Factor VIII mutations in 42 Moldovan haemophilia A families, including 12 that are novel.

Haemophilia A (HA) is a bleeding disorder caused by mutations within the X-linked F8 gene. A series of 42 unrelated Moldovan patients with HA had their disease-causative mutation determined to provide clinically valuable genotyping information for a historically underserved population and to utilize factor VIII (FVIII) structural information to analyse the effects of haemophilic missense substitutions. DNA samples were analysed to detect intron 22 and intron 1 inversions followed by heteroduplex analysis of PCR-amplified fragments containing all exonic sequences. Missense sites identified by DNA sequencing were visualized in the recently described crystal structures of human FVIII. Of the 26 different point mutations, 12 were novel. Gel electrophoresis identified samples with a second major DNA band that migrated abnormally; these amplified products were sequenced. Thirteen intron 22 inversions and one intron 1 inversion were found. Two patients had large, partial gene deletions and there were six frameshift, two non-sense, two splicing and 16 missense genotypes. Two subjects with an intron 22 inversion and one with a large, partial gene deletion developed an alloimmune inhibitor. Their localization suggests intra- and possibly inter-molecular interactions that are important for the structural integrity and/or procoagulant function of FVIII.

Effect of prenatal exposure to bisphenol A on the serum testosterone concentration of rats at birth.

In the rat, testosterone (T) in the neonatal period plays an important role in sexual differentiation and there is a serum T surge in male rats 2 hours after birth. Pregnant female rats were exposed to various doses of bisphenol A (BPA) from gestational day 1 (GD1) through 2 hours after parturition. About half of the BPA-exposed and control dams were subjected to cesarean section on GD22. The male fetuses on GD22 were immediately sacrificed and blood was collected. The other half of the BPA-treated and control dams delivered at GD23 (parturition day). The male pups were sacrificed 2 hours after birth. Serum T concentration was determined by radioimmunoassay (RIA). The BPA concentration in the fetal serum on GD22 increased inversely to the T levels in the serum. The T concentration in the pups' serum 2 hours after birth decreased inversely to the BPA concentration in the serum. However, there were no differences in the serum T concentration among the various doses of BPA. These results suggest that exposure to BPA in utero inhibits the T surge in the neonatal period and we speculate that exposure to BPA in utero disrupts the endocrine environment in the neonatal male.

Genetic diagnosis of haemophilia and other inherited bleeding disorders.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

Severe hemophilia A due to a 1.3 kb factor VIII gene deletion including exon 24: homologous recombination between 41 bp within an Alu repeat sequence in introns 23 and 24.

Partial or complete factor (F)VIII gene deletions are found in about 5% of families with severe hemophilia A. Relatively few deletions have been well characterized and, of these, recombination occurred between either common repeat elements or non-homologous sequences. In evaluating a family with severe hemophilia A, an exon 24 deletion was suspected when no fragment was obtained on attempted PCR amplifications. A combination of the 5' primer of exon 23 and the 3' primer of exon 25 fragments was used with prolonged extension times to amplify a normal 2.9 kb fragment that included exons 23 through 25; the patient's amplified product was 1.6 kb indicating a 1.3 kb deletion. A mixture of normal and patient DNA showed both sized fragments as did that from an obligate carrier. Carrier detection was applied to two women at risk; one was and one was not a carrier. Sequencing the proband's 1.6 kb fragment revealed that a 1328 bp deletion occurred between homologous sequences of 287 and 285 bp in introns 23 and 24, respectively; these share 85% identity. Blast nucleotide search revealed that these represent Alu elements. Comparison with an alignment of each of the two homologous sequences further localized recombination to a 41-bp segment. However, a simple recombination event would not account for the proband's sequence. The most likely explanation is that the homologous recombination was accompanied by incomplete mismatch repair.

Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997.

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus-positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa-group with long electropherotype (e-type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN-group with a short e-type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4-96.3%, amino acid identity) and 116E from India (92-92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996-1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1-14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes.

