Abnormally located SSEA1+/SOX9+ endometrial epithelial cells with a basalis-like phenotype in the eutopic functionalis layer may play a role in the pathogenesis of endometriosis.

STUDY QUESTION: Is endometriosis associated with abnormally located endometrial basalis-like (SSEA1+/SOX9+) cells in the secretory phase functionalis and could they contribute to ectopic endometriotic lesion formation? SUMMARY ANSWER: Women with endometriosis had an abnormally higher number of basalis-like SSEA1+/SOX9+ epithelial cells present in the stratum functionalis and, since these cells formed 3D structures in vitro with phenotypic similarities to ectopic endometriotic lesions, they may generate ectopic lesions following retrograde menstruation. WHAT IS KNOWN ALREADY: Endometrial basalis cells with progenitor potential are postulated to play a role in the pathogenesis of endometriosis and SSEA1 and nuclear SOX9 (nSOX9) mark basalis epithelial cells that also have some adenogenic properties in vitro. Induction of ectopic endometriotic lesions in a baboon model of endometriosis produces characteristic changes in the eutopic endometrium. Retrograde menstruation of endometrial basalis cells is proposed to play a role in the pathogenesis of endometriosis. STUDY DESIGN, SIZE, DURATION: This prospective study included endometrial samples from 102 women with and without endometriosis undergoing gynaecological surgery and from six baboons before and after induction of endometriosis, with in vitro assays examining the differentiation potential of human basalis-like cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a University Research Institute. SSEA1 and SOX9 expression levels were examined in human endometrial samples from women aged 18-55 years (by immunohistochemistry (IHC) and qPCR) and from baboons (IHC). The differential gene expression and differentiation potential was assessed in freshly isolated SSEA1+ endometrial epithelial cells from women with and without endometriosis (n = 8/group) in vitro. In silico analysis of selected published microarray datasets identified differential regulation of genes of interest for the mid-secretory phase endometrium of women with endometriosis relative to that of healthy women without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: Women with endometriosis demonstrated higher number of basalis-like cells (SSEA1+, nSOX9+) in the functionalis layer of the eutopic endometrium compared with the healthy women without endometriosis in the secretory phase of the cycle (P < 0.05). Induction of endometriosis resulted in a similar increase in basalis-like epithelial cells in the eutopic baboon endometrium. The isolated SSEA1+ epithelial cells from the eutopic endometrium of women with endometriosis had higher expression of OCT4, NANOG, FUT4 mRNA (P = 0.05, P = 0.007, P = 0.018, respectively) and they differentiated into ectopic endometriotic gland-like structures in 3D culture, but not into mesodermal lineages (adipose or bone cells). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Small sample size. Bioinformatics analysis and results depends on the quality of published microarray datasets and the stringency of patient selection criteria employed. Differentiation of SSEA-1+ cells was only examined for two mesodermal lineages (adipogenic and osteogenic). WIDER IMPLICATIONS OF THE FINDINGS: Since endometrial epithelial cells with SSEA1+/nSOX9+ basalis-like phenotype generate endometriotic gland-like structures in vitro, they may potentially be a therapeutic target for endometriosis. An in depth analysis of the function of basalis-like eutopic endometrial epithelial cells might provide insights into their potential deregulation in other disorders of the endometrium including heavy menstrual bleeding and endometrial cancer where their function may be aberrant. STUDY FUNDING/COMPETING INTEREST(S): We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., C.E.G.) and R01 HD083273 from the National Institutes of Health (A.T.F.). We also acknowledge the support of Liverpool Women's Hospital Foundation Trust (J.D.), Institute of Translational Medicine (L.D.S., H.A.L., A.J.V., D.K.H.), University of Liverpool, the National Health and Medical Research Council of Australia ID 1042298 (C.E.G.) and the Victorian Government Operational Infrastructure Support Fund. All authors declare no conflict of interest.

Using heterogeneity to predict inhibitory network model characteristics.

