Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1-CXCR1/2-pathway.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

Salivary gland homogenates of Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma leakage via PAC1 receptor activation.

OBJECTIVES: Experiments were designed to determine if salivary gland homogenates (SGH) of the sand fly Lutzomyia longipalpis, the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage and to what extent these effects could be due to PAC1 receptor stimulation. METHODS: Using FITC-dextran as a plasma marker, intravital microscopy of the hamster cheek pouch (HCP) and a digital camera were used to assess arteriolar diameter and fluorescence of a selected area (5 mm(2)) representative of the HCP microcirculation. RESULTS: Cheek pouches prepared for intravital microscopy and exposed to topical application of SGH, maxadilan or PACAP-38 developed maximal dilation of arterioles in the range of 20-60 mum within 10 min, and this effect lasted for 30-90 min. The increase in fluorescence intensity induced by each of these compounds was due to plasma leakage from postcapillary venules. The mutant peptide of maxadilan (M-65), a PAC1 receptor antagonist, inhibited both dilation and plasma leakage induced by SGH or maxadilan. Plasma leakage induced by SGH was modestly inhibited by the bradykinin B(2) receptor antagonist HOE-140, but not by the antihistamine mepyramine or the nitric oxide synthase inhibitor L-NA. CONCLUSIONS: SGH of L. longipalpis and its vasodilatory peptide maxadilan caused long-lasting arteriolar dilation and plasma leakage in the cheek pouch via PAC1 receptor activation.

Longchain n-3 polyunsaturated fatty acids and microvascular reactivity: observation in the hamster cheek pouch.

Previous experiments in our laboratory, using the hamster cheek pouch microcirculation, have shown that precapillary vessels exhibit spontaneous rhythmic luminal variations, termed vasomotion, a myogenic activity sustained by a balance between membrane currents among which polarizing K(+) currents play an important role. In these microvessels, endothelium-derived relaxing factors (EDRFs) seem to regulate arteriolar diameter [via nitric oxide (NO) and cyclic GMP] and vasomotion [probably via endothelium-derived hyperpolarizing factor (EDHF)]. Fish or fish oil diet can decrease the risk of cardiovascular diseases, probably by modifying the conductance of selective ion channels, such as K(+) and/or Ca(++), and/or increasing the production of vasodilators, such as NO. To investigate its effect on microvascular reactivity, using the same preparation and an intravital microscope coupled to a closed circuit TV system, male hamsters were treated for 14 days, twice a day, with 0.4 mL/100 g body weight with fish or olive oil. An attempt was also undertaken to record in arterioles, in vivo, the membrane potential of smooth muscle cells during their vasomotor activity combining conventional microelectrode and intravital microscopy techniques. The effects of topical application of two vasodilators, acetylcholine [endothelium-dependent one, NO release and membrane hyperpolarization via Ca(++)-activated K(+) channels (K(Ca))] and sodium nitroprusside (endothelium-independent, NO donor and no change on membrane potential) and two vasoconstrictors which elicited membrane depolarization via Ca(++) channels, phenylephrine (alpha(1)-adrenergic receptor agonist) and serotonin (5-hydroxi-tryptamine) on mean internal diameter of arterioles and venules, arteriolar blood flows, spontaneous arteriolar vasomotion frequency and amplitude and functional capillary density (FCD, number of capillaries with flowing red blood cells per unit area of tissue) were determined. Anesthesia was induced by sodium pentobarbital (i.p.) and maintained with alpha-chloralose through the femoral vein. In the presence of vasomotion, the membrane potentials are slowly oscillating by about 20 mV around values of approximately -50 mV in perfect synchrony with vasomotor movements and depolarizing phases coincide with vasoconstrictions while polarizing ones with vasodilatations. Comparing all parameters, in control conditions, only the spontaneous vasomotion frequency was significantly higher (2.37 times higher) on the group treated with fish oil and persisted as such throughout all experiments. With topical application of the drugs mentioned above, the group treated with fish oil showed, for each drug concentration, a balance towards vasodilatation with consequent increase on arteriolar blood flow and on FCD, compared with the olive oil treated one. No significant changes on mean arterial pressure, spontaneous arteriolar vasomotion amplitude or venular diameter could be detected in the two groups. Our results support the concept that, in the hamster cheek pouch microcirculation, fish oil supplementation activates K(+) channels which act as the EDHF and might also increase the production of vasodilators, probably NO.

