serum lipid levels and the severity of ED. PATIENTS AND METHODS: In all, 199 consecutive men attending an ED clinic were assessed for risk factors for ED; patients completed the International Index of Erectile Function (IIEF)-15 questionnaire and provided venous blood samples for assaying fasting total cholesterol and total triglyceride levels. The proportion of newly diagnosed hyperlipidaemia in patients presenting with ED was calculated and related to patient age, total IIEF score and severity of ED. RESULTS: Using a threshold of 5.0 mmol/L, there was newly diagnosed hypercholesterolaemia in 40% of the men, while there was undiagnosed hypertriglyceridaemia (>2 mmol/L) in 29% of the population. There was no clear correlation between patient age and the fasting lipid levels, and no association between total IIEF-15 score or severity of ED and serum cholesterol and triglyceride levels. CONCLUSION: This study shows the high prevalence of undiagnosed hypercholesterolaemia and hypertriglyceridaemia in men presenting with ED. The opportunity to screen for and treat these risk factors has long-term benefits in preventing cardiovascular disease in this group of patients.

APE1 and XRCC1 protein expression levels predict cancer-specific survival following radical radiotherapy in bladder cancer.

INTRODUCTION: Radiotherapy offers the potential of bladder preservation in muscle-invasive bladder cancer, but only a proportion of tumors respond, and there are no accurate predictive methods. The ability of tumor cells to repair DNA damage induced by ionizing radiation influences radiosensitivity. We therefore investigated the prognostic value of the DNA repair proteins APE1 and XRCC1 in patients with muscle-invasive bladder cancer treated by radical radiotherapy. MATERIALS AND METHODS: The tumors of 90 patients with muscle-invasive transitional cell carcinoma and known clinical outcomes were immunostained with APE1 and XRCC1 antibodies. Levels of protein expression were assessed as a percentage of tumor cells with positive nuclear staining (1,000 cells per tumor). RESULTS: The median percentage of nuclear staining for APE1 was 98.7% (range, 42.2-100%) and for XRCC1 was 96.5% (range, 0.6-99.6%). High expression levels of APE1 or XRCC1 (> or = 95% positivity) were associated with improved patient cancer-specific survival (log-rank, P = 0.02 and 0.006, respectively). In a multivariate Cox regression model, APE1 and XRCC1 expression and hydronephrosis were the only independent predictors of patient survival. CONCLUSIONS: Expression levels of both APE1 and XRCC1 proteins were strongly associated with patient outcome following radiotherapy, separating patients with good outcome from the 50% with poor outcome (82% and 44%, 3-year cause-specific survival, respectively). If prospectively validated, this simple test could be incorporated into clinical practice to select patients likely to respond to radiotherapy and consider alternative forms of therapy for those unlikely to respond.

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1.

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.

Evidence for functional localization of the proenkephalin-processing enzyme, prohormone thiol protease, to secretory vesicles of chromaffin cells.

The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephalin-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.

Alpha1-antichymotrypsin-like proteins I and II purified from bovine adrenal medulla are enriched in chromaffin granules and inhibit the proenkephalin processing enzyme "prohormone thiol protease".

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.

The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

Acquisition versus loss of Helicobacter pylori infection in Japan: results from an 8-year birth cohort study.

Studies of the pattern of change in the epidemiology of Helicobacter pylori infection are scarce. A longitudinal cohort study consisted of 644 children and adults, and two independent cross-sectional surveys were conducted in rural Japan between 1986 and 1994. The anti-H. pylori IgG seroconversion rates were 1.1% and 1% per year for children and adults, respectively. The seroreversion rate per year was 1.8% for children and 1.5% for adults. The cohort study was confirmed by the two cross-sectional studies. H. pylori prevalence fell in all age groups in both children (odds ratio [OR] = 0.5, 95% confidence interval [CI] = 0.2-1.0, P = .05) and adults (OR = 0.4, 95% CI = 0.3-0.6, P = .001). The rate of loss of H. pylori infection was greater than the acquisition. Data regarding acquisition and loss of H. pylori infection are critical to understanding the epidemiology of the infection and to developing treatment and vaccination strategies.

Myocardial alpha-thrombin receptor activation induces hypertrophy and increases atrial natriuretic factor gene expression.

The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.

Reversed phase HPLC analysis of proenkephalin-related and prodynorphin-related end-products in the brain of a urodele amphibian, Ambystoma tigrinum.

