Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature.

Although the use of nonviral vectors for gene therapy offers distinct advantages including the lack of significant inflammatory and immune responses, the levels of expression in vivo remain much lower than those obtained with their viral counterparts. One reason for such low expression is that unlike many viruses, plasmids have not evolved mechanisms to target to the nucleus of the nondividing cell. In the absence of mitosis, plasmids are imported into the nucleus in a sequence-specific manner, and we have shown in cultured cells by transfection and microinjection experiments that the SV40 enhancer mediates plasmid nuclear import in all cell types tested (Dean et al., 1999, Exp Cell Res 253: 713-722). To test the effect of this import sequence on gene transfer in the intact animal, we have recently developed an electroporation method for DNA delivery to the intact mesenteric vasculature of the rat. Plasmids expressing luciferase or GFP from the CMV immediate-early promoter/enhancer and either containing or lacking the SV40 enhancer downstream of the reporter gene were transferred to the vasculature by electroporation. When transfected into actively dividing populations of smooth muscle or epithelial cells, the plasmids gave similar levels of expression. By contrast, the presence of the SV40 sequence greatly enhanced gene expression of both reporters in the target tissue. At 2 days post-transfer, plasmids with the SV40 sequence gave 10-fold higher levels of luciferase expression, and at 3 days the difference was over 40-fold. The presence of the SV40 sequence did not simply increase the rate of nuclear import and expression, since expression from the SV40-lacking plasmid did not increase beyond that seen at day 2, the time of maximum expression for either plasmid. In situ hybridization experiments confirmed that the increased gene transfer and expression was indeed due to increased nuclear localization of the delivered SV40 sequence-containing plasmid. Based on these findings, the ability to target DNA to the nucleus can increase gene transfer in vivo and inclusion of the SV40 sequence into plasmids will enhance nonviral gene delivery.

Gene transfer to intact mesenteric arteries by electroporation.

The purpose of the present study was to develop a rapid, reproducible method of nonviral gene transfer to the intact vasculature. Male Sprague-Dawley rats were anesthetized, a midline abdominal incision was made and segmental branches of the superior mesenteric artery were dissected free of surrounding mesentery. A specially designed electroporation probe was placed around the neurovascular bundle and the electroporation chamber filled with a solution containing the firefly luciferase expressing plasmid (pCMV-Lux-DTS) or the green fluorescent protein expressing plasmid (pEGFP-N1). Vessels were electroporated with eight 10-ms pulses of 200 V/cm. Sixty seconds after electroporation, the DNA solution was removed, the intestine returned to the abdomen and the abdominal wall closed with suture and metal wound clips. Six hours to 5 days later, rats were sacrificed and electroporated vessels were recovered. Luciferase activity of the blood vessels was monitored. Gene expression was detected as early as 6 h postelectroporation, peaked at 1-3 days with levels up to 1 ng of reporter gene product per vessel segment and returned towards baseline by day 5. Histological analysis of blood vessel segments revealed green fluorescent protein-positive cells throughout the thickness of the vessel wall (endothelial cells to adventitia). Responses of electroporated vessels to vasoconstricting stimuli were indistinguishable from those of control vessels at either 2 or 40 days posttreatment. The results of this study provide evidence that electroporation is an effective means for introducing naked DNA into the blood vessel wall and form the basis for future studies on targeted gene therapy to the intact vasculature.

Differential effects of furosemide on porcine bronchial arterial and airway smooth muscle.