Impaired testicular function in rats with diet-induced hypercholesterolemia and/or streptozotocin-induced diabetes mellitus.

Hypercholesterolemia and diabetes mellitus are known to be accompanied by reproductive dysfunction. In this study, we investigated the effects of hypercholesterolemia, hyperglycemia, and these conditions combined, on testosterone (T) and testicular luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding. Sprague-Dawley rats (8 weeks old) were divided into four groups: Group 1 was the control, group 2 was fed standard chow containing 2% cholesterol (C-diet), group 3 was administered streptozotocin (STZ, 65 mg/kg, i.p.), group 4 was treated with both the C-diet and STZ. After 4 weeks, rats were sacrificed. Serum glucose was significantly higher in the STZ group (304% that of controls) and the C-diet plus STZ group (345%), but there was no difference between the C-diet group (89%) and the control group. Serum cholesterol was significantly higher in the C-diet group (206% that of controls), the STZ group (452%) and the C-diet plus STZ group (2042%). Serum T, testicular T, and LH/hCG binding were significantly lower in the C-diet group (49%, 52%, and 81% that of controls, respectively), the STZ group (15%, 32%, and 72%) and the C-diet plus STZ group (8%, 21%, and 57%). These results suggest that hypercholesterolemia is an independent risk factor for testicular dysfunction and that the reduction of serum and testicular T levels is due at least in part to a reduction in testicular LH/hCG binding in rats with hypercholesterolemia, hyperglycemia, and these conditions combined. It is further suggested that the reduction in LH/hCG binding is mainly related to a rise in serum cholesterol levels.

Ancestral residues stabilizing 3-isopropylmalate dehydrogenase of an extreme thermophile: experimental evidence supporting the thermophilic common ancestor hypothesis.

Ancestral amino acid residues were inferred for 3-isopropylmalate dehydrogenase (IPMDH), and were introduced into the enzyme of an extreme thermophile, Sulfolobus sp. strain 7. The thermostability of the mutant enzymes was compared with that of the wild type enzyme. At least five of the seven mutants tested showed higher thermal stability than the wild type IPMDH. The results are compatible with the hyperthermophilic universal ancestor hypothesis. The results also provide a new method for designing thermostable enzymes. The method only relies on the first dimensional structures of homologous enzymes that can be obtained from genetic databases.

Effects of PR-350, a newly developed radiosensitizer, on dihydropyrimidine dehydrogenase activity and 5-fluorouracil pharmacokinetics.

This study was designed to investigate the effects of PR-350, a newly developed radiosensitizer, on dihydropyrimidine dehydrogenase (DPD) activity and 5-fluorouracil (5-FU) pharmacokinetics in 8-week-old male Sprague-Dawley rats. In an in vitro study with hepatic cytosol, DPD activity was dose-dependently reduced by PR-350 at 0.5, 1.0, and 2.0 mmol/l to 75.5%, 64.9%, and 61.5%, respectively, of the control values. In an ex vivo study, DPD activities in hepatic cytosols obtained from animals which had received PR-350 over 4 days (200 mg/kg per day) were not significantly different from those in animals which had not. In an in vivo study, none of the pharmacokinetic parameters obtained from the plasma concentration-time profile of 5-FU were significantly altered by single i.v. injections of PR-350 (50, 100, or 200 mg/kg). However, (E)-5-(2)-(bromovinyl)uracil (BVU), a DPD inhibitor, significantly increased the half-life and area under the curve of 5-FU to 238.1% and 323.2%, respectively, of the control values. Administration of PR-350 over 4 days (200 mg/kg per day) did not affect either of these parameters. The administration of PR-350 significantly reduced the clearance (73.5% of control) and volume of distribution (71.0% of control) of 5-FU, but the alterations were much less than those caused by BVU. These results suggest that the effect of PR-350 on 5-FU pharmacokinetics is much less than that of BVU and that the enhancement of 5-FU toxicity by PR-350 is less than we initially anticipated.

Some factor VIII exon 14 frameshift mutations cause moderately severe haemophilia A.