From modeling studies it has been known for >10 years that purely inhibitory networks can produce synchronous output given appropriate balances of intrinsic and synaptic parameters. Several experimental studies indicate that synchronous activity produced by inhibitory networks is critical to the production of population rhythms associated with various behavioral states. Heterogeneity of inputs to inhibitory networks strongly affect their ability to synchronize. In this paper, we explore how the amount of input heterogeneity to two-cell inhibitory networks affects their dynamics. Using numerical simulations and bifurcation analyses, we find that the ability of inhibitory networks to synchronize in the face of heterogeneity depends nonmonotonically on each of the synaptic time constant, synaptic conductance and external drive parameters. Because of this, an optimal set of parameters for a given cellular model with various biophysical characteristics can be determined. We suggest that this could be a helpful approach to use in determining the importance of different, underlying biophysical details. We further find that two-cell coherence properties are maintained in larger 10-cell networks. As such, we think that a strategy of "embedding" small network dynamics in larger networks is a useful way to understand the contribution of biophysically derived parameters to population dynamics in large networks.

Information given to patients about adverse effects of radiotherapy: a survey of patients' views.

AIMS: In practice, clinicians vary markedly in the amount of information they give to patients before consent for investigation or treatment is obtained. We present a study to evaluate the amount of information patients feel that they should be given. MATERIALS AND METHODS: Between October 2001 and February 2002, 82 adults were enrolled into the study before commencing treatment with radiotherapy. Participants were interviewed with the aid of a questionnaire, and responses were analysed to detect differences related to age, sex, disease site, treatment intent and social class. RESULTS: The distribution of responses to the interview was large. For a mild side-effect, 23 patients (28%) wanted to be informed if the risk of the side-effect was as small as 0.1%, whereas 25 patients (31%) would only want to be informed if there was either a 50% or a 100% chance of it occurring. For severe side-effects, 36 (44%) wanted to be informed of a 0.1% risk, whereas 13 (16%) only wanted to be informed if the risk was either 50% or 100%. There was no association with sex, treatment intent (radical or palliative), social class or disease site. Information requirements tended to be greater in people under 60 years. This reached statistical significance (P = 0.007) for severe side-effects, where younger patients were more likely to want to be informed of a side-effect if there was a 10% or less chance of it occurring. CONCLUSIONS: Information needs varied widely within our survey population. It is difficult to predict how much information patients feel they need before giving informed consent. Therefore, a patient-centred approach must involve tailoring information to individual patient requirements.

A novel continuous infusional 5-fluorouracil-based chemotherapy regimen compared with conventional chemotherapy in the neo-adjuvant treatment of early breast cancer: 5 year results of the TOPIC trial.

BACKGROUND: To compare the efficacy of continuous infusional 5-fluorouracil (5-FU)-based chemotherapy against conventional bolus chemotherapy in the preoperative treatment of patients with large operable early breast cancer. PATIENTS AND METHODS: Four hundred and twenty-six women with histologically proven 3 cm invasive early breast cancer were randomised to receive pre-operative infusional 5-FU 200 mg/m(2) by daily 24 h continuous infusion via a Hickman line for 18 weeks with epirubicin 60 mg/m(2) intravenous (i.v.) bolus on day 1 and cisplatin 60 mg/m(2) i.v. bolus on day 1, both repeating 3-weekly (infusional ECisF), or conventional bolus doxorubicin 60 mg/m(2) i.v. on day 1 and cyclophosphamide 600 mg/m(2) i.v. on day 1, both repeating 3-weekly (AC), both schedules for six courses. Patients subsequently had local therapy (surgery or radiotherapy or both) and tamoxifen 20 mg orally daily as appropriate. RESULTS: The 5 year results for AC and infusional ECisF, respectively, were as follows: overall response, 75% and 77%; complete clinical remission, 31% and 34%; pathological complete remission (pathCR), 16% for both; and pathCR with residual ductal carcinoma in situ (DCIS), 25% and 24%. Mastectomy rates were 37% and 34%, respectively. Five-year overall survival was 74% for AC and 82% for infusional ECisF (hazard ratio 0.76, 95% confidence interval 0.51-1.13; P = 0.18). Both treatments were well tolerated. Grade III/IV lethargy, vomiting, alopecia and plantar-palmar erythema were significantly greater for infusional ECisF; grade III/IV leucopenia was significantly greater for AC. CONCLUSIONS: Preoperative continuous infusional 5-FU-based chemotherapy is no more active than conventional AC for early breast cancer; with a median 5 year follow-up, the infusion-based schedule shows a non-significant trend towards improved survival.

The role of desaturases in cold-induced lipid restructuring.