Furosemide attenuates airway obstruction in asthmatic subjects when administered as an aerosol pretreatment. This protective effect of furosemide could be related to relaxation of bronchial smooth muscle or to increased bronchial blood flow. To determine whether furosemide dilates bronchial smooth muscle, isometric contractile responses in distal bronchi from young pigs were studied. In bronchial smooth muscle rings that were precontracted with 10(-5) M acetylcholine, significant relaxation occurred with 10(-8) to 3 x 10(-6) M isoproterenol but not with 10(-8) to 10(-3) M furosemide. In contrast, bronchial arteries that were precontracted with either 10(-4) M norepinephrine or 10(-8) M vasopressin significantly relaxed in response to 10(-4) to 3 x 10(-3) M and 10(-3) to 3 x 10(-3) M furosemide, respectively. We conclude that furosemide, under the described experimental conditions, relaxes airway vascular smooth muscle but not bronchial smooth muscle. These results are consistent with previous suggestions that inhaled furosemide increases blood flow to airway tissues (Gilbert IA, Lenner KA, Nelson JA, Wolin AD, and Fouke JM. J Appl Physiol 76: 409-415, 1994).

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells.

OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.

Effect of milrinone on small mesenteric artery vasoconstriction: role of K(+) channels.

We examined whether milrinone-mediated attenuation of small mesenteric artery vasoconstriction results predominantly from the activation of vascular smooth muscle K(+) channels. Resistance arteries (approximately 150 micrometers) were dissected from rat mesentery and were mounted on a wire myograph. Isometric force development in response to increasing concentrations of norepinephrine (NE) was monitored before and after treatment with the type 3 phosphodiesterase inhibitor milrinone. Milrinone significantly reduced NE-induced vasoconstriction, attenuating both NE sensitivity and maximal tension generation. Inhibition of ATP-sensitive K(+) channels or voltage-gated K(+) channels did not prevent the milrinone-induced attenuation of NE responses. Blockade of inwardly rectifying K(+) channels or Ca(2+)-sensitive K(+) channels prevented the milrinone-mediated reduction in NE sensitivity, but this effect was apparently due to direct enhancement of vasoconstrictor responsiveness rather than interference with the mechanism of milrinone action. In addition, milrinone elicited substantial relaxation in vessels preconstricted with 100 mM KCl. This effect was mimicked by the adenylyl cyclase activator forskolin and was reversed by the Rp diastereomer of cAMP, which is a cAMP-dependent protein kinase (PKA) inhibitor. Our results indicate that cAMP/PKA-mediated impairment of vasoconstriction may occur without the contribution of K(+) channel regulation.

Effects of IBMX on norepinephrine-induced vasoconstriction in small mesenteric arteries.

The present study assesses the effects of the phosphodiesterase inhibitor IBMX on norepinephrine (NE)-induced constriction of small mesenteric arteries. Arteries ( approximately 150 micrometer) were dissected from rats and mounted on a wire myograph for isometric force measurement. NE concentration effect curves were generated after exposure to 500 microM IBMX for 60 min. IBMX significantly reduced NE-induced tension development. Studies were also conducted following sarcoplasmic reticulum (SR) depletion (ryanodine, 10 microM) or L-type Ca2+ channel blockade [(+)-BAY K 8644, 10 microM] in the presence and absence of IBMX. Both SR depletion and L-channel blockade reduced NE-induced tension generation, consistent with incomplete Ca2+ mobilization. IBMX significantly attenuated NE responses in ryanodine and (+)-BAY K 8644-treated vessels. Finally, treatment of NE-stimulated vessels with IBMX (500 microM) caused a reduction in vascular tension that was greater than the concomitant reduction in cytosolic Ca2+ concentration ([Ca2+]i), indicating that a portion of the IBMX-mediated relaxation is Ca2+-independent. These data suggest that IBMX attenuation of NE responsiveness not only involves a reduction in [Ca2+]i but also a significant decrease in Ca2+ sensitivity.

Effects of alpha-adrenergic stimuli on mesenteric collecting lymphatics in the rat.

