Biomedical subjects

D Sun

Publications and source records attributed to D Sun.

At least 55 records · Page 3Linked to original sources

PubMed2001

Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases.

Recent studies of the p53 homologue p63 indicate that this gene is preferentially expressed in basal and immature cervical squamous epithelium. This study correlated p63 expression with morphologic phenotype and human papillomavirus (HPV) type in a wide range of cervical neoplasms. Two hundred fifty cases of cervical carcinoma, including squamous cell carcinoma (SCCA; n = 178), adenocarcinoma (ADCA; n = 28), adenosquamous carcinoma (ASCA; n = 8), neuroendocrine carcinoma (NECA; n = 15), and other variant or mixed types (n = 21) were studied. Ninety-seven percent of SCCA, 0% of ADCA, and 0% of SCUC showed strong (>75% v <30%) positivity for p63 (P<.001). p63 sharply distinguished SCCA (p63+) from ADCA (p63-), Large-cell, poorly differentiated carcinomas were distinguished as putative glandular (glassy cell) or squamous (lymphoepithelial-like or spindle cell) types based on p63 staining. Eight (73%) of 11 neuroendocrine tumors tested were chromogranin positive; all showed no or low (<30%) levels of p63 immunostaining. Absence of p63 was also associated with a subset of nonneuroendocrine undifferentiated carcinomas. Transitions from squamous to columnar or undifferentiated morphology coincided with loss of p63 expression. A strong association between HPV 16 and p63 positivity was identified because of the colocalization of both within tumors of squamous phenotype. p63 is a powerful marker for squamous differentiation and, when diffusely expressed, excludes a glandular or neuroendocrine differentiation. p63 may be useful for differentiating pure squamous or glandular from adenosquamous carcinomas, tracking shifts in differentiation within tumors, supporting (by its absence) the diagnosis of neuroendocrine carcinomas, and clarifying the spectrum of poorly differentiated carcinomas lacking either squamous or neuroendocrine differentiation.

Adenocarcinoma↗

PubMed2001

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy.

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.

Animals↗

PubMed2001

Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.

Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions.

Aedes↗

PubMed2001

Extracellular calmodulin stimulates light-independent RbcS-GUS expression in suspension-cultured cells of transgenic tobacco.

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

Calmodulin↗

PubMed2001

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.

Amino Acid Sequence↗

PubMed2001

h-Caldesmon expression effectively distinguishes endometrial stromal tumors from uterine smooth muscle tumors.

Distinction of endometrial stromal neoplasms from cellular smooth muscle tumors of the uterus is sometimes difficult. Immunohistochemistry is often not helpful because muscle actins and desmin are expressed in both neoplasms. This study's goal was to determine whether h-caldesmon, a smooth muscle-specific isoform of a calcium, calmodulin, and actin binding protein, could effectively distinguish endometrial stromal tumors from uterine smooth muscle tumors. The authors analyzed immunohistochemical expression in 24 endometrial stromal neoplasms (21 sarcomas and three nodules), 29 leiomyosarcomas, 32 leiomyomas (10 "usual," 14 cellular leiomyoma, and eight "highly cellular" types), 40 myometria, and 25 endometria. h-Caldesmon was diffusely positive in all myometria, leiomyomata, and leiomyosarcomas. Of note, 16 leiomyosarcomas (55%) were positive for h-caldesmon in more than 50% of tumor cells. In five "highly cellular" leiomyomas, h-caldesmon expression was markedly decreased or absent in areas morphologically resembling endometrial stromal tumors, raising the possibility that these tumors may be mixed smooth muscle-endometrial stromal neoplasms. In contrast, h-caldesmon expression was absent in all endometria and endometrial stromal neoplasms apart from accompanying small vessels. Desmin was diffusely positive in all myometria and leiomyomata. The fraction of cells expressing desmin was greater than that of h-caldesmon in only 10% of leiomyosarcomas. Focal desmin expression was also present in eight of 25 (32%) endometria and 12 of 24 (50%) endometrial stromal neoplasms. h-Caldesmon appears to be a more sensitive and specific marker of smooth muscle differentiation in the uterus than desmin and may be a useful tool for distinguishing and classifying uterine mesenchymal tumors.

Biomarkers↗

PubMed2001

Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia.

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

Biomarkers↗

PubMed2001

Discrimination of complete hydatidiform mole from its mimics by immunohistochemistry of the paternally imprinted gene product p57KIP2.

