Tenckhoff catheter; fracture
The incidence of prostate cancer is much lower in Asian men than in Western men. This study investigated whether prostate cancer is associated with prostatitis, benign prostatic hyperplasia (BPH), and other medical conditions in the low-incidence population.
From the claims data obtained from the universal National Health Insurance of Taiwan, we identified 1184 patients with prostate cancer diagnosed from 1997 to 2008. Controls comprised 4736 men randomly selected from a cancer-free population. Both groups were 50 years of age or above. Medical histories between the two groups were compared.
Multivariate logistic regression analysis showed that prostatitis and BPH had stronger association with prostate cancer than the other medical conditions tested. Compared with men without prostatitis and BPH, a higher odds ratio (OR) for prostate cancer was associated with BPH (26.2, 95% confidence interval (CI) 20.8–33.0) than with prostatitis (10.5, 95% CI=3.36–32.7). Men with both conditions had an OR of 49.2 (95% CI=34.7–69.9).
Men with prostate cancer have strong association with prostatitis and/or BPH. Prostatitis interacts with BPH, resulting in higher estimated relative risk of prostate cancer in men suffering from both conditions.
benign prostatic hyperplasia; prostatitis; diabetes; interaction; prostate cancer
This study compared the odds ratio (OR) of surgical site infection (SSI) within 30 days after operation with general anaesthesia (GA) or neuraxial anaesthesia (NA) in Taiwanese women undergoing Caesarean delivery (CD).
An epidemiologic design was used. The study population was based on the records of all deliveries in hospitals or obstetric clinics between January 2002 and December 2006 in Taiwan. Anonymized claim data from the Taiwan National Health Insurance Research Database (NHIRD) were analysed. Women who received CD were identified from the NHIRD by Diagnosis-Related Group codes. The mode of anaesthesia was defined by order codes. Multivariate logistic regression was used to estimate the OR and associated 95% confidence interval (CI) of post-CD SSIs for GA when compared with NA. The outcome was whether a woman had been diagnosed as having an SSI during the hospitalization or was re-hospitalized within 30 days after CD for the treatment of SSIs using five or 81 International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes.
Among the 303 834 Taiwanese women who underwent CD during the 5 yr observation period, the 30 day post-CD SSI rate was 0.3% or 0.9% based on five or 81 ICD-9-CM codes. The multivariate-adjusted OR of having post-CD SSIs in the GA group was 3.73 (95% CI, 3.07–4.53) compared with the NA group (P<0.001) using five ICD-9-CM codes for the definition of SSI.
GA for CD was associated with a higher risk of SSI when compared with neuraxial anaesthesia.
anaesthesia; Caesarean section; general anaesthesia; neuraxial anaesthesia; surgical site infection
Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK−/− mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.
DAPK; MARK/PAR-1; tau phosphorylation; neurodegeneration; microtubules
Gonadotrophin-releasing hormone (GnRH) neurones control the onset and maintenance of fertility. Aberrant development of the GnRH system underlies infertility in Kallmann syndrome [KS; idiopathic hypogonadotropic hypogonadism (IHH) and anosmia]. Some KS patients harbour mutations in the fibroblast growth factor receptor 1 (Fgfr1) and Fgf8 genes. The biological significance of these two genes in GnRH neuronal development was corroborated by the observation that GnRH neurones were severely reduced in newborn transgenic mice deficient in either gene. In the present study, we hypothesised that the compound deficiency of Fgf8 and its cognate receptors, Fgfr1 and Fgfr3, may lead to more deleterious effects on the GnRH system, thereby resulting in a more severe reproductive phenotype in patients harbouring these mutations. This hypothesis was tested by counting the number of GnRH neurones in adult transgenic mice with digenic heterozygous mutations in Fgfr1/Fgf8, Fgfr3/Fgf8 or Fgfr1/Fgfr3. Monogenic heterozygous mutations in Fgfr1, Fgf8 or Fgfr3 caused a 30–50% decrease in the total number of GnRH neurones. Interestingly, mice with digenic mutations in Fgfr1/Fgf8 showed a greater decrease in GnRH neurones compared to mice with a heterozygous defect in the Fgfr1 or Fgf8 alone. This compounding effect was not detected in mice with digenic heterozygous mutations in Fgfr3/Fgf8 or Fgfr1/Fgfr3. These results support the hypothesis that IHH/KS patients with digenic mutations in Fgfr1/Fgf8 may have a further reduction in the GnRH neuronal population compared to patients harbouring monogenic haploid mutations in Fgfr1 or Fgf8. Because only Fgfr1/Fgf8 compound deficiency leads to greater GnRH system defect, this also suggests that these fibroblast growth factor signalling components interact in a highly specific fashion to support GnRH neuronal development.