The present study examined the effects of alpha 1- and alpha 2-adrenergic stimuli on rat mesenteric collecting lymphatics in vivo. Sprague-Dawley rats were anesthetized, and the mesentery was prepared for intravital microscopic study. Mesenteric collecting lymphatic diameter was continuously monitored by using a computerized video tracking system, and indexes of lymphatic pumping (e.g., contraction frequency, stroke volume, ejection fraction, and muscle shortening velocity) were determined from the diameter record. Contractile activity was monitored before and during the administration of various adrenergic agonists and antagonists. The receptor antagonists prazosin (alpha 1) and yohimbine (alpha 2) did not significantly alter baseline diameter or contractile activity, which suggests that lymphatics possess no basal adrenergic tone. Norepinephrine and phenylephrine (01-1.0 microM) produced dose-dependent increases in frequency and decreases in diameter. Lymphatic pump flow increased in direct proportion to frequency, because stroke volume did not change. The changes in lymphatic pumping produced by 1 microM norepinephrine were completely blocked by prazosin or phentolamine and only partially blocked by yohimbine. The alpha 2-adrenoceptor agonist (alpha-methyl-norepinephrine) produced no changes in lymphatic activity. This latter observation suggests that a role for postjunctional alpha 2-adrenoceptors in modulating mesenteric lymphatic smooth muscle is unlikely. The results of these studies support the existence of alpha-adrenoceptors on lymphatic smooth muscle. It is concluded that conditions characterized by increased sympathetic outflow may augment lymphatic function through alpha 1- but not alpha 2-adrenoceptors.

Vascular reactivity following ischemia/reperfusion.

Data in the literature supports the hypothesis that reactive oxygen species generated in the vascular wall alter vascular regulation. At present the majority of the literature tends to suggest that oxidant induced damage on the smooth muscle cell impair vasoconstriction. However, direct action of oxidants on the smooth muscle cell impair vasoconstrictor function. Differences in studies in the literature are likely to be reconciled when the target sites of reactive oxygen species are considered. Future research in this area should lead to a more comprehensive understanding on the impact of these pathways on vasoregulation in postischemic tissue.

Altered vascular norepinephrine responses in portal hypertensive intestine: role of PKA and guanylate cyclase.

The purpose of the present study was to determine whether selective blockade of adenosine 3',5'-cyclic monophosphate (cAMP)- or guanosine 3',5'-cyclic monophosphate (cGMP)-mediated events modulated norepinephrine responses in intestinal microvessels of normal and portal hypertensive rats. Vascular norepinephrine responses were evaluated before and after inhibition of cAMP-dependent protein kinase [protein kinase A(PKA)] with Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) or guanylate cyclase with LY-83583. Male Sprague-Dawley rats were divided into two groups: those with portal hypertension by portal vein stenosis and normal controls. The small intestine was prepared for microcirculatory studies. Arteriolar diameter and erythrocyte velocity were monitored, and microvascular flow was calculated from velocity and diameter data. The preparation was challenged with incremental concentrations of norepinephrine before and after addition of Rp-cAMPS (50 microM) or LY-83583 (30 microM). Arteriolar diameter and blood flow were significantly elevated in portal hypertensive rats; norepinephrine responses were significantly depressed. LY-83583 did not alter arteriolar diameter, blood flow, or norepinephrine responsiveness in normal or portal hypertensive rats. Rp-cAMPS did not affect arteriolar diameter, blood flow, or norepinephrine responsiveness in normal rats. However, in portal hypertensive rats, Rp-cAMPS reduced blood flow by approximately 20% (P < 0.05) and completely restored vascular norepinephrine responses to normal. The results indicate that cAMP- but not cGMP-dependent events are primarily responsible for the loss of microvascular norepinephrine responsiveness in portal hypertensive intestine.

Hypoxia/reoxygenation selectively impairs alpha 1b-adrenoceptor function in small mesenteric arteries.

