Venular Leaky Sites Research Articles

Generation of Superoxide Anion Impairs Histamine-Induced Increases in Macromolecular Efflux

The first goal of this study was to determine the effect of generation of superoxide anion using pyrogallol on histamine-induced increases in macromolecular efflux. We used intravital microscopy and fluorescein isothiocyanate–dextran (FITC–dextran; MW 70K) to examine macromolecular extravazation from postcapillary venules in the hamster cheek pouch in response to histamine before and following topical application of vehicle or pyrogallol. Extravazation of macromolecules was quantitated by counting venular leaky sites. Histamine elicited reproducible increases in venular leaky sites before and during infusion of vehicle. In contrast, topical application of pyrogallol (0.5 mM) abolished histamine-induced increases in formation of venular leaky sites. Our second goal was to examine whether pyrogallol-induced inhibition of venular leaky site formation could be reversed by superoxide dismutase. Application of superoxide dismutase (300 U/ml) to the cheek pouch in the presence of pyrogallol restored histamine-induced increases in venular leaky sites. Thus, the generation of superoxide anion alters histamine-induced increases in macromolecular efflux. These results support the concept that disease states that produce oxidative stress may impair agonist-induced increases in microvascular permeability via inactivation of nitric oxide.

Superoxide Dismutase Restores Impaired Histamine‐Induced Increase in Venular Macromolecular Efflux During Diabetes Mellitus

The goal of this study was to determine the role of oxygen radicals in impaired histamine-induced increases in venular macromolecular efflux from the hamster cheek pouch. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; MW = 70K) to examine macromolecular extravasation from post-capillary venules in nondiabetic and diabetic (2-4 weeks after injection of streptozotocin) hamsters in response to histamine. Increases in extravasation of macromolecules were quantitated by counting venular leaky sites and by calculating clearance (ml/s x 10(-6)) of FITC-dextran-70K. In nondiabetic hamsters, superfusion with histamine (1.0 and 5.0 microM) increased venular leaky sites from 0 +/- 0 to 17 +/- 6 and 35 +/- 6 per 0.11 cm2, respectively. In addition, clearance of FITC-dextran-70K increased during superfusion with histamine. In contrast, superfusion with histamine did not increase the formation of venular leaky sites (0 +/- 0) or clearance of FITC-dextran-70K in diabetic hamsters. Next, we examined whether alterations in histamine-induced increases in macromolecular efflux in diabetic hamsters may be related to the production of oxygen radicals. We examined whether exogenous application of superoxide dismutase (150 U/ml) could restore impaired histamine-induced increases in macromolecular extravasation in diabetic hamsters. Application of superoxide dismutase did not alter histamine-induced increases in venular leaky sites or clearance of FITC-dextran-70K in nondiabetic hamsters. However, application of superoxide dismutase restored histamine-induced increases in leaky site formation and clearance of FITC-dextran-70K in diabetic hamsters towards that observed in nondiabetic hamsters. These findings suggest that oxygen radical formation appears to contribute to impaired macromolecular efflux in response to histamine during short-term diabetes mellitus.

Nicotine impairs histamine-induced increases in macromolecular efflux: role of oxygen radicals.

Nicotine, a major component of cigarettes and smokeless tobacco, has toxic effects on endothelium and impairs reactivity of resistance arterioles in response to agonists that stimulate the synthesis and/or release of nitric oxide. However, the effect of nicotine on nitric oxide synthase-dependent increases in macromolecular transport is not known. Thus our first goal was to determine the effect of nicotine on histamine-induced increases in macromolecular efflux. We used intravital microscopy and FITC dextran (mol wt 70, 000) (FITC-dextran-70K) to examine macromolecular extravasation from postcapillary venules in response to histamine before and after intravenous infusion of vehicle or nicotine. Extravasation of macromolecules was quantitated by counting venular leaky sites and calculating clearance (ml/s x 10(-6)) of FITC-dextran-70K. Histamine elicited reproducible increases in venular leaky sites and clearance in hamsters infused with vehicle. In contrast, nicotine infusion inhibited histamine-induced increases in macromolecular efflux. Histamine (1.0 and 5.0 micro M) elicited 19 +/- 2 and 34 +/- 4 vs. 3 +/- 1 and 11 +/- 5 leaky sites per 0.11 cm2, before vs. after nicotine infusion, respectively (P < 0.05). Histamine-induced clearance of FITC-dextran-70K was also impaired after infusion of nicotine. Our second goal was to examine whether alterations in histamine-induced increases in macromolecular efflux by nicotine may be related to the production of oxygen radicals. Application of superoxide dismutase (150 U/ml) to the hamster cheek pouch restored histamine-induced increases in venular leaky sites and clearance of FITC-dextran-70K during infusion of nicotine. Thus nicotine alters agonist-induced increases in microvascular permeability, via the formation of oxygen radicals, to presumably inactivate nitric oxide.

