Stereotactic navigation using registration based on intra-abdominal landmarks in robotic-assisted lateral pelvic lymph node dissection.

BACKGROUND: We carried out robot-assisted lateral pelvic lymph node dissection (LPLND) for rectal cancer with a stereotactic navigation system. The purpose of this study was to evaluate the accuracy and feasibility of the system. METHODS: We constructed a navigation system based on the Polaris Spectra optical tracking device (Northern Digital Inc., Canada) and the open-source software 3D Slicer (version 3.8.1; http://www.slicer.org ). We used the landmark-based registration method for patient-to-image registration. Body surface landmarks and intra-abdominal landmarks were used. We evaluated the time required for registration and target registration error (TRE; the distance between corresponding points after registration) for the root of the superior gluteal artery the root of the obturator or superior vesical artery, and the obturator foramen during minimally invasive LPLND for rectal cancer. Five patients who had LPLND for rectal cancer at the University of Tokyo Hospital between September 2020 and May 2021 were enrolled. RESULTS: The mean time required for registration was 49 s with the body surface landmarks and 88 s with the intra-abdominal landmarks. The mean TRE improved markedly when the registration was performed using intra-abdominal landmarks. The mean TRE of the root of the superior gluteal artery, the root of the obturator or superior vesical artery, and the obturator foramen were 55.8 mm, 53.4 mm, and 55.2 mm with the body surface landmarks and 11.8 mm, 10.0 mm, and 12.6 mm with the intra-abdominal landmarks, respectively. There were no adverse events related to the registration process. CONCLUSIONS: When stereotactic navigation systems are used for minimally invasive LPLND, the use of intra-abdominal landmarks for registration is feasible and may allow simpler and more accurate navigation than the use of body surface landmarks.

Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide.

L-arginine-dependent synthesis of nitrite (NO2-) and nitrate (NO3-) by macrophages correlates with and is required for their execution of nonspecific cytotoxicity toward some tumor cells and microbes. However, the bioactive L-arginine metabolites responsible for cytotoxicity are unknown. Mammalian endothelial cells have recently been shown to release nitric oxide (NO.); we therefore determined if this reactive metabolite was synthesized by activated murine macrophages. Macrophage-derived NO. was detected by two independent methods: a bioassay for NO.-mediated relaxation of preconstricted rings of rabbit aorta; and a spectroscopic measurement of the reaction of NO. with clostridial ferredoxin, an Fe-S protein. After activation with IFN-gamma and LPS, macrophages continuously secreted a substance that relaxed rabbit aortic rings denuded of endothelium. Production of the vasorelaxant was enhanced by 0.5 mM L-arginine and inhibited reversibly by NG-methylated L-arginine analogs that block macrophage NO2-/NO3- synthesis. The vasorelaxant was scavenged by ferrous myoglobin, was labile, and was neither NO2- nor a cyclooxygenase metabolite. Activated M phi also secreted a substance that bleached Fd, a reaction carried out by NO. and NO2, but not NO2-. Macrophage bleaching of Fd correlated directly with time, cell number, and concomitant NO2-/NO3- production, required L-arginine, and was independent of reactive oxygen intermediates. Thus, activated murine M phi release NO. and/or a closely related, highly reactive nitrogen oxide such as NO2, during their conversion of L-arginine to NO2-/NO3-. NO. and NO2 may mediate L-arginine-dependent pathologic effects of M phi, as well as physiologic effects not previously considered for this widely distributed cell type.

A high-resolution, three-dimensional thin endoscope for fetal surgery.

BACKGROUND: Fetal surgery is receiving considerable attention. However, surgeons must have great skill to perform this surgery. For assisting with the operation, the three-dimensional (3D) endoscope is very useful because it allows the surgeon depth perception. However, the diameter of existing 3D endoscopes is approximately 10 mm. Therefore, the authors have developed a high-resolution, thin, 3D endoscope for use in fetal surgery. METHODS: The authors' system uses two 1/10-in. micro charge-coupled device (CCD) cameras at the tip of the endoscope and achieves a diameter of 5.4 mm. The endoscope's angle of convergence is 2.6 masculine, which very closely approximates the angle of convergence for humans. Thus, the surgeon experiences little visual fatigue. The view angle is 87 masculine. RESULTS: The authors compared image quality and depth perception between their system and conventional 3D and 2D endoscopes. Theoretical investigation of image quality allowed the surgeon to distinguish a line 0.2 to 0.25 mm wide. Furthermore, the depth perception with the thin 3D endoscope was almost the same as with an 11-mm normal 3D endoscope. In addition, with the 3D endoscope, a higher percentage of questions were answered correctly in the depth perception evaluation experiment in a water environment than with the 2D instrument. CONCLUSION: According to these experiments, the thin 3D endoscope has a sufficiently high image quality and depth perception even in a water environment.

Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.

We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype interferon sensitivity determining region appears to be interferon-resistant strains. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment.

Contrast-enhanced multi-shot echo-planar FLAIR in the depiction of metastatic tumors of the brain: comparison with contrast-enhanced spin-echo T1-weighted imaging.

BACKGROUND: The usefulness of fast fluid-attenuated inversion-recovery (FLAIR) sequences after administration of contrast medium (f-FLAIR (+)) has been shown in depicting brain tumors including metastases and meningeal carcinomatosis. Contrast-enhanced multi-shot echo-planar FLAIR (Ms-EPI-FLAIR (+)), comprising combined sequences of f-FLAIR (+) and Ms-EPI, may provide the advantages of f-FLAIR (+) along with rapid acquisition. PURPOSE: To compare Ms-EPI-FLAIR (+) with post-contrast spin-echo T1-weighted imaging (SE-T1WI (+)) in the depiction of brain metastases. MATERIAL AND METHODS: In 14 patients with metastatic tumors of the brain, spin-echo precontrast T1-weighted imaging (SE-T1WI (-)), fast spin-echo T2-weighted imaging (FSE-T2WI), fast-FLAIR, SE-T1WI (+), and Ms-EPI-FLAIR (+) were acquired. For qualitative evaluation of SE-T1WI (+) and Ms-EPI-FLAIR (+), receiver operating characteristic (ROC) analysis was performed in two different readers. For quantitative analysis, the intensity ratios (intensity of tumor divided by intensity of peritumoral region) in SE-T1WI (+) and Ms-EPI-FLAIR (+) were compared. RESULTS: Although pre-contrast f-FLAIR detected 84 of 106 tumors, Ms-EPI-FLAIR (+) detected 98 of 106 tumors. In the ROC analysis for observers A and B, Az values in SE-T1WI (+) did not differ from values in Ms-EPI-FLAIR (+). Quantitatively, the intensity ratio in Ms-EPI-FLAIR (+) also did not differ from that in SE-T1WI (+). CONCLUSION: Detectability of brain metastases with Ms-EPI-FLAIR (+) is almost similar to that with SE-T1WI (+). Ms-EPI-FLAIR (+) could be an alternative to SE-T1WI (+) in the depiction of brain metastases.

Transversely isotropic properties of porcine liver tissue: experiments and constitutive modelling.

Knowledge of the biomechanical properties of soft tissue, such as liver, is important in modelling computer aided surgical procedures. Liver tissue does not bear mechanical loads, and, in numerical simulation research, is typically assumed to be isotropic. Nevertheless, a typical biological soft tissue is anisotropic. In vitro uniaxial tension and compression experiments were conducted on porcine cylindrical and cubical liver tissue samples respectively assuming a simplistic architecture of liver tissue with its constituent lobule and connective tissues components. With the primary axis perpendicular to the cross sectional surface of samples, the tissue is stiffer with tensile or compressive force in the axial direction compared to that of the transverse direction. At 20% strain, about twice as much force is required to elongate a longitudinal tissue sample than that of a transverse sample. Results of the study suggest that liver tissue is transversely isotropic. A combined strain energy based constitutive equation for transversely isotropic material is proposed. The improved capability of this equation to model the experimental data compared to its previously disclosed isotropic version suggests that the assumption on the fourth invariant in the constitutive equation is probably correct and that anisotropy properties of liver tissue should be considered in surgical simulation.

Image-guided laparoscopic pelvic lymph node dissection using stereo visual tracking free-hand laparoscopic ultrasound.

Laparoscopic pelvic lymph node dissection is a delicate operation because pelvic arteries, which should be located first to guide the dissection, are often concealed by tissues and cannot be identified in the endoscopic view. Consequently, arteries can be damaged if they are not located accurately. To improve dissection safety and efficiency, we have developed an image-guided navigation system to provide pelvic artery position information by registering a 3D artery model extracted from CT images to a 3D model reconstructed from free-hand laparoscopic ultrasound images. The ultrasound probe is tracked using a proposed stereo vision-based tracking strategy that can simplify the system and reduce setup time. The artery is segmented from 2D ultrasound images using a local phase-based snakes framework. The accuracy of the proposed navigation system was estimated in a phantom experiment (the TRE error was 1.58 ± 0.70 mm), and the feasibility of the proposed navigation system was confirmed in an animal experiment.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro.