The present study examined whether hypoxia/reoxygenation (H/R) attenuates norepinephrine (NE) effectiveness in small arteries by interfering with function of alpha 1a- and/or alpha 1b- adrenoceptor subtypes. Small mesenteric arteries (approximately 150 microns) were obtained from rats mounted on a small vessel myograph in oxygenated physiological salt solution (PSS), and the relationship between NE concentrations and contractile tension was assessed. Hypoxia was induced by bubbling the vessels with 95% N2-5% CO2 for 15 min. Vessels were then reoxygenated for 30 min, and NE responses were reevaluated. Superoxide dismutase (SOD) and catalase (CAT) were added to the PSS in one group of vessels to investigate the role of reactive oxygen metabolites. In other groups, alpha 1b-receptors were blocked with chloroethylclonidine and alpha 1a-receptors were blocked with 5-methylurapidil or WB-4101 to produce exclusive alpha 1a- or alpha 1b-responses to NE. H/R decreased the NE negative logarithm of the mean effective concentration (pD2: i.e., -log[EC50], where EC50 is mean effective concentration) from 6.26 +/- 0.24 to 5.84 +/- 0.12 (P < 0.05). SOD and CAT prevented the H/R-induced contractile dysfunction. alpha 1a-Receptor responses to NE were not altered by H/R. In contrast, alpha 1b-receptor responses were significantly attenuated after H/R. The results indicate that alterations in NE responsiveness after H/R are due to dysfunction of the alpha 1b signal transduction pathway.

Effects of bradykinin on lymphatic pumping in rat mesentery.

The effects of bradykinin on lymphatic pump activity of rat mesenteric collecting duct were studied, and the receptor subtype responsible for the bradykinin response was evaluated. Rats were anesthetized with intraperitoneal alpha-chloralose and urethan, and exteriorized mesenteries were studied using intravital microscopic techniques. The diameter of the collecting lymph vessels (approximately 100 microns) was continuously monitored and lymphatic pump parameters (end diastolic diameter, end systolic diameter, stroke volume index, ejection fraction, contraction frequency, and pump flow index) were calculated. Bradykinin (0.1-1.0 nM) did not affect end diastolic diameter, end systolic diameter, stroke volume index, and ejection fraction. Bradykinin increased lymphatic contraction frequency and pump flow index in a dose-dependent manner. Des-Arg9-[Leu8]bradykinin (B1 antagonist, 0.1 microM) had no effect on baseline lymphatic pumping but completely inhibited the bradykinin-induced increase in contraction frequency. N-acetyl-D-Arg-[Hyp3,Thi5,8,D-Phe7] bradykinin (B2 antagonist, 0.1 microM) significantly depressed lymphatic contraction frequency in baseline conditions but had no effect on bradykinin-induced increases in contraction frequency. These results indicate that bradykinin induces positive chronotropic but not inotropic effects on lymphatic pump activity through the stimulation of B1 receptors.

Hypoxia-reoxygenation impairs endothelium-dependent relaxation in isolated rat aorta.

The effects of hypoxia followed by reoxygenation on endothelium-dependent relaxation in isolated rat aorta were investigated. Acetylcholine (ACh, 3 nM-10 microM) and calcium ionophore A-23187 (3 nM-300 nM)-induced endothelium-dependent vasorelaxation of isolated rate aortic vessel rings was impaired after 15 min of hypoxia followed by 30 min of reoxygenation. Impairment of ACh-induced relaxation was prevented by pretreatment with the combination of superoxide dismutase (200 U/ml) and catalase (1,000 U/ml). Hypoxia-reoxygenation did not affect sodium nitroprusside (0.1 nM-1 microM)-induced endothelium-independent relaxation nor the dissociation constant of ACh to endothelial M3 muscarinic receptors. Propidium iodide staining of the vascular endothelium revealed a significant increase in the number of dead endothelial cells on the aortic vessel rings following hypoxia-reoxygenation, but not on those pretreated with superoxide dismutase and catalase. These results suggest that hypoxia-reoxygenation impairs endothelium-dependent relaxation of rat aorta by a mechanism that involves oxidant-mediated endothelial cell death.