fibroblast growth factor receptor; fibroblast growth factor 8; gonadotrophin-releasing hormone neurones; hypogonadotropic hypogonadism; Kallmann syndrome
To assess the magnitude of cigarette smuggling after the market opened in Taiwan.
Review of tobacco industry documents for references to smuggling activities related to Taiwan and government statistics on seizure of smuggled cigarettes.
The market opening in 1987 led to an increase in smuggling. Contraband cigarettes became as available as legal ones, with only a small fraction (8%) being seized. Being specifically excluded from the market‐opening, Japan entered the Taiwan market by setting up a Swiss plant as a legal cover for smuggling 10–20 times its legal quota of exports to Taiwan. Smuggling in Taiwan contributed to increased consumption of foreign brands, particularly by the young. Taiwan, not a member of the World Health Organization, was excluded from the East Asian 16‐member “Project Crocodile”, a regional anti‐smuggling collaborative effort to implement the Framework Convention on Tobacco Control.
Taiwan showed a sharp increase in smuggling after market liberalisation. Being excluded from the international community, Taiwan faces an uphill battle to fight smuggling alone. If Taiwan remained as its weakest link, global efforts to reduce tobacco use will be undermined, particularly for countries in the East Asian region.
industry documents; market opening; smuggling
Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity, and visual function. The authors hypothesise that many of these proteins originate from the meibomian gland. This study's aim was to begin to develop the proteomic methodology to permit the testing of their hypothesis.
Meibomian gland secretions were collected from the lower eyelids of adult volunteers and placed in a chloroform‐methanol mixture. Samples were partitioned in a biphasic system and non‐lipid phase materials were reduced, alkylated, and trypsin digested to obtain peptides for protein identification. This peptide mixture was separated by µ‐capillary reverse phase chromatography and the effluent examined by nano‐electrospray MS and data dependent MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra.
The methodological approach to date has permitted the identification of more than 90 proteins in human meibomian gland secretions. Proteins include the α2‐macroglobulin receptor, IgA α chain, farnesoid X activated receptor, interferon regulatory factor 3, lacritin precursor, lactotransferrin, lipocalin 1, lysozyme C precursor, potential phospholipid transporting ATPase IK, seven transmembrane helix receptor (also termed somatostatin receptor type 4), testes development related NYD‐SP21 (also termed high affinity IgE receptor β subunit), and TrkC tyrosine kinase.
These findings indicate that the meibomian gland secretes a number of proteins into the tear film. It is quite possible that these proteins contribute to the dynamics of the tear film in both health and disease.
meibomian gland; protein; tear film; proteomics
Aims: To assess the relations between exposure to traffic exhausts and indicators of oxidative DNA damage among highway toll station workers.
Methods: Cross-sectional study of 47 female highway toll station workers exposed to traffic exhausts and 27 female office workers as a reference group. Exposure assessment was based on average and cumulative traffic density and a biomarker of exposure, urinary 1-hydroxypyrene-glucuronide (1-OHPG). Urinary 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. Plasma nitric oxide (NO) was measured as an indicator of oxidative stress related to traffic exhaust exposure.
Results: The mean concentration of urinary 8-OHdG was substantially higher among the exposed non-smokers (13.6 µg/g creatinine) compared with the reference non-smokers (7.3 µg/g creatinine; difference 6.3, 95% CI 3.0 to 9.6). The mean concentration of NO among the exposed (48.0 µmol/l) was also higher compared with the reference non-smokers (37.6 µmol/l; difference 10.4, 95% CI –0.4 to 21.2). In linear regression adjusting for confounding, a change in log(8-OHdG) was statistically significantly related to a unit change in log(1-OHPG) (ß = 0.372, 95% CI 0.081 to 0.663).