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Laparoscopic cholecystectomy using a newly developed laparoscope manipulator for 10 patients with cholelithiasis.

BACKGROUND: Laparoscopic surgery has continued to gain popularity in almost all fields of abdominal surgery, and robotic systems have been introduced in general surgery. Naviot is a new remote-controlled laparoscope manipulator system controlled by the operator's hand. This study assessed its introduction into clinical practice. METHODS: A group of 10 consecutive patients with cholelithiasis underwent laparoscopic cholecystectomy assisted by the Naviot system (Naviot group). Another group of 41 patients who underwent laparoscopic cholecystectomy with a conventional human camera holder (human camera group) were selected for a comparison of their operative results with those of the Naviot group. RESULTS: The operative time of 89.3 +/- 27.1 min for the Naviot group was significantly longer than that of 74.8 +/- 28.1 min for the human camera group (p < 0.05). However, when the setup time for the Naviot system was excluded, the operative time was not significantly different from that for the human camera group. Other operative results showed no significant difference between the two groups. CONCLUSIONS: The authors believe that the new Naviot system is feasible for clinical use, and that it enables surgeons to perform solo gastrointestinal surgery.

Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrosis in genetically obese diabetic mice.

BACKGROUND: Obesity and insulin resistance are associated with accelerated macrovascular and microvascular coronary disease, cardiomyopathic phenomena, and increased concentrations and activity in blood of plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of fibrinolysis. METHODS AND RESULTS: To determine whether hypofibrinolysis in blood and tissues and its potential sequelae could be attenuated pharmacologically, we studied genetically modified obese mice. By 10 weeks of age, obese mice exhibited increases in left ventricular weight and glucose and immunoreactive insulin in blood. PAI-1 activity in blood measured spectrophotometrically was significantly elevated as well. The difference compared with values in lean controls widened by 20 weeks of age. Perivascular fibrosis in coronary arterioles and small coronary arteries was evident in obese mice 10 and 20 weeks of age, paralleling increases in PAI-1 and tissue factor expression evident by immunohistochemical image analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. Inhibition of ACE activity initiated in obese mice 10 weeks of age and continued for 20 weeks arrested the increase in PAI-1 activity in blood and in cardiac PAI-1 and tissue factor mRNA as well as coronary perivascular fibrosis. CONCLUSIONS: Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.

Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a membrane glycoprotein and a member of the immunoglobulin superfamily. It plays a central role in cell to cell-mediated immune responses and is a ligand for leukocyte function-associated antigen-1 (LFA-1). We report here that a newly discovered cytokine, interferon-gamma-inducing factor (IGIF) [H. Okamura et al. (1995) Nature 378, 88] recently proposed to be designated as IL-18, selectively up-regulates ICAM-1 expression in KG-1 cells, a human myelomonocytic cell line, in which IL-18 also enhances interferon-gamma production. IL-18 induced heterotypic aggregation between KG-1 and Peer T cells, which was blocked by anti-ICAM-1 and/or LFA-1 antibodies. Anti-interferon-gamma antibody did not block the IL-18-induced up-regulation of ICAM-1 on KG-1 cells. These results thus show that IGIF/IL-18, enhances ICAM-1 expression in KG-1 cells in an interferon-gamma-independent pathway, up-regulates ICAM-1 functions, and that IL-18 might play a potential role in immunoregulation by mediating immune cell infiltration into the tissues.

Vision-based endoscope tracking for 3D ultrasound image-guided surgical navigation.

This work introduces a self-contained framework for endoscopic camera tracking by combining 3D ultrasonography with endoscopy. The approach can be readily incorporated into surgical workflows without installing external tracking devices. By fusing the ultrasound-constructed scene geometry with endoscopic vision, this integrated approach addresses issues related to initialization, scale ambiguity, and interest point inadequacy that may be faced by conventional vision-based approaches when applied to fetoscopic procedures. Vision-based pose estimations were demonstrated by phantom and ex vivo monkey placenta imaging. The potential contribution of this method may extend beyond fetoscopic procedures to include general augmented reality applications in minimally invasive procedures.

Hemispheric representation of the central retina of commissurotomized subjects.