Acid extracts of the brain of a urodele amphibian, Ambystoma tigrinum, were screened with radioimmunoassays specific for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography a peak of enkephalin-sized immunoreactive material was detected near the total volume of the column. The enkephalin-sized immunoreactivity was further analyzed by reversed phase HPLC. This analysis detected peaks of authentic Met-enkephalin and Leu-enkephalin. However, the molar ratio of Met-enkephalin to Leu-enkephalin in the brain of this amphibian was approximately 80:1. These observations would suggest that the Leu-enkephalin detected in the brain of Ambystoma may be derived from a source other than the Proenkephalin precursor. Neither Met-enkephalin-RGL or Met-enkephalin-RF were detected by radioimmunoassay in brain extracts from this urodele. However, following digestion with trypsin and carboxypeptidase B, a novel peak of C-terminally extended Met-enkephalin was detected. Two peaks of Prodynorphin-related products were also detected following gel filtration chromatography. These immunoreactive forms were detected using antisera specific for alpha-neo-endorphin and dynorphin B(1-13). No immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17) or dynorphin A(1-8) were detected in this species. Reversed phase HPLC analysis indicated that the major form of urodele alpha-neo-endorphin eluted with the same retention time as synthetic mammalian alpha-neo-endorphin. Urodele dynorphin B(1-13)-related immunoreactivity eluted as a single peak, however, this form did not elute with the same retention time as synthetic mammalian dynorphin B(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulated secretion of atrial natriuretic factor from cultured ventricular myocytes.

We have investigated endothelin (ET)-regulated secretion of atrial natriuretic factor (ANF) from primary neonatal rat ventricular myocytes, where hormone release is thought to be constitutive. In a dose-dependent, nifedipine-sensitive manner, ET acutely enhanced ANF release by two- to fivefold over control cultures within 15 min of agonist exposure, demonstrating that ventricular myocytes display a primary characteristic of a regulated secretory cell type. Unlike atrial cultures, ET enhanced ANF release during the first 30 min of exposure; thereafter, secretion rates returned to control levels. KCl, however, effectively enhanced ANF release only during the first 15 min of exposure. Subcellular fractionation of ventricular culture homogenates did not reveal atrial-type dense secretory granules, and pulse-chase labeling experiments showed that the transit time of newly synthesized ANF was short in ventricular myocytes [time required for half of labeled ANF to be released from cells (t1/2) = 0.5-1.5 h) compared with atrial myocytes (t1/2 = 4 h). These results suggest that, whereas ventricular myocytes possess some of the characteristics of a constitutively secreting cell type (e.g., few, if any, dense secretory granules and rapid transit time for newly synthesized hormone); however, they also display the capacity for regulated secretion of ANF in response to the physiological agonist ET.

Clinical Report of 46 Intracranial Tumors with LINAC Based Stereotactic Radiosurgery / 대한치료방사선과학회지

Between July 1988 and December 1992, we treated 45 patients who had deep seated inoperable or residual and/or recurrent intracranial tumors using LINAC based stereotactic radiosurgery at the Department of Therapeutic Radiology, Kangnam St. Mary's Hospital, Catholic University Medical College. Treated intracranial tumors included pituitary tumors(n=15), acoustic neurinomas(n=8), meningiomas(n=7), gliomas(n=6), craniopharyngiomas(n=4), pinealomas(n=3), hemangioblastomas(n=2), and solitary metastatic tumor from lung cancer (n=1). The dimension of treatment field varied from 0.23 to 42.88 cm3(mean ; 7.26 cm3). The maximum tumor doses ranging from 5 to 35.5 Gy (mean; 29.9 Gy) were given, and depended on patients' age, target volume, location of lesion and previous history of irradiation. There were 22 male and 23 female patients. The age was varied from 5 to 74 years of age(a median age; 43 years). The mean duration of follow-up was 35 months (2~55 months). To date, 18(35.1%) of 46 intracranial tumors treated with SRS showed absent or decrease of the tumor by serial follow-up CT and/or MRI and 16(34.8%) were stationary, e.g. growth arrest. From the view point of the clinical aspects, 34(73.9%) of 46 tumors were considered improved status, that is, alive with no evidence of active tumor and 8(17.4%) of them were stable, alive with disease but no deterioration as compared with before SRS. Although there showed slight increase of the tumor in size according to follow-up imagings of 4 cases(pituitary tumor 1, acoustic neurinomas 2, pinealoma 1), they still represented clinically stable status. Clinically, two(4.4%) patients who were anaplastic astrocytoma(n=1) and metastatic brain tumor(n=1) were worsened following SRS treatment. So far, no serious complications were found after treatment. The minor degree headache which could be relieved by steroid or analgesics and transient focal hair loss were observed in a few cases. There should be meticulous long term follow-up in all cases.