Conclusions: Results indicate that exposure to traffic exhausts increases oxidative DNA damage. Urinary 8-OHdG is a promising biomarker of traffic exhaust induced oxidative stress.
Baicalein is a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, a medicinal herb that has been used since ancient times to treat bacterial infections. As little is known concerning its pharmacokinetics, this study focussed on its pharmacokinetics as well as the possible roles of the multidrug transporter P-glycoprotein on its distribution and disposition.Three microdialysis probes were simultaneously inserted into the jugular vein, the hippocampus and the bile duct of male Sprague–Dawley rats for sampling in biological fluids following the administration of baicalein (10, 30 and 60 mg kg−1) through the femoral vein. The P-glycoprotein inhibitor cyclosporin A was used to help delineate its roles.The study design consisted of two groups of six rats in parallel: control rats which received baicalein alone and the cyclosporin A treated-group in which the rats were injected cyclosporin A, a P-glycoprotein inhibitor, 10 min prior to baicalein administration.Cyclosporin A treatment resulted in a significant increase in elimination half-life, mean residence time and area under the concentration versus time curve (AUC) of unbound baicalein in the brain. However, AUC in the bile was decreased.The decline of baicalein in the hippocampus, blood and bile suggested that there was rapid exchange and equilibration between the peripheral compartment and the central nervous system. In addition, the results indicated that baicalein was able to penetrate the blood–brain barrier as well as undergoing hepatobiliary excretion.Although no direct transport studies were undertaken and multiple factors may affect BBB penetration and hepatobiliary excretion, strong association of the involvement of P-glycoprotein in these processes is indicated.
Baicalein; cyclosporin A; hepatobiliary excretion; microdialysis; P-glycoprotein; pharmacokinetics
Our objective in this study was to assess the effect of using two kinds of lead-free gasoline [including 92-lead-free gasoline (92-LFG) and 95-lead-free gasoline (95-LFG), rated according to their octane levels] to replace the use of premium leaded gasoline (PLG) on the emissions of polycyclic aromatic hydrocarbons (PAHs) and their corresponding benzo[a]pyrene equivalent (BaP(eq)) amounts from the gasoline-powered engine. The results show that the three gasoline fuels originally contained similar total PAHs and total BaP(eq) contents; however, we found significant differences in the engine exhausts in both contents. The above results suggest that PAHs originally contained in the gasoline fuel did not affect the PAH emissions in the engine exhausts. The emission factors of both total PAHs and total BaP(eq) obtained from the three gasoline fuels shared the same trend: 95-LFG > PLG > 92-LFG. The above result suggests that when PLG was replaced by 95-LFG, the emissions would increase in both total PAHs and total BaP(eq), but when replaced by 92-LFG would lead to the decreased emissions of both contents. By taking emission factors and their corresponding annual gasoline consumption rates into account, we found that both total PAH and total BaP(eq) emissions increased from 1994 to 1999. However, the annual increasing rates in total BaP(eq) emissions were slightly higher than the corresponding increasing rates in total PAHs.
OBJECTIVES: A 10 year extension of follow up (up to 1993) of 863 employees who had potential exposure to epichlorohydrin at two chemical plants between May 1948 and December 1965 was conducted to further evaluate the previously reported potential association between exposure to epichlorohydrin and heart disease. METHODS: The mortality observed was compared with that expected from the death rates from the local male population where these chemical plants are located. Workers were assigned to one of five exposure categories based on their job with the highest level of potential exposure. Vital status was ascertained to the end of 1993. RESULTS: Among diseases of particular interest, there were no excess deaths from heart disease (standardised mortality ratio (SMR) 63.3), lung cancer (SMR 63.8), or non-malignant respiratory disease (SMR 37.7) for employees with 20 or more years after first exposure. Based on the level of potential exposure to epichlorohydrin, mortality for heart disease was slightly higher (SMR 75.7, 95% confidence interval (95% CI) 51.8-106.7) in the moderate to heavy exposure group than in the none to light exposure group (SMR 59.5, 95% CI 37.7-89.3); this difference is well within the range of random variation. The SMR for heart disease was 90.4 among employees who had both probable exposure to allyl chloride and moderate to heavy exposure to epichlorohydrin, although it was 88.1 among employees who had moderate to heavy potential exposure to epichlorohydrin but no exposure to allyl chloride. CONCLUSIONS: This study does not support an association between exposure to epichlorohydrin and heart disease or lung cancer. There were no additional deaths from leukaemia in this update; the raised SMR for leukaemia noted in the previous study has substantially decreased from 500.0 to 161.3 (95% CI 33.2-471.0) and is not significant. The overall mortality and cancer mortality of employees potentially exposed to epichlorohydrin continued to be lower than that of the local population.