Factor VIII's exon 14 codes for its B domain that includes nearly one-third of its amino acid sequence that is not necessary for function. Frameshift mutations appear to occur more frequently within exon 14 than in other exons. To assess exon 14 frameshift mutations and their clinical correlates, a series of unrelated, severe or moderately severe haemophilia A patients were screened for heteroduplex formation in amplified exon 14 fragments. In 25 families, a frameshift mutation was identified by sequencing. Occurrence of haemophilia was isolated in 18 of these families. Moderate severity was noted in at least six out of 13 families with an A insertion or deletion at one of two sequences where the frameshift resulted in a sequence of 8-10 As. Inhibitors occurred in five of the other 12 families including one with an A insertion within a sequence of six As. Recurrent insertions into an A(8) (codons 1439-1441) or an A(9) (codons 1191-1194) sequence or of an A deletion from the A(9) sequence are common, recurrent causes of haemophilia A that may have a moderately severe phenotype.

Effects of estrogen on serum leptin levels and leptin mRNA expression in adipose tissue in rats.

OBJECTIVE: In this study, we examined changes in serum leptin levels during the estrus cycle and the role of estrogen in these changes. METHODS: We measured serum leptin levels during normal estrus cycles in intact rats and estradiol-17beta (E2)-induced artificial estrus cycles in ovariectomized rats. RESULTS: Serum leptin levels increased 1.6-fold from 4.2 +/- 0.2 ng/ml during diestrus stage 2 to 6.7 +/- 0.9 ng/ml during proestrus stage during the 4-day estrus cycle. During the E2-induced estrus cycle, serum leptin levels increased 2.3-fold from 2.3 +/- 0.1 ng/ml at estrus to 5.4 +/- 1.2 ng/ml at proestrus. E2 also increased serum leptin concentrations and leptin mRNA expression in adipose tissue of immature rats. DISCUSSION: These findings suggest that increased serum leptin induced by estrogen during proestrus may trigger the preovulatory release of luteinizing hormone. Furthermore, our findings indicate that estrogen has a positive effect on leptin production in adipose tissue.

Quantitation by (1)H-NMR of dolichol, cholesterol and choline-containing lipids in extracts of normal and phathological thyroid tissue.

Proton magnetic resonance spectroscopy at 1.9 T was used to quantify dolichols, cholesterols, choline-containing phospholipids and double bonds in unsaturated acyl chains in lipid extracts of four types of thyroid tissue [normal (n = 27), papillary cancer (n = 15), adenoma (n = 13) and Basedow disease (n = 6)]. In normal thyroid the mean concentrations of dolichol, cholesterol and phospholipids were 1.2, 3.6 and 2.1 micromol/g wet weight, respectively. The concentrations of these lipids exhibited positive mutual correlations and positive correlations with patient age. The increase in dolichol in elderly human thyroid may be due to the accumulation of lysosomes and may help to compensate for the decrease in the activity of lysosomal enzymes and in thyroid hormone production and release. Dolichol concentrations were significantly lower in papillary cancer (0.4 micromol/g) and Basedow disease (0.3 micromol/g) compared to normal thyroid (p < 0.01 and p < 0.05, respectively), while cholesterol was enhanced only in cancer tissue (10.7 micromol/g). Benign adenoma exhibited normal levels of both dolichol and cholesterol. These results suggest that the synthesis and accumulation of isoprenoids are normal in adenoma but not in cancer.

Rotavirus infection in children in Japan.