All organisms respond to environmental challenge by adaptive responses, although, in many cases, the underlying molecular mechanisms are not understood. In the case of membranes, the physical structure of membrane phospholipids is conserved in the face of cold, rigidifying conditions by the elevated proportions of unsaturated fatty acids. We have observed a clear positional specificity in this substitution and head group preferences in carp liver membranes. We have also demonstrated changes in the activity of lipid desaturases that mediate the unsaturation response, caused by both transcriptional and post-translational mechanisms. Another hepatic isoform has recently been discovered with sensitivity, not to cooling, but to dietary variations. Finally, we are testing the importance of desaturase inductions in the inducible cold tolerance of the whole animal.

Propofol attenuates capacitative calcium entry in pulmonary artery smooth muscle cells.

BACKGROUND: Depletion of intracellular Ca2+ stores results in capacitative Ca2+ entry (CCE) in pulmonary artery smooth muscle cells (PASMCs). The authors aimed to investigate the effects of propofol on CCE and to assess the extent to which protein kinase C (PKC) and tyrosine kinases mediate propofol-induced changes in CCE. METHODS: Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration was measured using fura 2 in PASMCs using a dual-wavelength spectrofluorometer. Thapsigargin (1 microM), a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was activated when extracellular Ca2+ (2.2 mM) was restored. RESULTS: Thapsigargin caused a transient increase in intracellular Ca2+ concentration (182+/-11%). Restoring extracellular calcium (to induce CCE) resulted in a peak (246+/-12% of baseline) and a sustained (187+/-7% of baseline) increase in intracellular Ca2+ concentration. Propofol (1, 10, 100 microM) attenuated CCE in a dose-dependent manner (peak: 85+/-3, 70+/-4, 62+/-4%; sustained: 94+/-5, 80+/-5, 72+/-5% of control respectively). Tyrosine kinase inhibition (tyrphostin 23) attenuated CCE (peak: 67+/-4%; sustained: 74+/-5% of control), but the propofol-induced decrease in CCE was still apparent after tyrosine kinases inhibition. PKC activation (phorbol 12-myristate 13-acetate) attenuated CCE (peak: 48+/-1%; sustained: 53+/-3% of control), whereas PKC inhibition (bisindolylmaleimide) potentiated CCE (peak: 132+/-11%; sustained: 120+/-4% of control). Moreover, PKC inhibition abolished the propofol-induced attenuation of CCE. CONCLUSION: Tyrosine kinases activate and PKC inhibits CCE in PASMCs. Propofol attenuates CCE primarily via a PKC-dependent pathway. CCE should be considered a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.

Propofol increases myofilament Ca2+ sensitivity and intracellular pH via activation of Na+-H+ exchange in rat ventricular myocytes.

BACKGROUND: The objectives were to determine the extent and mechanism of action by which propofol increases myofilament Ca2+ sensitivity and intracellular pH (pHi) in ventricular myocytes. METHODS: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. Myocyte shortening (video edge detection) and pHi (2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, 500/440 ratio) were monitored simultaneously in individual cells field-stimulated (0.3 Hz) and superfused with HEPES-buffered solution (pH 7.4, 30 degrees C). RESULTS: Propofol (100 microM) reduced the Ca2+ concentration required for activation of myofibrillar actomyosin ATPase from pCa 5.7 +/- 0.01 to 6.6 +/- 0.01. Increasing pHi (7.05 +/- 0.03 to 7.39 +/- 0.04) with NH4Cl increased myocyte shortening by 35 +/- 12%. Washout of NH4Cl decreased pHi to 6.82 +/- 0.03 and decreased myocyte shortening to 52 +/- 10% of control. Propofol caused a dose-dependent increase in pHi but reduced myocyte shortening. The propofol-induced increase in pHi was attenuated, whereas the decrease in myocyte shortening was enhanced after pretreatment with ethylisopropyl amiloride, a Na+-H+ exchange inhibitor, or bisindolylmaleimide I, a protein kinase C inhibitor. Propofol also attenuated the NH4Cl-induced intracellular acidosis, increased the rate of recovery from acidosis, and attenuated the associated decrease in myocyte shortening. Propofol caused a leftward shift in the extracellular Ca2+-shortening relation, and this effect was attenuated by ethylisopropyl amiloride. CONCLUSIONS: These results suggest that propofol increases the sensitivity of myofibrillar actomyosin ATPase to Ca2+ (ie., increases myofilament Ca2+ sensitivity), at least in part by increasing pHi via protein kinase C-dependent activation of Na+-H+ exchange.