Superoxide Dismutase Restores Impaired Histamine-Induced Increase in Venular Macromolecular Efflux During Diabetes Mellitus

The goal of this study was to determine the role of oxygen radicals in impaired histamine-induced increases in venular macromolecular efflux from the hamster cheek pouch. We used intravital fluorescent microscopy and fluorescein isothio-cynate dextran (FITC-dextran; MW = 70 K) to examine macromolecular extravasation from post-capillary venules in nondiabetic and diabetic (2–4 weeks after injection of streptozoticin) hamsters in response to histamine. Increases in extravasation of macromolecules were quantitated by counting venular leaky sites and by calculating clearance (ml/s × 10−6) of FITC-dextran-70 K. In non-diabetic hamsters, superfusion with histamine (1.0 and 5.0 µM) increased venular leaky sites from 0 ± 0 to 17 ± 6 and 35 ± 6 per 0.11 cm2, respectively. In addition, clearance of FITC-dextran-70 K increased during superfusion with histamine. In contrast, superfusion with histamine did not increase the formation of venular leaky sites (0 ± 0) or clearance of FITC-dextran-70 K in diabetic hamsters. Next, we examined whether alterations in histamine-induced increases in macromolecular efflux in diabetic hamsters may be related to the production of oxygen radicals. We examined whether exogenous application of superoxide dismutase (150 U/ml) could restore impaired histamine-induced increases in macromolecular extravasation in diabetic hamsters. Application of superoxide dismutase did not alter histamine-induced increases in venular leaky sites or clearance of FITC-dextran-70 K in nondiabetic hamsters. However, application of superoxide dismutase restored histamine-induced increases in leaky site formation and clearance of FITC-dextran-70 K in diabetic hamsters towards that observed in nondiabetic hamsters. These findings suggest that oxygen radical formation appears to contribute to impaired macromolecular efflux in response to histamine during short-term diabetes mellitus.

L-arginine does not reverse impaired agonist-induced increases in macromolecular efflux during diabetes mellitus.

The first goal of this study was to determine the effect of diabetes mellitus on agonist-induced increases in venular macromolecular permeability of the hamster cheek pouch. The second goal was examine the role for an alteration in the availability of L-arginine to nitric oxide synthase in impaired agonist-induced increases in macromolecular permeability during diabetes. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mw = 70 K) to examine macromolecular extravasation from post-capillary venules in non-diabetic and diabetic (2 weeks after injection of streptozotocin) hamsters in response to histamine and substance P. Increases in extravasation of macromolecules were quantitated by counting the number of venular leaky sites and by calculating the clearance (ml/s x 10(-6)) of FITC-dextran-70 K. In non-diabetic hamsters, histamine (1.0 and 5.0 microM) and substance P (50 and 100 nM) increased permeability of the cheek pouch microcirculation to FITC-dextran-70 K. In contrast, histamine- and substance P-induced increases in macromolecular extravasation were markedly reduced in diabetic hamsters. Next, we investigated whether alterations in histamine- and substance P-induced changes in macromolecular extravasation in diabetic hamsters may be related to an alteration in the availability of L-arginine. We examined whether exogenous application of L-arginine (100 microM) could restore impaired histamine- and substance-P-induced increases in macromolecular extravasation in diabetic hamsters. We found that L-arginine potentiated agonist-induced increases in macromolecular extravasation in non-diabetic hamsters, but did not alter responses in diabetic hamsters. These findings suggest that short-term diabetes mellitus alters agonist-induced alterations in microvascular permeability. The mechanism of altered microvascular permeability during diabetes mellitus does not appear to be related to an impaired availability of L-arginine.