Currently, a high morbidity of rotavirus diarrhea has been seen in children in developed and developing countries. Improvement of the vaccines is necessary in order to reduce the burden of diarrhea caused by rotavirus. A survey of rotavirus infection from diarrheal stool specimens in children of seven regions in Japan was conducted from 1984 to 1999. The present study discusses the survey results and reviews the national and international data of more than 23 papers and congress proceedings about rotavirus infection in Japan. We analyze the prevalence of rotavirus infection in acute diarrheal in- and outpatients, the distribution of rotavirus G-serotypes and surveillance data for seasonality and age groups in Japan. The data indicated that rotavirus is the most important cause of diarrhea in Japan among young children, with the prevalence ranging from approximately 9.7 to 88%. The most common rotavirus strains belonged to serotype G1, specifically since 1993. Serotypes G2, G3 and G4 had also been documented to be predominantly based in the area and year before 1992. However, untypeable rotavirus strains had been found each year, with a prevalence up to 56.7% which suggests that rare serotypes (except G1-4) or new serotypes might exist. Unexpectedly, in Tokyo and Sapporo from 1998 to 1999, G9 was found to be the first most prevailing serotype with a high prevalence of 52.9 and 71.4%, respectively. Despite these data from different geographic areas, the year under investigation was relatively clear in respect to seasonality, with a peak of rotavirus activity in late winter (February) through early spring (March). Age distribution had also characterized that the infection was predominant among children aged 1-2 years of age, although it was also common in children of 2-3 years. In addition, mixed infection with bacteria was documented.

Hemophilic factor VIII C1- and C2-domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure.

Factor VIII C domains contain key binding sites for von Willebrand factor (vWF) and phospholipid membranes. Hemophilic patients were screened for factor VIII C-domain mutations to provide a well-characterized series. Mutated residues were localized to the high-resolution C2 structure and to a homology model of C1. Of 30 families found with mutations in the C domains, there were 14 missense changes, and 9 of these were novel. Of the missense mutations, 10 were associated with reduced vWF binding and 8 were at residues with surface-exposed side chains. Six of the 10 mutants had nearly equivalent factor VIII clotting activity and antigen level, suggesting that reduced vWF binding could cause hemophilia by reducing factor VIII stability in circulation. When the present series was combined with previously described mutations from an online international database, 11 C1 and C2 mutations in patients with mild or moderately severe hemophilia A were associated with antibody-inhibitor development in at least one affected individual. Of these substitutions, 6 occurred at surface-exposed residues. As further details of the C1 structure and its interface with C2 become available, and as binding studies are performed on the plasma of more patients with hemophilic C-domain mutations, prediction of surface binding sites should improve, allowing confirmation by site-specific mutagenesis of surface-exposed residues.

Regulation of inwardly rectifying K(+) channel in cultured opossum proximal tubule cells by protein phosphatases 1 and 2A.

The inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cells is activated at least in part by protein kinase A (PKA). In this study, we examined the effects of protein serine/threonine phosphatase types 1 (PP-1) and 2A (PP-2A) on activity of the K(+) channel using the patch-clamp technique. In cell-attached patches, channel activity was enhanced by the application of okadaic acid (OA, 1 microM), a membrane-permeable inhibitor of PP-1 and PP-2A, to the bath solution. This enhancement was abolished by the pretreatment of cells with KT5720 (200 nM), a specific inhibitor of PKA. In inside-out patches, channel activity which could be maintained in the presence of ATP (3 mM) in the bath solution was also increased by the addition of OA (1 microM), and the OA-induced increase in channel activity was partially prevented in the presence of KT5720 (200 nM). Direct application of either PP-1 (1 U/ml) or PP-2A (1 U/ml) to the cytoplasmic surface of the patch membrane inhibited channel activity maintained by ATP (3 mM) in inside-out patches. Moreover, channel activity stimulated by PKA (20 nM) in the presence of ATP (3 mM) was also inhibited by the application of either PP-1 (1 U/ml) or PP-2A (1 U/ml). These results indicate that the OA-sensitive protein phosphatase is involved in the regulation of channel activity, and suggest that both PP-1 and PP-2A are candidates responsible for the inhibition of channel activity through dephosphorylation of the PKA-mediated protein phosphorylation.

Lymphoepithelial cyst of the pancreas with sebaceous differentiation.