Pulmonary vascular effects of propofol at baseline, during elevated vasomotor tone, and in response to sympathetic alpha- and beta-adrenoreceptor activation.

BACKGROUND: This in vivo study had two primary objectives. The first goal was to determine whether the pulmonary vascular effects of propofol depend on the preexisting level of vasomotor tone, and the second was to investigate the effects of propofol on the pulmonary vascular responses to sympathetic alpha- and beta-adrenoreceptor activation. METHODS: Thirty-one mongrel dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LPQ) relation. Left lung autotransplantation (LLA) was also performed in eight additional dogs to induce a long-term increase in pulmonary vascular resistance. LPQ plots were measured on separate days in the conscious state and during propofol anesthesia. LPQ plots were measured at baseline and when vasomotor tone was acutely increased with the alpha agonist, phenylephrine, or the thromboxane mimetic, U46619. In separate experiments, cumulative dose-response curves to alpha- (phenylephrine) and beta- (isoproterenol) adrenoreceptor agonists were generated in conscious and propofol-anesthetized dogs. RESULTS: Compared with the conscious state, propofol had no effect on the baseline LPQ relation in normal or post-LLA dogs. However, propofol caused pulmonary vasoconstriction (P < 0.05) when vasomotor tone was acutely increased with either phenylephrine or U46619 in normal or post-LLA dogs. The pulmonary vasoconstrictor response to alpha-adrenoreceptor activation was potentiated (P < 0.05) during propofol anesthesia, whereas the pulmonary vasodilator response to beta-adrenoreceptor activation was not altered. CONCLUSION: These results indicate that the pulmonary vascular response to propofol anesthesia is tone-dependent. During sympathetic activation, propofol may favor alpha-adrenoreceptor-mediated vasoconstriction over beta-adrenoreceptor-mediated vasodilation.

Propofol potentiates phenylephrine-induced contraction via cyclooxygenase inhibition in pulmonary artery smooth muscle.

BACKGROUND: The authors previously demonstrated in vivo that the pulmonary vasoconstrictor response to the a agonist phenylephrine is potentiated during propofol anesthesia compared with the conscious state. The current in vitro study tested the hypothesis that propofol potentiates phenylephrine-induced contraction by inhibiting the synthesis and/or activity of vasodilator metabolites of the cyclooxygenase pathway. METHODS: Canine pulmonary arterial rings were suspended for isometric tension recording. Intracellular calcium concentration ([Ca2+]i) was measured in pulmonary arterial strips loaded with acetoxylmethyl ester of fura-2. After phenylephrine-induced contraction, propofol (10(-7) to 10(-4) M) was administered in the presence or absence of the cyclooxygenase inhibitor ibuprofen (10(-5) M). The effects of propofol on the arachidonic acid and prostacyclin relaxation-response curves were assessed. The amount of 6-keto prostaglandin F1alpha (stable metabolite of prostacyclin) released from pulmonary vascular smooth muscle in response to phenylephrine was measured with enzyme immunoassay in the presence or absence of propofol and ibuprofen. RESULTS: Propofol potentiated phenylephrine-induced contraction in pulmonary arterial rings in a concentration-dependent and endothelium-independent manner. In endothelium-denuded strips, propofol (10(-4) M) increased tension by 53+/-11%, and increased [Ca2+]i by 56+/-9%. Ibuprofen also potentiated phenylephrine-induced contraction but abolished the propofol-induced increases in tension and [Ca2+]i. Propofol had no effect on the relaxation response to prostacyclin, whereas propofol and ibuprofen attenuated the relaxation response to arachidonic acid to a similar extent. Phenylephrine markedly increased 6-keto prostaglandin F1alpha production, and this effect was virtually abolished by propofol and ibuprofen. CONCLUSION: These results suggest that propofol potentiates alpha-adrenoreceptor-mediated pulmonary vasoconstriction by inhibiting the concomitant production of prostacyclin by cyclooxygenase.

Inhibitory effects of etomidate and ketamine on endothelium-dependent relaxation in canine pulmonary artery.