Effects of hypoxia/reoxygenation on aortic vasoconstrictor responsiveness.

The purpose of the present study was to assess the effects of hypoxia/reoxygenation (H/R) on vasoconstrictor effectiveness, in vitro. Aortic rings were obtained from rats and placed on isometric force transducers in oxygenated Krebs buffer (95% O2/5% CO2, PO2 > 500 torr). Cumulative concentration/effect relationships to norepinephrine, G-protein activation by AlICl3/NaF, depolarization by KCl or BayK-8644, mobilization of intracellular calcium by caffeine, and protein kinase C activation by l-indolactam were evaluated. Hypoxia (PO2 < 5 torr) was induced by rapidly bubbling the Krebs buffer with 95% N2/5% CO2 for 15 min. Vessel rings were reoxygenated for 30 min and concentration/effect relationships reevaluated. The dissociation constant (KA) for norepinephrine was also determined. The pD2 for maximal norepinephrine responsiveness decreased from 7.7 to 7.3 following H/R. Maximal tension generation was significantly decreased following H/R. Endothelium denudation or nitric oxide synthesis inhibition did not prevent the right shift in norepinephrine concentration/effect relationship caused by H/R. The combination of superoxide dismutase and catalase prevented the dextral shift in the concentration/effect curve. The dissociation constant for norepinephrine increased from 0.16 to 0.32 microM following H/R, suggesting decreased affinity of adrenergic receptor. H/R did not alter AlCl3/NaF, KCl, BayK-8644 or l-indolactam-induced vasoconstriction. Caffeine-induced vasoconstriction was significantly impaired following H/R, suggesting that release of calcium from the sarcoplasmic reticulum is compromised. These results suggest that H/R leads to an endothelium independent, oxidant-mediated decrease in vascular norepinephrine responsiveness that may be related to defects in the mobilization of intracellular calcium from the sarcoplasmic reticulum pool.

Effects of propranolol on intestinal microcirculation of normal and portal hypertensive rats.

BACKGROUND/AIMS: The goal of the present study was to compare the efficacy of locally and systemically administered propranolol in normal and prehepatic portal hypertensive rats, and to test the hypothesis that beta-adrenoceptor blockade reduces intestinal arteriolar diameter by allowing unopposed alpha-adrenergic activity. METHODS: The small intestine was prepared for in vivo microcirculatory studies and transferred to an intravital microscope where arteriolar diameter and erythrocyte velocity were continuously monitored. First order arteriolar (1A) blood flow was calculated from the product of mean velocity and microvessel cross-sectional area. In separate experiments, diameter responses of 2A and 3A were monitored. Once steady-state conditions were achieved, the preparation was challenged by topically applied doses of propranolol (0.01-100.00 microM) in the presence and absence of the alpha-receptor antagonist, phentolamine. In a separate group of experiments, the effects of systemically administered propranolol (10 mg/kg body weight) were evaluated before and after local alpha-adrenoceptor blockade. RESULTS: Propranolol produced significant vasoconstriction and decreased blood flow in both normal and portal hypertensive rats. Portal hypertensive arterioles exhibited an attenuated response to propranolol. Local administration of phentolamine completely blocked the propranolol-induced diameter changes. Comparison of equivalent concentrations of local and systemic propranolol indicated that both routes of administration were equally effective. CONCLUSION: The results of the present study suggest that the cardiovascular actions of propranolol are predominantly mediated through blockade of peripheral beta 2-adrenoceptor.

Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.

Nonreceptor-mediated intestinal vasoconstriction in portal hypertensive rats.