Activation of protein kinase C does not participate in disruption of the blood-brain barrier to albumin during acute hypertension

The blood-brain barrier minimizes the entry of macromolecules into brain tissue. During acute increases in arterial blood pressure, disruption of the blood-brain barrier occurs primarily in cerebral venules and veins. Mechanisms by which increases in cerebral venous pressure produce disruption of the blood-brain barrier during acute hypertension are not clear. The goal of this study was to determine the role of activation of protein kinase C in disruption of the blood-brain barrier during acute hypertension. We examined the microcirculation of the cerebrum in vivo. Permeability of the blood-brain barrier was quantitated by the formation of venular leaky sites and clearance of fluorescent-labeled albumin (FITC-albumin) before and during phenylephrine-induced acute hypertension. In addition, we examined changes in pial arteriolar and pial venular pressure before and during phenylephrine-induced acute hypertension. We compared responses of the blood-brain barrier to acute hypertension in control (untreated) rats and in rats treated with inhibitors of protein kinse C; calphostin C (0.1 μM) or sphingosine (1.0 μM). Under control conditions, no venular leaky sites were visible and clearance of FITC-albumin was minimal in all groups. Phenylephrine infusion increased systemic arterial, pial arteriolar and pial venular pressures, and increased the formation of venular leaky sites and clearance of FITC-albumin by a similar magnitude in all groups. The findings of the present study suggest that inhibition of protein kinase C does not significantly alter the formation of venular leaky sites and/or clearance of FITC-albumin during acute hypertension. Thus, disruption of the blood-brain barrier during acute hypertension does not appear to be influenced by activation of protein kinase C.

Role of nitric oxide in disruption of the blood-brain barrier during acute hypertension.

The goal of this study was to determine the role of nitric oxide in disruption of the blood-brain barrier during acute hypertension. We examined the microcirculation of the cerebrum in vivo. Permeability of the blood-brain barrier was quantitated by the formation of venular leaky sites and clearance of fluorescent-labeled albumin (FITC-albumin) before and during phenylephrine-induced acute hypertension. We compared disruption of the blood-brain barrier during acute hypertension in untreated rats and in rats treated for 1 h with topical application of NG-monomethyl-L-arginine (L-NMMA; 100 microM) or NG-nitro-L-arginine methyl ester (L-NAME; 100 microM). Under control conditions, no venular leaky sites were visible and clearance of FITC-albumin was minimal in untreated rats and in rats treated with topical application of nitric oxide synthase inhibitors. Phenylephrine (20 micrograms/kg/min for 5 min) infusion increased systemic arterial pressure by a similar magnitude in all groups of rats and produced disruption of the blood-brain barrier in venules. However, the magnitude of disruption of the blood-brain barrier during acute hypertension was significantly less in rats treated with L-NMMA (52% reduction in the clearance of FITC-albumin) and L-NAME (47% reduction in clearance of FITC-albumin). The findings of the present study suggest that synthesis/release of nitric oxide contributes to disruption of the blood-brain barrier during acute hypertension.

Effects of endothelin receptor antagonists on bradykinin-induced increases in macromolecular efflux

The goal of this study was to determine the effects of endothelin receptor antagonists on agonist-induced increases in macromolecular extravasation in the hamster cheek pouch in vivo. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mol wt = 70 K) to examine extravasation from postcapillary venules in response to bradykinin and endothelin before and following application of inhibitors of endothelin receptors (ETAB and ETA). Increases in extravasation of macromolecules were quantitated by counting the number of venular leaky sites. Bradykinin (0.5 and 1.0 microM) and endothelin-1 (0.01 and 0.1 nM) produced a dose-related increase in the number of venular leaky sites and superfusion of PD 142893 (ETAB antagonist), and PD 147953 and BQ-123 (ETA antagonists) significantly decreased bradykinin- and endothelin-induced responses. Addition of calcium to the superfusate restored bradykinin-induced increases in venular leaky sites in the presence of endothelin receptor antagonism. Thus, the findings of the present study suggest that endothelin receptor antagonists abrogate bradykinin- and endothelin-induced increases in macromolecular efflux from postcapillary venules. The mechanism for the effects of endothelin receptor antagonists appears to be related to inhibition of the ETA receptor which, in turn, alters the mobilization of calcium across venular endothelium.