Detection of prodynorphin end products in lizard, turtle, and alligator brain extracts.

Heterologous radioimmunoassays (RIAs) for the mammalian prodynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8), and dynorphin B(1-13) were used to screen brain extracts obtained from representatives of the major surviving orders of reptiles: Chelonia (Pseudemys scripta), Squamata (Anolis carolinensis), and Crocodylia (Alligator mississippiensis). Methanol/acid extracts of whole brains obtained from each species were separately fractionated by gel filtration chromatography and reversed-phase HPLC. In all three species, an immunoreactive form of alpha-neo-endorphin was detected with the same retention time as synthetic mammalian alpha-neo-endorphin following reversed-phase HPLC analysis. In all three species, reversed-phase HPLC analysis revealed a novel form of dynorphin B(1-13)-related immunoreactivity. With the available immunological probes, dynorphin A products were only detected in the Anolis brain extracts. Both dynorphin A(1-17) and dynorphin A(1-8) were detected in this species.

The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes.

Cardiac myocytes store the 126-amino acid precursor of atrial natriuretic factor (pro-ANF), yet the mature, bioactive 28-amino acid peptide, ANF-(99-126), and the resulting N-terminal product, ANF-(1-98), are the forms of the hormone that are released by the heart and found in the circulation. Although previous studies have shown that the maturation of ANF takes place in the heart, it is not known whether it occurs in or on the myocyte concurrently with secretion, or whether cleavage takes place postsecretionally on either the myocyte surface or the surface of a nonmuscle cardiac cell. To address these questions, experiments were carried out in the present study using primary atrial cultures that had been prepared such that greater than 90% of the cells were myocytes. Reversed-phase and ion-exchange HPLC, coupled with immunoprecipitation of biosynthetically labeled ANF, showed that the stored peptide, pro-ANF, was cleaved between residues 98 and 99 such that ANF-(1-98) and (99-126) accumulated in the medium. Coupling biosynthetic labeling with timed secretion experiments showed that the extent of ANF processing was not dependent on the time after secretion; maximal levels of processing were observed at all secretion times examined. Additionally, the processing-competent myocyte-enriched cultures were unable to cleave exogenously added pro-ANF. These results indicate that the myocyte is the cell type responsible for pro-ANF maturation and that this cleavage event takes place cosecretionally.

Protein kinase C and calmodulin kinase are required for endothelin-stimulated atrial natriuretic factor secretion from primary atrial myocytes.

Endothelin (ET), a potent stimulator of atrial natriuretic factor (ANF) secretion in atrial myocyte cultures, has been hypothesized to act via the stimulation of protein kinase C (PKC). This study was carried out in order to determine if ET activates PKC in atrial cultures and whether this activation fully accounts for the effects of ET on ANF secretion. By monitoring the phosphorylation of p80 upon exposure to phorbol ester or ET, it was shown that ET activated PKC in atrial cultures, but to a lesser extent than phorbol ester. In contrast, ET stimulated ANF secretion to a level five times greater than phorbol ester, indicating that PKC activation alone does not fully account for the effects of ET on ANF secretion. Down-regulation of PKC or exposure to the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) resulted in a 50% decrease in ET-stimulated ANF secretion. Interestingly, increasing calcium influx with BAY K 8644 stimulated ANF secretion but did not effect the phosphorylation of p80, indicating a PKC-independent pathway of ANF secretion. Similarly, a component of ET-stimulated secretion that required calcium influx was independent of PKC activation but was sensitive to the Ca2+/calmodulin kinase (CaMK) inhibitor KN-62. Complete inhibition of ET-mediated ANF secretion was obtained only in the presence of both H7 and KN-62. These results demonstrate that ET activates PKC in atrial myocyte cultures and that the full effects of ET on ANF secretion require both PKC and Ca2+/calmodulin kinase activities.

Studies of ANF processing and secretion using a primary cardiocyte culture model.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.