Previous studies have demonstrated that receptor-mediated vasoconstriction is impaired in chronic portal hypertension (PH). Furthermore, it has been suggested that altered vasoconstrictor effectiveness in chronic PH is due to a defect in the intracellular events associated with smooth muscle activation and not to impaired coupling of vasoconstrictors with vascular smooth muscle receptors. The present study was designed to determine whether nonreceptor-mediated vasoconstrictor responses are impaired in the PH intestinal microcirculation. Specifically, we examined the effects of aluminum fluoride-induced activation of G proteins, KCl-induced depolarization, caffeine-induced release of intracellular Ca2+, and l-indolactam-induced activation of protein kinase C on the intestinal microcirculation of normal (Norm, n = 39) and PH (n = 42) rats. The small intestine was prepared for microcirculatory studies and transferred to a video microscope. First-order arteriolar (1A) diameter and red cell velocity were measured on-line. Blood flow was calculated from the product of velocity and microvessel cross-sectional area. After a control period, the microvasculature was exposed to a solution containing aluminum chloride plus sodium fluoride, potassium chloride, caffeine, or l-indolactam. Maximal decreases in arteriolar diameter produced by aluminum fluoride, KCl, caffeine, and l-indolactam were significantly greater in Norm rats when compared with PH rats. Changes in arteriolar blood flow were also larger in Norm than in PH rats. The results of the present study provide the first direct evidence of an impaired response to second-messenger activation in the PH circulation.

Effects of reactive oxygen metabolites on norepinephrine-induced vasoconstriction.

Aortic rings, 4 mm in length, were obtained from rats and placed on isometric force transducers in oxygenated Krebs buffer. Following a period of stabilization, the cumulative dose response relationship to norepinephrine was assessed. The vessels were washed and allowed to return to baseline in Krebs buffer containing xanthine (0.5 mM). Xanthine oxidase (0.1 U/ml) was then added to the bath and vessels incubated for 30 min. The vessels were resuspended in Krebs buffer and cumulative dose-response curves to norepinephrine reevaluated. The results indicate that generation of reactive oxygen metabolites by xanthine/xanthine oxidase decreases the pD2 from 7.80 +/- 0.04 to 7.40 +/- 0.09 with the endothelium intact. Removal of the endothelium did not attenuate the contractile dysfunction, indicating that endothelial-derived metabolites were not mediating the loss of vasoconstrictor effectiveness. Maximal tension development did not differ between normal and oxidized vessel rings. Introduction of oxypurinol (0.2 mg/ml) to the bath prevented the loss of constrictor responsiveness, thereby confirming that all of the oxidants were derived from the xanthine/xanthine oxidase reaction. Superoxide dismutase (200 U/ml) partially prevented the loss of norepinephrine responsiveness produced by xanthine oxidase-derived radicals. The pD2 in the SOD + xanthine/xanthine oxidase-treated vessels rings (7.19 +/- 0.11) was significantly lower than control vessel rings (7.49 +/- 0.04) and significantly higher than xanthine/xanthine oxidase-treated vessels (6.89 +/- 0.06). Catalase (1000 U/ml) also partially attenuated the loss of vascular norepinephrine responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of lymphatic myogenic activity and respiratory movements to pleural lymph flow.

In 11 anesthetized spontaneously breathing rabbits, we studied the contribution to total pleural lymph flow of myogenic activity of pleural lymphatics ("intrinsic mechanism") and the effect due to mechanical action of respiratory movements ("extrinsic mechanism"). Isoncotic saline solution (5 ml) containing 100 microCi of 125I-lactate dehydrogenase (LDH) was injected into right pleural space; in all but three control rabbits, injectate contained 1 mM amiloride in dimethyl sulfoxide to induce relaxation of smooth muscle tone. At 3 h, rabbits were killed and pleural fluid was collected and its volume measured. LDH radioactivity in pleural liquid and parietal pleural tissue was counted. In control rabbits, net pleural liquid flow (Jnet) at 3 h was -0.17 +/- 0.04 (SD) ml.kg-1.h-1; LDH concentration (C) and quantity (Q) decreased by 40.3 and 51.1% of initial value, respectively; total pleural lymphatic flow (Jl), calculated from LDH clearance, was 0.58 +/- 0.01 ml.kg-1.h-1. In amiloride-treated rabbits, Jnet was 0.01 +/- 0.1 ml.kg-1.h-1, C decreased by 34.4% and Q by 33.1%, and Jl averaged 0.39 +/- 0.02 ml.kg-1.h-1. C in parietal pleura, rich in lymphatics, was 13-fold higher in control than in amiloride-treated animals. The significant decrease of pleural lymphatic flow observed with amiloride (-40% relative to control) resulted from impairment of intrinsic mechanism, whereas, at comparable breathing frequencies, extrinsic mechanism remained unaltered. The direct effect of topical application of 1 mM amiloride was confirmed on exposed mesenteric collecting lymphatic ducts (data from 5 rats): amiloride reduced lymph flow by 40% by decreasing stroke volume without greatly affecting contraction rate of lymphatic walls.