BACKGROUND: The authors recently demonstrated that acetylcholine-induced pulmonary vasorelaxation had two primary components, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). The goal was to investigate the effects of etomidate and ketamine on the NO- and EDHF-mediated components of pulmonary vasorelaxation in response to acetylcholine, bradykinin, and the calcium ionophore, A23187. METHODS: Canine pulmonary arterial rings with an intact endothelium were suspended in organ chambers for isometric tension recording. The effects of etomidate and ketamine (10(-5) M and 10(-4) M) on vasorelaxation responses to acetylcholine, bradykinin, and A23187 were assessed in phenylephrine-contracted rings. The NO- and EDHF-mediated components of relaxation were assessed using a NO synthase inhibitor (N-nitro-L-arginine methylester [L-NAME]: 10(-4) M) and a Ca2+-activated potassium channel inhibitor (tetrabutylammonium hydrogen sulfate [TBA]: 10(-3) M) in rings pretreated with a cyclooxygenase inhibitor (ibuprofen: 10(-5) M). Intracellular calcium concentration ([Ca2+]i) was measured in cultured bovine pulmonary artery endothelial cells loaded with acetoxylmethyl ester of fura-2. RESULTS: Etomidate and ketamine attenuated pulmonary vasorelaxation in response to acetylcholine and bradykinin, whereas they had no effect on the response to A23187. The relaxant responses to acetylcholine and bradykinin were attenuated by L-NAME or TBA alone and were abolished by combined inhibition in rings pretreated with ibuprofen. Etomidate and ketamine further attenuated both L-NAME-resistant and TBA-resistant relaxation. These anesthetics also inhibited increases in endothelial [Ca2+]i in response to bradykinin, but not A23187. CONCLUSION: These results indicate that etomidate and ketamine attenuated vasorelaxant responses to acetylcholine and bradykinin by inhibiting both NO- and EDHF-mediated components. Moreover, our results suggest that these anesthetics do not directly suppress NO or EDHF activity, but rather inhibit the endothelial [Ca2+]i transient in response to receptor activation.

Estimation of current leakage in left and right ventricular conductance volumetry using a dynamic finite element model.

Leakage of electric current through cardiac structures surrounding the ventricle is a primary source of error during ventricular volume measurements using a conductance catheter. This error can be represented as a leakage volume, VL. VL is generally estimated by a saline-bolus method, and is assumed constant throughout the cardiac cycle. However, dynamic changes in ventricular volume and cardiac wall thickness could change VL. To estimate VL, a dynamic finite element model of the heart was developed based on MR images. Conductance measurements were simulated using a modeled conductance catheter, and true VL was calculated. VL varied from 22.7 ml (end-systole) to 26.4 ml (end-diastole) in the left ventricle and from 19.9 ml (end-systole) to 26.9 ml (end-diastole) in the right ventricle. The saline-bolus method underestimated VL in both the left (VL = 19.4 ml) and the right (VL = 4.1 ml) ventricular volume measurements. VL increased linearly with the ratio of blood to tissue resistivity, and changed minimally with catheter position. These results indicate that VL has to be estimated dynamically throughout the cardiac cycle to obtain accurate cardiac volume measurements. The results also show that the saline bolus method does not estimate current leakage accurately, especially in the right ventricular volume measurement.

A comparison of periodontal disease in HIV-infected children and household peers: a two year report.

PURPOSE: The purpose of this study was to compare the incidence and progression of periodontal disease in HIV-infected children to HIV-negative household peers. This paper reports the findings after two years. METHODS: Children diagnosed as HIV-infected and their household peers were recruited from the Children's Hospital AIDS Program in Newark NJ. A periodontal examination was performed at baseline and at six-month intervals for two years. A total of 121 subjects were examined two years after baseline (68 HIV-infected and 53 controls). These children ranged in age from 2-15 years at baseline. RESULTS: Plaque assessment (PHP-M) in HIV-infected cases showed a seven-fold increase over controls for the period. However, there were no significant differences between the two groups in changes over the two years for Bleeding on Probing, Gingival Index or Pocket Depths. There was virtually no recession or pathologic mobility in either group. One-fourth of the HIV-infected group exhibited Linear Gingival Erythema at both baseline and year two. Although the number of subjects with LGE did not increase, there was an increase in the severity of LGE at year 2. CONCLUSION: This study suggests that in a medically well-controlled HIV-infected population, with the exception of the prevalence of Linear Gingival Erythema, the periodontal findings are similar to their HIV-negative household peers and to the general pediatric population.

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle.

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.

