Paul A. Murray

Spindle Cell Conversion by Kaposi's Sarcoma-Associated Herpesvirus: Formation of Colonies and Plaques with Mixed Lytic and Latent Gene Expression in Infected Primary Dermal Microvascular Endothelial Cell Cultures

ABSTRACT Angiogenic Kaposis sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.

Propofol and Ketamine Only Inhibit Intracellular Ca2+Transients and Contraction in Rat Ventricular Myocytes at Supraclinical Concentrations

Background The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation‐contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field‐stimulated ventricular myocytes. Methods Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura‐2, and placed on the stage of an inverted fluorescence microscope in a temperature‐regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field‐stimulated at 0.3 Hz. Results Baseline [Ca2+]i (mean +/‐ SEM) was 80 +/‐ 12 nM, and resting cell length was 112 +/‐ 2 micro meter. Field stimulation increased [Ca2+]i to 350 +/‐ 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose‐dependent decreases in peak [Ca2+]i and shortening. At 300 micro Meter, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose‐response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. Conclusions These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.

Entropy measures of heart rate variation in conscious dogs

Our goal was to determine the contributions of sympathetic and parasympathetic activity to entropy measures of heart rate variability (HRV). We compared our results with two commonly used methods to analyze HRV: standard deviation (SDNN) and power spectral analysis (HF norm). Beat-by-beat analysis of R-R intervals was performed in conscious dogs. The R-R intervals were analyzed with approximate entropy (ApEn) and entropy of symbolic dynamics (SymDyn) to assess the effects of reducing system complexity. This was achieved by pharmacologically inhibiting sympathetic, parasympathetic, and total autonomic nervous system regulation of heart rate. Three conditions were examined: rest, standing, and systemic hypotension. At rest or standing, sympathetic inhibition (propranolol) had no effect on ApEn or SymDyn, whereas parasympathetic (atropine) and combined (propranolol + atropine) inhibition reduced both entropy measures to near zero. Systemic hypotension reduced both entropy measures in intact dogs. When hypotension was induced after sympathetic inhibition, ApEn was increased compared with hypotension alone, whereas parasympathetic inhibition with hypotension resulted in near-zero ApEn. Changes in the entropy measures of HRV were directionally similar to changes in SDNN and HF norm. These results indicate that the entropy of R-R intervals reflects parasympathetic modulation of heart rate.

Attenuated Hypoxic Pulmonary Vasoconstriction during Isoflurane Anesthesia Is Abolished by Cyclooxygenase Inhibition in Chronically Instrumented Dogs

Background Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism whereby gas exchange is improved through the diversion of blood flow away from poorly oxygenated regions of the lung. The effect of isoflurane anesthesia on HPV is unclear. Using a chronically instrumented canine model, it was hypothesized that isoflurane anesthesia would attenuate HPV compared to the response measured in the same animal in the conscious state. Moreover, because volatile anesthetics increase the production of cyclooxygenase metabolites, it was hypothesized that attenuation of HPV during isoflurane anesthesia would be abolished by cyclooxygenase inhibition. Methods Left pulmonary vascular pressure-flow plots were generated in chronically instrumented dogs by measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure-left atrial pressure) and left pulmonary blood flow during inflation of a hydraulic occluder implanted around the right main pulmonary artery. In protocol 1 (n = 7), left pulmonary vascular pressure-flow plots were generated during normoxia and hypoxia (systemic arterial PO2 approximately 50 mmHg) in the conscious and isoflurane-anesthetized states. In protocol 2 (n = 7), left pulmonary vascular pressure-flow plots were generated during normoxia and hypoxia (1) in the conscious state, (2) in the conscious state after inhibition of the cyclooxygenase pathway with indomethacin, and (3) during isoflurane anesthesia after cyclooxygenase inhibition. Results In both the conscious and isoflurane-anesthetized states, the magnitude of HPV was dependent on the level of left pulmonary blood flow. Compared to the response measured in the conscious state, the magnitude of HPV was attenuated during isoflurane anesthesia over the empirically measured range of left pulmonary blood flow. Cyclooxygenase inhibition abolished the isoflurane-induced attenuation of HPV. Conclusions This is the first study to demonstrate that isoflurane anesthesia attenuates the magnitude of HPV compared to the response measured in the same animal in the conscious state. Cyclooxygenase inhibition potentiated the magnitude of HPV in both the conscious and isoflurane-anesthetized states, which indicates that vasodilator metabolites of the cyclooxygenase pathway modulate HPV under these conditions. Importantly, the finding that the magnitude of HPV is flow-dependent in both the conscious and isoflurane-anesthetized states may explain conflicting reports in the literature concerning the effects of isoflurane anesthesia on the HPV response.

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°C). Results Propofol (100 &mgr;m) 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+ (i.e., increases myofilament Ca2+sensitivity), at least in part by increasing pHivia protein kinase C–dependent activation of Na+–H+ exchange.