OBJECTIVES--This paper describes a study that was carried out in the primary nickel production industry to investigate the levels of personal exposure to aerosols containing nickel and the impact on exposure assessment of introducing new personal sampling techniques with performance consistent with the latest particle size-selective criteria. METHODS--Experiments were carried out at workplaces in mining, milling, smelting, and refining works to investigate the effect of changing from the current method of total aerosol (with the widely used 37 mm filter holder) to the new method of measuring inhalable aerosol (with the Institute of Occupational Medicine (IOM) inhalable aerosol sampler). RESULTS--The results show that inhalable aerosol exposure concentrations--for both overall aerosol and for total nickel--were consistently and significantly higher than the corresponding total aerosol concentrations. Weighted least squares linear regression yielded IOM/37 mm factors ranging from about 1.2 to 4.0. The exposure data for each company process were found to be log-normally distributed. CONCLUSIONS--The results suggest the possibility of generating a single pragmatic factor for each company process for converting current total aerosol exposures to new exposures based on the inhalability concept contained in the latest particle size-selective criteria for aerosol exposure assessment. Such data may be important in determining new occupational exposure limits for nickel.
OBJECTIVES--The study was undertaken to update a previous study of employees from a resins and plastics research and development facility and to further examine the mortality of these employees with particular emphasis on deaths due to pancreatic cancer. METHODS--This retrospective cohort study examined mortality from 1962 to 1992 for 257 men who were employed for at least one year during a 14 year period from 1962 to 1975 at a plastics and resins research and development facility. During the operative period, the primary activities involved applications and process development for polypropylene, polystyrene, epoxy resins, and to a lesser extent high density polyethylene. RESULTS--The cohort was young and was followed up for an average of 26 years. Although mortality for all causes among employees who worked at least one year at this facility was low (standardised mortality ratio (SMR) 0.74), the death rate from cancer was moderately higher than that of the general population (14 observed and 9.4 expected deaths). There were four observed and 0.5 expected deaths from pancreatic cancer among men who worked at this facility for at least one year, which resulted in a statistically increased SMR of 8.88 (95% confidence interval 2.42-22.74). All cases of pancreatic cancer had "laboratory" jobs, and their ages at death were relatively young compared with deaths in the general population from pancreatic cancer. Lung cancer mortality was high but not significant with seven observed and 3.5 expected deaths. There were no deaths due to non-malignant respiratory disease (1.9 expected). CONCLUSIONS--The increased cancer mortality was entirely due to excess deaths from pancreatic and lung cancers. No causative agent or process for these cases of pancreatic cancer has been identified. This study shows no increased colorectal cancer mortality as was found among another group of workers involved in the manufacture of polypropylene.
A retrospective mortality analysis and prospective morbidity and haematological analyses were performed for Shell Deer Park Manufacturing Complex (DPMC) male employees who worked in jobs with potential exposure to 1,3-butadiene from 1948 to 1989. 614 employees qualified for the mortality study (1948-89), 438 of those were still employed during the period of the morbidity study (1982-9), and 429 of those had haematological data available for analysis. Industrial hygiene data from 1979 to 1992 showed that most butadiene exposures did not exceed 10 ppm (eight-hour time weighted average (8 hour TWA)), and most were below 1 ppm, with an arithmetic mean of 3.5 ppm. 24 deaths occurred during the mortality study period. For all causes of death, the standardised mortality ratio (SMR) was 48 (95% confidence interval (95% CI) = 31-72), and the all cancer SMR was 34 (95% CI = 9-87). There were only two deaths due to lung cancer (SMR 42, 95% CI = 5-151) and none due to lymphohaematopoietic cancer (expected = 1.2). Morbidity (illness absence) events of six days or more for the 438 butadiene employees were compared with the rest of the complex. No cause of morbidity was in excess for this group; the all cause standardised morbidity ratio (SMbR) was 85 (95% CI = 77-93) and the all neoplasms SMbR was 51 (95% CI = 22-100). Haematological results for the 429 with laboratory data were compared with results for the rest of the complex. No significant differences occurred between the two groups and the distributions of results between butadiene and non-butadiene groups were virtually identical. These results suggest that butadiene exposures at concentrations common at DPMC in the past 10-20 years do not pose a health hazard to employees.