Vascular NE responsiveness in portal hypertension: role of portal pressure and portosystemic shunting.

Previous studies have suggested that the development of portal venous collaterals and subsequent portosystemic shunting is the key event responsible for the reduced vasoconstrictor effectiveness in chronic portal hypertension. The purpose of the present study was to test this hypothesis. Thirty-nine male Sprague-Dawley rats were divided into four groups: end-to-side portacaval shunt (PCS, n = 11), chronic prehepatic portal hypertension (CPH, n = 10), acute prehepatic portal hypertension (APH, n = 8), and sham-operated controls (Sham, n = 10). The small intestine was prepared for microcirculatory studies. First-order arteriolar diameter and erythrocyte velocity were measured on-line, and blood flow was subsequently calculated. Once steady-state values were obtained the preparation was topically exposed to incremental doses of norepinephrine. The half-maximal effective dosage (ED50) for maximal vasoconstriction (diameter response) was significantly increased in PCS (4.5 microM) and CPH (1.5 microM) compared with Sham (0.8 microM). However, the ED50 was significantly lower in APH (0.17 microM) than in Sham. Similarly the ED50 for maximal blood flow reduction was higher in PCS (2.4 microM) and CPH (1.2 microM) compared with Sham (0.2 microM). The results demonstrate that vascular norepinephrine responsiveness is reduced in both portacaval shunted and chronic portal hypertensive rats, but not in acute portal hypertension. These data indicate that portosystemic shunting, not portal pressure elevation, is the key event leading to the reduced vascular norepinephrine responsiveness observed in CPH conditions.

Capillary filtration during acute inflammation: role of adherent neutrophils.

Fluid filtration rate with respect to surface area (Jv/S) was measured in capillaries of rat mesentery by a micro-occlusion technique. Superfusion of the mesentery with 100 nM platelet-activating factor (PAF) caused a fivefold increase in Jv/S (control, mean +/- SE = 0.016 +/- 0.002 micron/s, n = 44 rats; PAF, 0.078 +/- 0.010, n = 10), whereas 20 nM leukotriene B4 (LTB4) had no effect (0.010 +/- 0.003, n = 8). These doses of PAF and LTB4 induced a similar level of leukocyte adherence to venular endothelium. Neutrophils play a role in PAF-mediated increases in Jv/S, since a significantly lower Jv/S was elicited by PAF superfusion in neutropenic rats (0.024 +/- 0.006, n = 7). Monoclonal antibodies (MAb) directed against either the leukocyte adhesion glycoprotein CD11/CD18 (0.037 +/- 0.006, n = 8) or the endothelial cell adhesion molecule P-selectin (0.025 +/- 0.004, n = 8) also attenuated PAF-induced capillary filtration, whereas a nonbinding form of the P-selection MAb had no inhibitory effect (0.066 +/- 0.024, n = 3). These results indicate that PAF, but not LTB4, enhances capillary fluid filtration rate. While neutrophils do not adhere to endothelium in capillaries exposed to PAF, they do appear to contribute significantly to the PAF-induced capillary fluid filtration.