The p57KIP2 protein is a cell cycle inhibitor and tumor suppressor encoded by a strongly paternally imprinted gene. We explored the utility of p57KIP2 as a diagnostic marker in hydatidiform mole, a disease likely the result of abnormal dosage and consequent misexpression of imprinted genes. Using a monoclonal antibody on paraffin-embedded, formalin-fixed tissue sections, the authors evaluated p57KIP2 expression in normal placenta and in 149 gestations including 59 complete hydatidiform moles, 39 PHMs, and 51 spontaneous losses with hydropic changes. p57KIP2 was strongly expressed in cytotrophoblast and villous mesenchyme in normal placenta, all cases of partial hydatidiform moles (39 of 39) and all spontaneous losses with hydropic changes (51 of 51). In contrast, p57KIP2 expression in cytotrophoblast and villous mesenchyme was absent or markedly decreased in 58 of 59 complete hydatidiform moles. In all gestations p57KIP2 was strongly expressed in decidua and in intervillous trophoblast islands, which served as internal positive controls for p57KIP2 immunostaining. p57KIP2 immunohistochemistry can reliably identify most cases of complete hydatidiform mole irrespective of gestational age and is thus a useful diagnostic adjunct, complementary to ploidy analysis, in the diagnosis of hydatidiform mole.

Abortion, Spontaneous↗

PubMed2001

Na+-K+-Cl- cotransporter in rat focal cerebral ischemia.

In cultured neurons, the authors previously demonstrated that the Na+-K+-Cl- cotransporter is significantly stimulated by elevated extracellular potassium and glutamate, which are important factors in cerebral ischemic damage. These findings led the authors to hypothesize that stimulation of the cotransporter after ischemia might result in Na+, K+, and Cl- influx, and might contribute to neuron damage. In the current study, the authors investigated such a role of the Na+-K+-Cl- cotransporter in focal cerebral ischemia. Cerebral ischemia was induced by 2-hour occlusion of the left middle cerebral artery (MCA) and 24-hour reperfusion in male spontaneously hypertensive rats (SHRs). Immunocytochemical staining and immunoblotting revealed an up-regulation of expression of the cotransporter protein in neurons in cortex at 24 hours of reperfusion. Artificial cerebral spinal fluid (aCSF) or 100 micromol/L bumetanide (a cotransporter inhibitor) in aCSF were continuously microdialyzed through a microdialysis probe into left cortices throughout 2-hour MCA occlusion and 24-hour reperfusion. Compared with the aCSF-treated group, infarction volume was significantly reduced in the bumetanide-treated group (25%, P < 0.05). In addition, brain water content in the bumetanide-treated brains was decreased by 70% (P < 0.05). These results strongly suggest that the Na+-K+-Cl- cotransporter may play an important role in cerebral ischemic neuronal damage.

Animals↗

PubMed2001

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Amino Acid Sequence↗

PubMed2001

Bayesian modeling of age-specific survival in nesting studies under Dirichlet priors.

There has been much work done in nest survival analysis using the maximum likelihood (ML) method. The ML method suffers from the instability of numerical calculations when models having a large number of unknown parameters are used. A Bayesian approach of model fitting is developed to estimate age-specific survival rates for nesting studies using a large class of prior distributions. The computation is done by Gibbs sampling. Some latent variables are introduced to simplify the full conditional distributions. The method is illustrated using both a real and a simulated data set. Results indicate that Bayesian analysis provides stable and accurate estimates of nest survival rates.

Age Factors↗

PubMed2001

Effects of calorie restriction on polymicrobial peritonitis induced by cecum ligation and puncture in young C57BL/6 mice.

Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-kappaB and NF-interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-A(b)) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P < 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-kappaB and NF-IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P < 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.

Aging↗

PubMed2001

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Amino Acid Motifs↗

PubMed2001

Nitric oxide-mediated arteriolar dilation after endothelial deformation.

Previously, we frequently observed dilation of arterioles after agonist-induced constrictions. We hypothesized that deformation of the endothelium during decreases in diameter of isolated arterioles elicits the release of nitric oxide (NO). In isolated arterioles of rat mesentery, phenylephrine (PE, 10(-7) M)-, U-46619 (10(-7) M)-, and KCl (50 mM)-induced constrictions were followed by potent dilations. Inhibition of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 2 x 10(-4) M) or removal of the endothelium significantly enhanced constriction and reduced the postconstriction dilation. In the presence of 80 mmHg of intraluminal pressure, an increase in extraluminal pressure (P(e)) to 75 mmHg for 20 s and 1 and 2 min decreased vessel diameter. After release of P(e), arterioles dilated as a function of the duration of diameter reduction by P(e). Removal of the endothelium or administration of L-NNA significantly diminished the post-P(e) dilations. In cultured mesenteric arteriolar endothelial cells (EC), PE, U-46619, or KCl did not increase, whereas ACh did increase, the production of NO, as measured by a fluorometric assay for nitrite. Furthermore, when EC, cultured on a stretched silicone membrane, were subjected to deformation by shortening the membrane to 50% of its original length, NO release increased significantly. Based on all of the above, we propose that deformation of EC per se elicits release of NO, a mechanism that modulates arteriolar constriction.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2001

Endothelial microtubule disruption blocks flow-dependent dilation of arterioles.