We recently encountered a patient with a lymphoepithelial cyst of the pancreas with sebaceous differentiation. We sought to compare the characteristics of this patient with those previously reported in order to foster a keener understanding of this rare clinical entity. After reviewing the present patient's case in detail, we conducted a comprehensive review of the English-language literature and analyzed the clinical characteristics of reported cases of lymphoepithelial cysts. Our patient was an asymptomatic 60-year-old man who presented with an incidental finding of a cystic lesion in the tail of the pancreas documented by computed tomography. The cyst was enucleated, and was found to contain keratinized material. It was lined by squamous epithelium with small sebaceous glands, and surrounded by lymphoid tissue with germinal centers. Of 33 reported cases, only 6 (18%) contained sebaceous glands. In all patients who underwent operation, the cysts were easily resected, and the outcome was favorable. Lymphoepithelial cyst of the pancreas is rare, and may be difficult to differentiate from cystic neoplasms preoperatively. Therefore resection is indicated. The diagnosis, however, can be confirmed by careful histologic review, and the prognosis is excellent.

Molecular epidemiology of astroviruses in Japan from 1995 to 1998 by reverse transcription-polymerase chain reaction with serotype-specific primers (1 to 8).

In addition to the serotype-specific primers described previously (1 to 7), a new serotype 8-specific primer has been designed, allowing detection of all astrovirus serotypes. A total of 1,382 diarrheal stool samples in 5 regions in Japan were examined by reverse transcription-polymerase chain reaction (RT-PCR). The incidence of astrovirus infection in all 5 regions was 5.9% (82 of 1,382 samples) and infection occurred mainly from November to April. Serotypes 1, 3, and 4 were detected in 66, 14, and 2 of the 82 positive samples, respectively. None of the other serotypes was detected. The highest detection rate was from 0 to 1 year old, 39.0%, and the next highest was from 1 to 2 years old, 34.1%. The primers provide a useful approach for study of the epidemiology of astroviruses.

Ursodeoxycholic acid prevents hepatic cytochrome P450 isozyme reduction in rats with deoxycholic acid-induced liver injury.

BACKGROUND/AIMS: Hydrophobic bile acids, such as deoxycholic acid produce cholestatic liver injury. Ursodeoxycholic acid has been shown to be useful in the treatment of cholestatic liver disease. METHODS: In this study, we investigated the effects of deoxycholic acid or ursodeoxycholic acid (1% of diet, for 14 days) and their combination (1% each) on expression of hepatic cytochrome P450 isozymes, their related enzyme activities and mRNA level in rats. RESULTS: Adding 1% deoxycholic acid to chow caused a marked increase in serum total bilirubin (47-fold) and total bile acid (8-fold) concentrations and in alkaline phosphatase (2.5-fold, p<0.01) and alanine aminotransferase activities (23.5-fold, p<0.01). Adding the same dose of ursodeoxycholic acid along with the deoxycholic acid mitigated both the rise in serum total bilirubin and bile acid concentrations and that in alkaline phosphatase and alanine aminotransferase activities, although the use of ursodeoxycholic acid alone did not affect any of the above. Feeding 1% deoxycholic acid caused a decrease (48% of control) in total cytochrome P450 content in hepatic microsomes. Addition of 1% ursodeoxycholic acid along with the 1% deoxycholic acid completely prevented the decrease in total cytochrome P450 content. Feeding ursodeoxycholic acid alone did not affect the total cytochrome P450 content. The expression of cytochrome P450 2B1, 2E1, 3A2, 2C6, 2C11 and 4A1 proteins in hepatic microsomes was decreased by deoxycholic acid (44, 51, 23, 59, 30 and 74% of control, respectively). Likewise, the activities of cytochrome P450 2B1 (pentoxyresorufin O-depentylation), 2E1 (aniline p-hydroxylation) and 3A2 (testosterone 6beta-hydroxylation) isozymes and the 3A2 mRNA levels in liver were decreased by deoxycholic acid. Addition of 1% ursodeoxycholic acid to 1% deoxycholic acid also prevented the decrease in these cytochrome P450 proteins, related enzyme activities and mRNA levels in liver. CONCLUSIONS: These results indicate that, in rats with deoxycholic acid-induced liver injury, ursodeoxycholic acid prevents the decrease in hepatic cytochrome P450 isozymes and suggest that ursodeoxycholic acid is useful for the treatment of liver injury in terms of aiding the normalization of the hepatic drug-metabolizing system.