It is controversial whether a stimulus projected within 1 to 3 degrees from the boundary between the right and left hemiretina is transmitted to only one cerebral hemisphere or to both cerebral hemispheres. In order to resolve this issue, letter- and word-stimuli were presented for 200 msec with a new type of tachistoscope, called the fundus tachistoscope, in and about the central retina, (i.e. fovea, 1.2 degrees in horizontal diameter) of the right eyes of two commissurotomized subjects (N.G. and A.A.). During stimulus presentation the subjects were attempting to fixate a fixation target. The fundus tachistoscope combined with image analysis of the fundus enables us to measure the position of the stimulus on the retina, relative to the foveal center, as well as whether or not the eye moved during stimulus presentation. The results indicate that the region of the right (temporal) hemiretina represented by both hemispheres in letter processing, if it exists, was estimated as less than 0.6 degrees from the foveal center. The two subjects frequently (27% in N.G. and 46% in A.A.) fixated the fixation target eccentrically, i.e. with a retinal point other than the foveal center, during fixation, namely stimulus presentation. Their eccentric fixations were small with magnitude almost all falling between 1.35 degrees right and 1.25 degrees left of the foveal center. It is therefore recommended that letter-stimuli be presented at least 2.0 degrees from the foveal center in ordinary tachistoscopic studies of cerebral hemispheric differences. Eye movements, which varied in 0.11 degrees and 1.43 degrees horizontally, occurred in about 8% of all the trials during fixation. On the average of the two subjects, the eye movements caused or worsened eccentric fixation in only about one third of the trials, and corrected eccentric fixation in about two thirds of the trials.

Diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type II diabetic rat model: a possible cause for erectile dysfunction in diabetes.

Erectile dysfunction (ED) is commonly experienced in men with diabetes mellitus. Vascular endothelial growth factor (VEGF) has been extensively documented for its pathogenic significance in different complications of diabetes. We hypothesized that expressions of VEGF, its receptors and its signaling pathway Akt may be drastically altered in diabetic penile tIssues and their alterations may modulate penile expression of the molecules that are believed to play a role in diabetic ED. Otsuka Long-Evans Fatty (OLETF) rats, a type II (non-insulin-dependent) diabetes mellitus, were used at the insulin-resistant stage of type II diabetes (20 weeks of age). We determined protein and mRNA expressions of VEGF, its receptors, Akt, nitric oxide synthase isoforms, and apoptosis-related molecules in the penis using immunohistochemistry, Western blotting, in situ hybridization, and real-time quantitative PCR analyses. The penile sections were also submitted to the Tdt-mediated dUTP nick end labeling assay for apoptosis. OLETF rats showed marked reductions in penile expression of VEGF, its two receptors and Akt. In OLETF rat penises, endothelial and neuronal nitric oxide synthase isoforms were expressed less abundantly. Furthermore, while anti-apoptotic markers, Bcl-2 and phosphorylated Bad, were down-regulated, pro-apoptotic markers, active caspase-3 and Bax, were up-regulated, resulting in the appearance of apoptotic cells in the penile tIssues of OLETF rats. The VEGF signaling system would work less well in diabetic penile tIssues as a result of the reduced expression, leading to diminished endothelial production of nitric oxide and apoptosis-related erectile tIssue damage. We propose that the abnormalities of the VEGF signaling system in the penis may play a role in the pathophysiology of diabetic ED.

Aromatase is abundantly expressed by neonatal rat penis but downregulated in adulthood.

Although synthesis of estrogen by male gonads has been well documented for over half a century, it is only recently that the role of estrogen in male reproductive events has gained appreciation. We recently reported abundant expression of estrogen receptor (ER)-alpha and -beta in different cell types of the rat penis, whose levels diminished with advancing age. The present study, which builds on data from the ER study, was designed to determine whether the penis is capable of generating its own local estrogen by examining evidence of the expression of aromatase, a microsomal enzymatic complex which irreversibly converts androgens to estrogens, using immunohistochemistry, Western blotting, in situ hybridization and real-time PCR analyses. Secondly, the effects of sex steroid hormones on penile aromatase were examined. Discrete aromatase immunoreactive cells were localized in primordial corpus cavernosum, corpus spongiosus and os penis, blood vessels and sensory corpuscle of glans penis. In situ hybridization signals corresponded with immunohistochemical findings. Western blot, enzyme immunoassay and real-time PCR analyses of rat penile samples revealed an age-dependent expression of aromatase and estrogen, with levels at week 1 almost resembling those of the ovary, but they decreased sharply by week 8, and decreased further by week 35. This expression pattern was strikingly similar to that of ER-alpha reported previously. Testosterone and diethylstilbesterol administered prenatally upregulate levels of aromatase mRNA and protein, and estrogen postnatally. Dihydrotestosterone upregulated aromatase mRNA and protein, but not estrogen. We conclude that estrogen acts via ER in a paracrine and/or autocrine manner to regulate penile events, particularly during development, and that estrogen synthesis is regulated by estrogen and androgens.

Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening.

Bilateral neurectomy of the pelvic nerve (BLPN) that carries uterine cervix-related sensory nerves induces dystocia, and administration of its vasoactive neuropeptides induces changes in the cervical microvasculature, resembling those that occur in the ripening cervix. This study was designed to test the hypothesis that (a) the cervix of pregnant rats expresses vascular endothelial growth factor (VEGF) and components of the angiogenic signaling pathway [VEGF receptors (Flt-1, KDR), activity of protein kinase B, Akt (phosphorylated Akt), and endothelial nitric oxide synthase (eNOS)] and von Willebrand Factor (vWF) and that these molecules undergo changes with pregnancy, and (b) bilateral pelvic neurectomy (BLPN) alters levels of VEGF concentration in the cervix. Using RT-PCR and sequencing, two VEGF isoforms, 120 and 164, were identified in the rat cervix. VEGF, VEGF receptor-1 (Flt-1), eNOS, and vWF immunoreactivities (ir) were localized in the microvasculature of cervical stroma. Their protein levels increased during pregnancy but decreased to control levels by 2 days postpartum. VEGF receptor-2 (KDR)-ir was confined to the epithelium of the endocervix. BLPN downregulated levels of VEGF by a third. Therefore, the components of the angiogenic signaling pathway are expressed in the cervix and change over pregnancy. Furthermore, angiogenic and sensory neuronal factors may be important in regulating the dynamic microvasculature in the ripening cervix and may subsequently play a role in cervical ripening and the birth process.

Alterations in endothelium-dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin-induced diabetic rats.

1. The aim of this study was to determine whether endothelium-dependent hyperpolarization and relaxation are altered during experimental diabetes mellitus. Membrane potentials were recorded in mesenteric arteries from rats with streptozotocin-induced diabetes and age-matched controls. The resting membrane potentials were not significantly different between control and diabetic mesenteric arteries (-55.3 +/- 0.5 vs -55.6 +/- 0.4 mV). However, endothelium-dependent hyperpolarization produced by acetylcholine (ACh; 10(-8)-10(-5) M) was significantly diminished in amplitude in diabetic arteries compared with that in controls (maximum -10.4 +/- 1.1 vs -17.2 +/- 0.8mV). Furthermore, the hyperpolarizing responses of diabetic arteries were more transient. 2. ACh-induced hyperpolarization observed in control and diabetic arteries remained unaltered even after treatment with 3 x 10(-4) M N(G)-nitro-L-arginine (L-NOARG), 10(-5) M indomethacin or 60 u ml (-1) superoxide dismutase. 3. Endothelium-dependent hyperpolarization with 10(-6) M A23187, a calcium ionophore, was also decreased in diabetic arteries compared to controls (-8.3 +/- 1.4 vs -18.0 +/- 1.9 mV). However, endothelium-independent hyperpolarizing responses to 10(-6) M pinacidil, a potassium channel opener, were similar in control and diabetic arteries (-20.0 +/- 1.4 vs - 19.2 +/- 1.1 mV). 4. The altered endothelium-dependent hyperpolarizations in diabetic arteries were almost completely prevented by insulin therapy. Endothelium-dependent relaxations by ACh in the presence of l0(-4) M L-NOARG and 10(-5) M indomethacin in diabetic arteries were also reduced and more transient compared to controls. 5. These data indicate that endothelium-dependent hyperpolarization is reduced by diabetes, and this would, in part, account for the impaired endothelium-dependent relaxations in mesenteric arteries from diabetic rats.

Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery.

1. In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dependent hyperpolarization by releasing endothelium-derived hyperpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh-induced hyperpolarization observed in rat mesenteric artery. 2. The phospholipase A2 inhibitor quinacrine (30 microM) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine. 3. The imidazole antimycotic agents ketoconazole (50 microM), clotrimazole (30 microM) and miconazole (10 microM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 microM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 microM), an inhibitor of all arachidonic acid metabolic pathways, altered ACh-induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 microM), diedthyldithiocarbamate (50 microM), metyrapone (20 microM) and troleandomycin (10 microM). 4. Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 microM), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh-induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg-1 day-1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh. 5. Arachidonic acid in concentrations up to 100 microM had no detectable effect on smooth muscle membrane potential. 11, 12-Epoxyeicosatrienoic acid (EET, 10 microM), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium-independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 microM), in contrast to the response to ACh. 6. These results suggest that the contribution of a cytochrome P450-derived metabolite of arachidonic acid to ACh-induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.

Thapsigargin- and cyclopiazonic acid-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.