Synergistic interaction between endothelium‐derived NO and prostacyclin in pulmonary artery: potential role for K+ATP channels

The aim of the present study was to assess interactions between nitric oxide (NO) and prostacyclin (PGI2) during endothelium‐dependent relaxations evoked by bradykinin, calcium ionophore (A23187) and acetylcholine in canine isolated pulmonary artery. Relaxations to low concentrations of bradykinin and A23187 were abolished by combined inhibition of NO‐synthase (by NΩ‐nitro‐L‐arginine methyl ester L‐NAME, 30 μM) and cyclo‐oxygenase (indomethacin, 10 μM), suggesting mediation by NO and PGI2. The individual contributions of NO and PGI2 to the dilator responses were quantified by use of areas above the separate indomethacin‐insensitive and L‐NAME‐insensitive components of the concentration‐effect curves, respectively. Individually, NO and PGI2 accounted for only 53±5% and 16±9% of total bradykinin‐induced relaxation, and 46±10% and 20±9% of total A23187‐induced relaxation, suggesting that NO and PGI2 acted synergistically to cause endothelium‐dependent relaxation. Relaxation to low concentrations of acetylcholine was abolished by L‐NAME but not affected by indomethacin, suggesting the response was mediated solely by NO with no interaction from PGI2. Glibenclamide (1 μM), an inhibitor of ATP‐sensitive potassium (K+ATP) channels, inhibited responses to bradykinin or A23187 but did not affect relaxations evoked by acetylcholine. Glibenclamide did not affect endothelium‐independent relaxations to PGI2 or the NO‐donor, 3‐morpholinosydnonimine (SIN‐1). With bradykinin, glibenclamide attenuated total relaxation by 49±8%, but did not alter the individual NO and PGI2‐mediated components of the response. Glibenclamide abolished the synergistic interaction between endothelium‐derived NO and PGI2. At high concentrations, bradykinin, A23187 or acetylcholine caused endothelium‐dependent relaxation that was insensitive to L‐NAME + indomethacin. With bradykinin or A23187, this component of relaxation was inhibited by glibenclamide, whereas with acetylcholine, glibenclamide had no effect. The synergistic interaction between endothelium‐derived NO and PGI2 in canine pulmonary artery is mediated by activation of K+ATP channels, presumably by an endothelium‐derived hyperpolarizing factor (EDHF). The pattern of endothelial dilator mediators and the presence of this synergistic interaction is dependent on the nature of the endothelial stimulus.

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.

Phenylephrine-Induced Ca2+ Oscillations in Canine Pulmonary Artery Smooth Muscle Cells

Modulation of [Ca2+]i in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of alpha 1-adrenoceptors with phenylephrine (PE) on [Ca2+]i in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs pretreated with propranolol (5 mumol/L) were continuously superfused with PE at 37 degrees C on the stage of an inverted fluorescence microscope, and [Ca2+]i was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca2+]i were 96 +/- 4 nmol/L. PE (10 mumol/L) stimulated oscillations in [Ca2+]i at a frequency of 1.35 +/- 0.07/min, which reached a peak [Ca2+]i of 650 +/- 26 nmol/L (n = 69 cells). The oscillations lasted for > 30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca2+]i oscillations increased in a dose-dependent (3 x 10(-8) to 10(-4) mol/L) manner. Pretreatment with the alpha 1-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca2+ abolished the repetitive [Ca2+]i oscillations induced by PE. The voltage-operated Ca2+ channel blockers nifedipine (1 mumol/L) and verapamil (1 mumol/L) had no effect on the [Ca2+]i oscillations. In contrast, inhibition of phospholipase C with U73122 (10(-7) to 10(-5) mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10(-9) to 10(-7) mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 mumol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 mumol/L) had no effect. In freshly dispersed PASMCs, PE (10 mumol/L) induced oscillations in [Ca2+]i similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca2+]i oscillations in PASMCs via stimulation of alpha 1-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca2+ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca2+ and intracellular Ca2+ stores indicates that both Ca2+ influx and intracellular Ca2+ release play a role in the maintenance of the oscillations.

Inhibitory Effects of Etomidate and Ketamine on Endothelium-dependent Relaxation in Canine Pulmonary Artery

BackgroundThe 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. MethodsCanine 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. ResultsEtomidate 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. ConclusionThese 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.

Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II.

Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (α, δ, ε, ζ, ι/λ, and μ) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (α, δ, ζ, ι/λ, and μ) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-α from the cytosol to the nuclear envelope and of PKC-δ to the myofilaments. In contrast, cytosolic PKC-ζ did not translocate, but nuclear PKC-ζ was upregulated. Translocation of PKC-α and PKC-δ and upregulation of PKC-ζ in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The α and ζ isoforms may act to regulate nuclear events, whereas PKC-δ may be involved in modulating contraction via actions on the myofilaments.Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha, delta, epsilon, zeta, iota/lambda, and mu) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha, delta, zeta, iota/lambda, and mu) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-zeta was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The alpha and zeta isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.