The cytoskeleton is believed to have an important role in the structural and functional integrity of endothelial cells. The role of the endothelial cytoskeleton, specifically microtubules, in the mediation of flow-induced dilation of arterioles has not yet been studied. Thus the aim of our study was to investigate the role of microtubules in the endothelial mechanotransduction of flow-induced dilation of isolated gracilis arterioles of the rat. The active diameter of arterioles at a constant perfusion pressure (80 mmHg) was approximately 63 microm, whereas their passive diameter (Ca(2+)-free solution) was approximately 119 microm. At a constant pressure, increases in flow of the perfusate solution (from 0 to 10 and from 10 to 20 microl/min) elicited increases in diameter up to approximately 95 microm (approximately a 53% increase). Intraluminal administration of nocodazole at concentrations of 5 x 10(-9) and 5 x 10(-8) M had no discernible effects on the structure of endothelial microtubules or on flow-induced dilation, whereas it disassembled microtubules and eliminated flow-induced dilation at a concentration of 5 x 10(-7) M. At this higher concentration, however, the basal diameter and dilations to acetylcholine (10(-8) M), sodium nitroprusside (10(-7) M), arachidonic acid (5 x 10(-6) M), and prostaglandin E2 (10(-8) M) were unaffected. Colchicine (5 x 10(-7) M) also disassembled microtubules and eliminated flow-induced dilation. We concluded that, in isolated arterioles, the integrity of the endothelial cytoskeleton is essential for the transduction of the shear stress signal that results in the release of endothelial factors evoking dilation.

Animals↗

PubMed2001

Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation.

Flow-induced dilation of gracilis muscle arterioles was examined in both genders of control rats and rats chronically treated with N(omega)-nitro-L-arginine methyl ester (L-NAME). After L-NAME treatment (4 wk), systolic blood pressure was significantly increased compared with control, whereas the plasma concentration of nitrate/nitrite was significantly reduced. Isolated and pressurized arterioles dilated significantly in response to increases in flow (0-25 microl/min). Flow-induced dilation was comparable in arterioles of control and L-NAME-treated rats but was significantly greater in female than in male rats. L-NAME + indomethacin, which abolished flow-induced dilation in arterioles of male control rats, inhibited the dilation by only ~75% in female control rats. The residual portion of the response was eliminated by additional administration of miconazole, an inhibitor of cytochrome P-450. Indomethacin did not affect the dilation in female L-NAME-treated rats but completely inhibited the response in male L-NAME-treated rats. The indomethacin-insensitive, flow-induced dilation in female L-NAME-treated arterioles was abolished by miconazole, 6-(2-proparglyoxyphenyl)hexanoic acid, or charybdotoxin. Thus an augmented release of endothelial prostaglandins accounts for the preserved flow-induced dilation in arterioles of male rats, whereas a metabolite of cytochrome P-450 is responsible for the maintenance of flow-induced dilation in female rats, suggesting important differences in the adaptation of the endothelium of arterioles from male and female rats to the lack of nitric oxide (NO) synthesis.

Animals↗

PubMed2001

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice.

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

Role of endothelial [Ca2+]i in activation of eNOS in pressurized arterioles by agonists and wall shear stress.

In cultured endothelial cells, Ca2+-dependent and -independent activation of nitric oxide (NO) synthesis to agonists and flow/wall shear stress (WSS) has been demonstrated. However, the presence and function of these pathways are less well known in microvessels that can be exposed to a high level of WSS. We hypothesized that the role of changes in endothelial intracellular calcium concentration ([Ca2+]i) is different in agonist- and WSS-induced release of NO. Thus changes in endothelial [Ca2+]i and diameter of intact pressurized (approximately 100 microm at 80 mmHg) gracilis skeletal muscle arterioles of rats were measured by fluorescent videomicroscopy. Acetylcholine (ACh) and increases in WSS (by increasing intraluminal flow) elicited dilations (maximum 91 +/- 2% and 34 +/- 4%) that could be inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME), a NO synthase blocker. In diameter-clamped arterioles, ACh caused substantial increases in the endothelial calcium fluorescence ratio (ER(Ca), maximum 43 +/- 5%), which was significantly greater than changes in ER(Ca) (maximum approximately 10%) to increases in WSS. The Ca(2+) ionophore A-23187 also substantially increased ER(Ca) (maximum 38 +/- 5%) and elicited significant L-NAME-sensitive arteriolar dilations (maximum 45 +/- 7%). Intraluminal administration of the tyrosine kinase inhibitor genistein had no effect on dilations induced by ACh or the NO donor sodium nitroprusside, whereas it eliminated WSS-induced dilations. Collectively, our data suggest that, in endothelium of skeletal muscle arterioles, NO synthesis is activated by shear stress without a substantial increase in [Ca2+]i, most likely by activation of tyrosine kinase pathways, whereas NO release by ACh and A-23187 is associated with substantial increases in [Ca2+]i.

Animals↗