Serotypes of human rotaviruses in 7 regions of Japan from 1984 to 1997.

Human rotavirus (HRV) serotypes were studied from diarrheal stool specimens in children in 7 regions of Japan (Sapporo, Tokyo, Maizuru, Osaka, Kagawa, Kurume, and Saga) from 1984 to 1997 by enzyme immunoassay (EIA) with serotype-specific monoclonal antibodies against serotypes 1, 2, 3, and 4. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was conducted for analysis of "others" which included nonserotypable and mixed-serotype rotavirus specimens by EIA. In 3756 rotavirus-positive specimens, serotype 1 was detected in 2649 (70.5%), serotype 2 in 362 (9.6%), serotype 3 in 232 (6.2%) and serotype 4 in 196 (5.2%). Overall, serotype 1 was predominant from 1984 to 1997, although there were a few cases in which serotype 2, 3 and 4 became predominant based on area and year. The frequency of serotype 1 has gradually increased since 1993. Twenty two, 2, 3 and 1 among 57 specimens of "others" by EIA from Tokyo, Maizuru, Sapporo and Kurume in 1995-1996 and 1996-1997 were determined as serotypes 1, 2, 3, and 9 by RT-PCR, respectively.

Non-invasive estimation of human left ventricular end-diastolic pressure.

Sato et al. (Electronic Letters 32, 949-950, 1996) reported that one can obtain a non-invasive estimate of left ventricular (LV) pressure at around end-diastole in an isolated canine preparation. In this study we examined whether this method can be applied to humans. Using the method proposed by Kanai et al. (IEEE. Trans. UFFC, 43, 791-810,1996), we detected small amplitude LV vibration from an ultrasonic pulse Doppler signal reflected from the interventricular septum in five patients (44-63 y.o., male;4, female;1). We measured the oscillation frequency of the LV wall through the wavelet transform of small amplitude LV vibration, and calculated LV pressure at around end-diastole from the values of oscillation frequency, internal radius and wall thickness using Mirsky's equation. The estimated LV pressures at around end-diastole were similar to end-diastolic pressure measured directly by cardiac catheterization. These results show the possibility that this method allows for the non-invasive estimate of LV pressure at around end-diastole, and furthermore provides the basis for future clinical applicability of this technique.

Activation of inwardly rectifying K+ channel in OK proximal tubule cells involves cGMP-dependent phosphorylation process.

The inwardly rectifying K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cell is activated by cyclic AMP-dependent protein kinase (PKA). In this study, we further examined the involvement of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent process in modulation of this K+ channel by using the patch-clamp technique. In cell-attached patches, channel activity was increased by the application of either N2, 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (DBcGMP, 100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 100 microM), and it was inhibited by KT5823 (10 microM), a membrane-permeable specific inhibitor of cGMP-dependent protein kinase (PKG). The effect of DBcGMP on channel activity was abolished by the pretreatment of cells with KT5823 (10 microM), but it was observed in the presence of KT5720 (200 nM), a specific inhibitor of PKA. Furthermore, atrial natriuretic peptide (ANP, 10 nM) increased channel activity, which was also prevented by the application of KT5823 (10 microM). In inside-out patches, ATP (3 mM) was required to maintain channel activity, which was inhibited by KT5823 (10 microM), but it was not increased by cGMP (100 microM) alone. The channel activity was increased by the coapplication of PKG (500 U/ml) and cGMP (100 microM). These results suggest that cGMP activates the inwardly rectifying K+ channel in OKP cells through PKG-mediated phosphorylation processes independent of PKA-mediated processes, and that ANP is an agonist which stimulates PKG-mediated processes in the proximal tubule cell. Furthermore, it is suggested that the ATP-dependent channel activity in inside-out patches is maintained at least in part by PKG, which is the membrane-bound catalytic domain.