Nitric oxide accounts for histamine-induced increases in macromolecular extravasation

The goal of this study was to determine the role of nitric oxide in histamine-induced increases in macromolecular extravasation in the hamster cheek pouch in vivo. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mol wt = 70,000 K) to examine extravasation from postcapillary venules in response to histamine before and after application of an enzymatic inhibitor of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 1.0 microM). Increases in extravasation of macromolecules were quantitated counting the number of venular leaky sites. Histamine (1.0 and 5.0 microM) increased the number of venular leaky sites from zero (basal conditions) to 11 +/- 1 and 21 +/- 2/0.11 cm2, respectively. Superfusion of L-NMMA (1.0 microM) and LY-83583 (1.0 microM) significantly decreased histamine-induced formation of venular leaky sites, whereas L-arginine (100 microM) potentiated histamine-induced formation of venular leaky sites. In contrast, superfusion of NG-monomethyl-D-arginine (1.0 microM) did not inhibit the formation of venular leaky sites in response to histamine. Thus the findings of the present study suggest that production of nitric oxide, and subsequent activation of guanylate cyclase, plays an important role in macromolecular efflux in vivo in response to histamine.

Role of nitric oxide in leukotriene C4-induced increases in microvascular transport.

The goal of this study was to determine the role of nitric oxide in alterations in macromolecular transport of the hamster cheek pouch in vivo in response to leukotriene C4. We used intravital fluorescent microscopy to examine the transport of macromolecules across the hamster cheek pouch in response to leukotriene C4 before and after application of an enzymatic inhibitor of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 1.0 microM). Increases in transport of macromolecules across the hamster cheek pouch were quantitated by the formation of venular leaky sites and clearance of fluorescein isothiocyanate-dextran (FITC-dextran; mol wt = 70 K). Leukotriene C4 (1.0 and 3.0 nM) produced an increase in the number of venular leaky sites and clearance of FITC-dextran-70K. Superfusion of L-NMMA (1.0 microM) significantly decreased leukotriene C4-induced increases in venular leaky sites and clearance of FITC-dextran-70K. In addition, superfusion of LY-83583 (10 microM) significantly decreased leukotriene C4-induced increases in venular leaky sites. In contrast, superfusion of NG-monomethyl-D-arginine (D-NMMA; 1.0 microM), indomethacin (10 mg/kg iv), or diphenhydramine hydrochloride; 15-20 mg/kg iv) did not significantly alter leukotriene C4-induced increases in venular leaky sites. Thus these findings suggest that production of nitric oxide and subsequent activation of guanylate cyclase play an important role in formation of venular leaky sites and clearance of FITC-dextran-70K in response to application of leukotriene C4.

Cigarette smoke extract potentiates bradykinin-induced increases in microvascular permeability

The first goal of this study was to determine whether cigarette smoke extract (CSE) increases microvascular permeability of the hamster cheek pouch in vivo. The second goal was to determine whether CSE potentiates bradykinin-induced increases in vascular permeability in the hamster cheek pouch. Using intravital microscopy, we examined the permeability of the hamster cheek pouch to fluorescein isothiocyanate-dextran (mol wt 70,000). Increases in permeability were quantitated by counting the number of postcapillary venular leaky sites per 0.11 cm2. Superfusion of CSE (1, 5, and 10%) did not produce venular leaky sites and, thus, did not alter macromolecular permeability. Superfusion of bradykinin (0.1, 0.5, and 1.0 microM) produced a dose-related increase in the number of venular leaky sites. Formation of leaky sites in response to bradykinin was potentiated by CSE. To determine whether potentiation of bradykinin-induced leaky site formation by CSE was related to products released via the cyclooxygenase pathway, we examined the effects of pretreatment with indomethacin (10 mg/kg i.v.). Indomethacin did not alter the potentiating effect of CSE on bradykinin-induced leaky site formation. These findings suggest that CSE does not alter basal permeability of the hamster cheek pouch microcirculation in vivo. However, CSE potentiates bradykinin-induced increases in microvascular permeability. The mechanism of CSE-induced potentiation of microvascular permeability does not appear to be related to substances produced via the cyclooxygenase pathway.

Role of nitric oxide in modulating permeability of hamster cheek pouch in response to adenosine 5?-diphosphate and bradykinin

The goal of this study was to determine the role of the synthesis and release of nitric oxide in modulating alterations in microvascular permeability of the hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. We used intra-vital fluorescent microscopy to examine the permeability of the hamster cheek pouch to agonists before and following application of enzymatic inhibitors of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 0.01, 0.1, and 1.0 microM) and NW-nitro-L-arginine methyl ester (L-NAME; 0.01, 0.1, and 1.0 microM). Increases in permeability of the hamster cheek pouch were quantitated by the formation of microvascular leaky sites. ADP and bradykinin produced an increase in the number of venular leaky sites, and superfusion of L-NMMA and L-NAME significantly decreased ADP- and bradykinin-induced increases in microvascular permeability. To determine the specificity of nitric oxide blockade on microvascular permeability, we examined changes in permeability in response to adenosine, and examined the effects of D-NMMA on microvascular permeability. Adenosine-induced increases in permeability were not altered by treatment with L-NMMA, and D-NMMA did not inhibit ADP-induced increases in microvascular permeability. Thus, these findings suggest that production of nitric oxide, in response to application of ADP and bradykinin, has a role in modulating macromolecular permeability of the hamster cheek pouch in vivo.