Propofol attenuates acetylcholine-induced pulmonary vasorelaxation: role of nitric oxide and endothelium-derived hyperpolarizing factors.

BACKGROUND: The mechanism by which propofol selectively attenuates the pulmonary vasodilator response to acetylcholine is unknown. The goals of this study were to identify the contributions of endogenous endothelial mediators (nitric oxide [NO], prostacyclin, and endothelium-derived hyperpolarizing factors [EDHFs]) to acetylcholine-induced pulmonary vasorelaxation, and to delineate the extent to which propofol attenuates responses to these endothelium-derived relaxing factors. METHODS: Canine pulmonary arterial rings were suspended for isometric tension recording. The effects of propofol on the vasorelaxation responses to acetylcholine, bradykinin, and the guanylyl cyclase activator, SIN-1, were assessed in phenylephrine-precontracted rings. The contributions of NO, prostacyclin, and EDHFs to acetylcholine-induced vasorelaxation were assessed in control and propofol-treated rings by pretreating the rings with a NO synthase inhibitor (l-NAME), a cyclooxygenase inhibitor (indomethacin), and a cytochrome P450 inhibitor (clotrimazole or SKF 525A) alone and in combination. RESULTS: Propofol caused a dose-dependent rightward shift in the acetylcholine dose-response relation, whereas it had no effect on the pulmonary vasorelaxant responses to bradykinin or SIN-1. Cyclooxygenase inhibition only attenuated acetylcholine-induced relaxation at high concentrations of the agonist. NO synthase inhibition and cytochrome P450 inhibition each attenuated the response to acetylcholine, and combined inhibition abolished the response. Propofol further attenuated acetylcholine-induced relaxation after NO synthase inhibition and after cytochrome P450 inhibition. CONCLUSION: These results suggest that acetylcholine-induced pulmonary vasorelaxation is mediated by two components: NO and a cytochrome P450 metabolite likely to be an EDHF. Propofol selectively attenuates acetylcholine-induced relaxation by inhibiting both of these endothelium-derived mediators.

Propofol selectively attenuates endothelium-dependent pulmonary vasodilation in chronically instrumented dogs.

BACKGROUND: The objective was to investigate the effects of propofol anesthesia on the pulmonary vascular response to endothelium-dependent and -independent vasodilators, compared with the responses measured in the conscious state. METHODS: Twenty-six conditioned, male, mongrel dogs were instrumented long-term to measure the left pulmonary vascular pressure-flow relation. Pressure-flow plots were measured on separate days in conscious and propofol-anesthetized (5.0 mg/kg plus 0.5 mg. kg-1. min-1 intravenously) dogs at baseline, after preconstriction with the thromboxane mimetic U46619, and during the cumulative intravenous administration of endothelium-dependent (acetylcholine and bradykinin) and -independent (proline-nitric oxide) vasodilators. RESULTS: Propofol had no effect on the baseline pressure-flow relation compared with the conscious state. A lower (P < 0.05) dose of U46619 was necessary to achieve the same degree of preconstriction during propofol anesthesia. The pulmonary vasodilator responses to bradykinin and proline-nitric oxide were similar in the conscious and propofol-anesthetized states. In contrast, the pulmonary vasodilator response to acetylcholine was markedly attenuated (P < 0.01) during propofol anesthesia. The intralipid vehicle for propofol had no effect on the acetylcholine dose-response relation. CONCLUSION: These results suggest that propofol causes a specific defect in the signal transduction pathway for acetylcholine-induced pulmonary vasodilation. This defect involves the endothelial and not the vascular smooth muscle component of the response.

Pulmonary vasoregulation by endothelin in conscious dogs after left lung transplantation.

We tested the hypothesis that regulation of the pulmonary circulation by endogenous endothelin (ET) during normoxia and hypoxia was altered in conscious dogs 1 mo after left lung autotransplantation (LLA). Sham-operated control and post-LLA dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LP-Q) relationship. LP-Q plots were generated on separate days during normoxia and hypoxia (arterial PO(2) approximately 50 Torr) in the intact condition, after selective ET(A)-receptor inhibition (BQ-485), and after combined ET(A+B)-receptor inhibition (bosentan). Although LLA resulted in a chronic increase in pulmonary vascular resistance, the ET-receptor antagonists had no effect on the LP-Q relationship during normoxia in either group. The magnitude of hypoxic pulmonary vasoconstriction (HPV) was flow dependent in both groups, and the HPV response was potentiated post-LLA compared with control. ET(A)-receptor inhibition attenuated the HPV response to the same extent in both groups. ET(A+B)-receptor inhibition attenuated the HPV response to a greater extent than did ET(A)-receptor inhibition alone, and this effect was greater post-LLA compared with control. Plasma ET-1 concentration only increased during hypoxia in the LLA group. These results indicate that ET does not regulate the baseline LP-Q relationship in either group. Both ET(A)- and ET(B)-receptor activation mediate a component of HPV in conscious dogs, and the vasoconstrictor influence of ET(B)-receptor activation is enhanced post-LLA.

Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.

Binding, partial agonism, and potentiation of alpha(1)-adrenergic receptor function by benzodiazepines: A potential site of allosteric modulation.

Benzodiazepines, a class of drugs commonly used to induce anesthesia and sedation, can attenuate intracellular calcium oscillations evoked by alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation in pulmonary artery smooth muscle cells. We postulated a direct action of benzodiazepines in modulating alpha(1)-AR function at the receptor level. Benzodiazepines bound to each of the cloned alpha(1)-AR subtypes (alpha(1a)-, alpha(1b)-, or alpha(1d)-AR) on COS-1 cell membranes transiently transfected to express a single population of alpha(1)-AR subtype. The ability of benzodiazepines to alter alpha(1)-AR signal transduction was investigated by measuring total inositol phosphate generation in rat-1 fibroblast cells, stably transfected to express a single alpha(1)-AR subtype. By themselves, benzodiazepines displayed partial agonism. At alpha(1b)-ARs and alpha(1d)-ARs, the maximal inositol phosphate response to phenylephrine was potentiated almost 2-fold by either midazolam or lorazepam (100 microM). At alpha(1a)-ARs, diazepam, lorazepam, and midazolam all increased the maximal response of the partial agonist clonidine at these receptors, whereas the response to the full agonist phenylephrine was unaltered or inhibited. The potentiating actions of midazolam and its partial agonism at alpha(1)-ARs was blocked by the addition of 1 microM prazosin, an alpha(1)-AR antagonist, and not by a gamma-aminobutyric acid(A)-receptor antagonist. These studies show that benzodiazepines modulate the function of alpha(1)-ARs in vitro, and this is the first report of a potential allosteric site on alpha(1)-ARs that may be therapeutically useful for drug design.

Ketamine preserves and propofol potentiates hypoxic pulmonary vasoconstriction compared with the conscious state in chronically instrumented dogs.

BACKGROUND: The authors tested the hypothesis that ketamine and propofol anesthesia would alter the magnitude of hypoxic pulmonary vasoconstriction compared with the conscious state. In addition, they assessed the extent to which cyclooxygenase pathway inhibition and adenosine triphosphate-sensitive potassium channel inhibition modulate hypoxic pulmonary vasoconstriction in the conscious state, and whether these pathways are altered during propofol anesthesia. METHODS: Twenty conditioned, male mongrel dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow relationship. Pressure-flow plots were measured during normoxia and hypoxia (systemic arterial PO2 reduced to about 60 and about 50 mm Hg) on separate days in the conscious state, during ketamine anesthesia, and during propofol anesthesia. The effects of indomethacin and glibenclamide on the magnitude of hypoxic pulmonary vasoconstriction were also assessed in the conscious and propofol-anesthetized states. RESULTS: Neither ketamine nor propofol had an effect on the baseline pressure-flow relationship during normoxia compared with the conscious state. Hypoxia resulted in stimulus-dependent pulmonary vasoconstriction (P<0.01) in the conscious state. Compared with the conscious state, the magnitude of hypoxic pulmonary vasoconstriction was preserved during ketamine but was potentiated (P<0.01) during propofol anesthesia. Indomethacin enhanced (P<0.01) hypoxic pulmonary vasoconstriction in both the conscious and propofol-anesthetized states. In contrast, glibenclamide only enhanced (P<0.01) hypoxic pulmonary vasoconstriction in the conscious state and had no effect during propofol anesthesia. CONCLUSION: Hypoxic pulmonary vasoconstriction is preserved during ketamine anesthesia but is potentiated during propofol anesthesia. The potentiated response during propofol anesthesia appears to be caused by inhibition of adenosine triphosphate-sensitive potassium channel-mediated pulmonary vasodilation.