Early diabetes mellitus is characterized by impaired responses to pressor hormones and pressor receptor downregulation. The present study examined the effect of elevated extracellular glucose concentrations on angiotensin II (AII) and arginine vasopressin (AVP) receptor kinetics in cultured rat vascular smooth muscle cells (VSMC). Scatchard analysis of [3H]AVP and 125I-AII binding to confluent VSMC showed that high glucose concentrations (20 mM) similarly depressed AVP and AII surface receptor Bmax but did not influence receptor Kd. This receptor downregulation was not reproduced by osmotic control media containing either L-glucose or mannitol. Receptor downregulation was maximal at a glucose concentration of 15-20 mM and required 24-48 h for a maximum effect. Normalization of the extracellular glucose concentration allowed complete recovery of AVP and AII binding within 48 h. Receptor downregulation was associated with depressed AVP and AII-stimulated intracellular signaling and cell contraction. High glucose concentrations induced a sustained activation of protein kinase C (PKC) in VSMC, which was prevented by coincubation with H-7. H-7 also markedly attenuated glucose-induced downregulation of AVP and AII receptors on VSMC. This study demonstrates a novel cellular mechanism whereby high extracellular glucose concentrations directly and independently downregulate pressor hormone receptors and their function on vascular tissue via glucose-stimulated PKC activation.
This study examined the morbidity experience from 1981 to 1988 of a prospective cohort of 3422 refinery and petrochemical plant employees from the Shell Deer Park manufacturing complex. The morbidity data for this study, which include all illness and absence records in excess of five days, were extracted from the morbidity section of the Shell health surveillance system. Standardised morbidity ratios (SMRs) of disease prevalence in this cohort were calculated using an internal comparison group of all manufacturing employees of the Shell Oil Company. Among production employees, the overall morbidity was statistically significantly higher (SMR = 109) than that of the comparison group. Illness due to hypertension (SMR = 144), haemorrhoids (SMR = 149), diseases of the nervous system (SMR = 120), respiratory system (SMR = 108), and digestive system (SMR = 117) were also raised for this group. The increased risk due to these medical conditions does not appear to be associated with occupational factors. Lymphatic and haematopoietic tissue neoplasms were raised (SMR = 124), but were based on only four cases.
Results for a prospective morbidity study of 14,170 refinery and chemical workers from 1981 through 1988 are presented. Illness/absence data for this study were extracted from the morbidity section of the Shell Health Surveillance System which includes records of all illness/absences in excess of five days. Age adjusted annual morbidity frequency rates and annual durations of absence are presented by age, sex, job, and work status. Generally, rates and durations of absence were highest for older age groups, women, and production workers. Increased risk was associated with the presence of known disease risk factors. Overall, 48% of the employees had at least one illness/absence in excess of five days during the eight year period. Twelve per cent of the employees had four or more absences, which accounted for 54% of the total number of absences and 52% of the total work days lost. Among men, the five most common conditions accounted for 72% of all illness/absences. In descending order they were injuries (25%), respiratory illnesses (17%), musculoskeletal disorders (14%), digestive illnesses (9%), and heart disease (7%). Similar patterns were noted among women. These findings may be useful in setting priorities and directing efforts such as health education programmes and other strategies for the prevention of disease.
The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.