Effect of prostaglandin E1 on leukotriene C4-induced increases in vascular permeability of hamster cheek pouch.

Our goal was to determine whether there is a synergistic effect of prostaglandin E1 (PGE1) on leukotriene C4 (LTC4)-induced increases in vascular permeability of the hamster cheek pouch in vivo. Changes in permeability were quantified by counting venular leaky sites (LS) and calculating clearance (CLR) of fluorescein isothiocyanate (FITC)-dextran 70kDa, during suffusion of the cheek pouch with LTC4 in the absence or presence of PGE1. LTC4 produced a dose-related increase in LS and CLR, and PGE1 (0.01 microM) significantly potentiated the effect of LTC4. During suffusion with LTC4 (15.0 nM) in the absence of PGE1, LS increased from 0 to 19 +/- 3/0.11 cm2 and CLR increased from 0.3 +/- 0.1 to 1.0 +/- 0.2 x 10(-6) ml/sec. During superfusion with LTC4 (15.0 nM) in the presence of PGE1, LS increased from 0 to 40 +/- 3/0.11 cm2, and CLR increased from 0.4 +/- 0.1 to 2.1 +/- 0.5 x 10(-6) ml/sec. To determine whether the effect of PGE1 on LTC4-induced increases in vascular permeability was related to the vasodilatory effect of PGE1, we examined the effect of isoproterenol (ISO). In contrast to that observed with PGE1, ISO decreased LTC4-induced increases in LS and CLR. Our data suggest that there is a synergistic effect of PGE1 on LTC4-induced increases in venular permeability that is not mediated by a vasodilatory action of PGE1.

Leukotriene B4-induced permeability increase in postcapillary venules and its inhibition by three different antiinflammatory drugs

Leukotriene B4 (LTB4) is a potent chemotactic and chemokinetic substance that causes leukocyte accumulation and increases vascular permeability. We have used the hamster cheek pouch model to study the effect of some different antiinflammatory drugs on the LTB4-induced microvascular permeability. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular leakage, which was measured as the number of venular leaky sites and as efflux of the vascular tracer. Topical application of LTB4 (10(-8) M, 5 min) resulted in a reversible increase of the vascular permeability and could be repeated with 40-min intervals without any significant reduction in permeability response. The LTB4-induced response could be inhibited by budesonide (a glucocorticoid) given locally, by iloprost (a prostacyclin analog) given intravenously and by a combined local and intravenous treatment with terbutaline (a beta 2-receptor agonist) but not by local terbutaline treatment only.

Permeability of blood-brain barrier to various sized molecules.

We studied disruption of the blood-brain barrier (BBB) by acute hypertension and a hyperosmolar solution. The goals were to determine whether 1) disruption of the BBB occurs primarily in arteries, capillaries, or veins, and 2) transport of different-sized molecules is homogeneous or size dependent. Sprague-Dawley rats were studied using intravital fluorescent microscopy of pial vessels and fluorescein-labeled dextrans (FITC-dextran, mol wt = 70,000, 20,000, and 4,000 daltons). The site of disruption was determined by the appearance of microvascular leaky sites. Transport of different-sized molecules was calculated from clearance of FITC-dextran. During gradual hypertension and osmotic disruption, all leaky sites were venular. Rapid hypertension produced venular leaky sites and, in some experiments, diffuse arteriolar extravasation of FITC-dextran. Clearance of different-sized molecules was homogeneous during acute hypertension. In contrast, clearance of molecules during osmotic disruption was size dependent. The findings suggest that 1) venules and veins are the primary sites of disruption following acute hypertension and a hyperosmolar solution; 2) transport of different-sized molecules is homogeneous following acute hypertension, which suggests a vesicular mechanism; and 3) transport following hyperosmolar disruption is size dependent, which suggests that hyperosmolar disruption may involve formation of pores as well as vesicular transport.