This study examined the morbidity experience from 1981 to 1988 of two cohorts (Shell cohort and Enterline cohort) of workers who had potential exposure to epichlorohydrin (ECH). The morbidity prevalence data for this study were extracted from the morbidity section of the Shell health surveillance system which included all illness and absence records in excess of five days. For both cohorts, the standardised morbidity ratios (SMRs) for all causes and all neoplasms were similar to an internal comparison group. There were no increases in heart disease morbidity for the Shell cohort (SMR = 97) or the Enterline cohort (SMR = 90). The SMRs for heart disease in the lower exposure group of the Shell cohort were 101 and 93 for the corresponding Enterline cohort. They were 92 and 87, respectively, in the higher exposure group. The increased risk of heart disease mortality reported by Enterline et al in workers more heavily exposed to ECH was not confirmed in this morbidity study. Morbidity from skin and subcutaneous tissue disorders, however, was found to be increased significantly in the Shell cohort. The SMR was 98 for the lower exposure group and 195 for the higher exposure group. A review of the original morbidity reports for each case suggested that factors unrelated to exposure to ECH such as the physical demands of a particular job, amount of time outside--for example, exposure to poison ivy--and other underlying medical conditions may be of greater importance than exposure to ECH.
The properties and sequence of an oligomeric antigen of Treponema pallidum are presented. Antigen C1-5 assembles into oligomers of 140,000 and greater. The nucleotide sequence predicts an open reading frame for a protein monomer of 19,400, confirmed by amino-terminal sequencing of the recombinant antigen.
The cellular mechanism of the vasodilatory action of atriopeptin III (APIII) on vasopressin (AVP)-induced Ca2+ mobilization and cell shape change in cultured vascular smooth muscle cells (VSMC) was studied. APIII (10(-8) M) attenuated the increase of intracellular free Ca2+, [Ca2+]i, induced by 10(-8) M AVP (234.0 +/- 14.8 vs. 310.0 +/- 28.4 nM, P less than 0.01). Similar results were obtained in 45Ca2+ efflux experiments. APIII (10(-7) M), however, did not alter AVP-induced inositol trisphosphate (IP3) production, although the levels of inositol-1-phosphate were significantly reduced. The effect of APIII to block or attenuate AVP-induced Ca2+ mobilization was associated with an inhibition of AVP-stimulated cell shape change. The effect of atrial natriuretic factor (ANF) on cell shape, however, occurred at lower ANF concentrations than the effect on the Ca2+ mobilization. APIII stimulated production of cyclic guanosine monophosphate (cGMP) in VSMC. The effect of APIII on AVP-stimulated Ca2+ mobilization was partially mimicked by the stable nucleotide 8-bromo cGMP and was not affected by the soluble guanylate cyclase inhibitor, methylene blue (10(-4) M). These results suggest that APIII exerts its vasodilatory effect, in part, by interference with vasopressor-stimulated Ca2+ mobilization in vascular smooth muscle cells, perhaps by stimulating particulate guanylate cyclase and cGMP. However, an effect of ANF on the contractile mechanism at a site independent of Ca2+ release is also suggested by the present results.
We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. At saturating concentrations, the binding of 125I-NAPA-DP-insulin to the isolated adipocytes at 16 degrees C was rapid (half-maximal in approximately 1 min and maximal in approximately 10 min) and approximately 25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis of the photolabeled cellular proteins by PAGE in the absence of disulfide reductants revealed the specific labeling of a major protein band of Mr 330,000 and two less intense bands of Mr 295,000 and 260,000. Upon reduction of disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled Mr-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin. When intact adipocytes photolabeled at 16 degrees C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16 degrees C all of the labeled insulin-receptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 37 degrees C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by approximately 30 min of incubation at 37 degrees C. Cellular processing of the internalized insulin-receptor complexes also occurred, since incubation at 37 degrees C (but not 16 degrees C) resulted in the generation of a Mr-115,000 component from the labeled receptors. Inclusion of chloroquine, a drug with lysosomotropic properties, in the incubation media caused a time-dependent increase (maximal increase of 50% above control by 2 h at 37 degrees C) in the intracellular pool of labeled receptors. In contrast to these findings in human adipocytes, no appreciable internalization of insulin-receptor complexes and no chloroquine effect was observed in cultures human IM-9 lymphocytes during a 1-h incubation at 37 degrees C. We concluded that in isolated human adipocytes: (a) the subunit structure of insulin receptors is the same as that reported for several other tissues, (b) insulin-receptor complexes are rapidly internalized and processed at physiologic temperatures, and (c) the cellular processing of insulin-receptor complexes occurs at one or more chloroquine-sensitive intracellular site(s).