Muscle arteriolar and venular reactivity in two models of hypertensive rats.

This study was designed to test if skeletal muscle fiber composition could influence vascular response in hypertensive rats. Muscle vessels were observed by intravital microscopy in anesthetized rats and changes in diameter were measured after local administration of endothelium-dependent and -independent vasodilators. Vascular reactivity was compared in two models of hypertension deoxicorticosterone acetate and salt load (DOCA-s) hypertensive rats and spontaneously hypertensive rats (SHR). The muscles used were: the fast-twitch glycolytic muscle, extensor digitorum longus (EDL), and the slow-twitch oxidative, soleus muscle. Maximal dilation induced by vasoactive drugs was of similar magnitude in EDL and soleus arterioles. Terminal arteriole reactivity to acetylcholine and adenosine was blunted in EDL (35% and 49% reduction, respectively) and soleus muscles (42% and 34% reduction, respectively) of SHR compared with Wistar Kyoto rats. Reactivity of DOCA-s rats to acetylcholine, adenosine, and sodium nitroprusside was reduced by 38%, 50%, 39% in EDL third- and fourth-order arterioles and by 30%, 38%, 38% in soleus fourth-order arterioles, respectively. These studies show that hypertension probably induced similar vascular changes in both muscles studied. Vascular reactivity is blunted for some vasodilator drugs and is more affected in DOCA-s rats. In addition, a preferential action for bradykinin was observed on upstream arterioles but not on venules. This effect was not observed for adenosine.

Effects of L-NA and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in hamster cheek pouch venules.

Our objective was to study how the topical application of a nitric oxide synthase inhibitor (l-NA, Nomega-nitro-L-arginine) and a nitric oxide donor, sodium nitroprusside (SNP), could modulate leukocyte adhesion (sticking) and microvascular permeability as altered by ischemia/reperfusion (I/R) and topically applied histamine after I/R. Golden hamsters were prepared for intravital microscopy. Ischemia was induced by an inflatable silicon rubber cuff mounted around the neck of the cheek pouch prepared for intravital microscopy. Saline, L-NA, sodium nitroprusside, and histamine were applied in the superfusion solution. FITC-dextran was injected iv 30 min before initiation of ischemia as a marker of microvascular permeability. L-NA 10(-5) M inhibited both the increase in number of sticking leukocytes and the increase in vascular permeability after I/R compared with the untreated control group of hamsters. SNP neutralized this effect of L-NA on leukocytes and vascular permeability and caused arteriolar dilation at the concentration used, 10(-6) M. Both SNP and L-NA + SNP enhanced the I/R-induced macromolecular leakage. The topical application of SNP and SNP + L-NA did not modify the response to histamine after I/R compared with the untreated control group. In hamsters not subjected to I/R, histamine-induced macromolecular leakage was inhibited by L-NA and L-NA + SNP but was unchanged by SNP. It is concluded that inhibition of nitric oxide formation by L-NA reduced both leukocyte adhesion in postcapillary venules and the increase in macromolecular leakage and that a NO donor such as SNP could enhance the macromolecular leakage response to I/R.

Effects of insulin and the combination of insulin plus metformin (glucophage) on microvascular reactivity in control and diabetic hamsters.

The purpose of this study was to determine the in vivo microvascular reactivity of arterioles (mean internal diameter range: 16.0 to 106.4 microm) and venules (mean internal diameter range: 24.0 to 117.3 microm) in the hamster cheek pouch to insulin and to the mixture insulin + metformin. Experiments were performed using an intravital microscope coupled to a closed-circuit TV system and a videotape. The TV monitor display was used to obtain arteriolar and venular internal diameter measurements by an image-shearing device. The studied drugs were applied topically, added to the superfusion solution, to avoid systemic effects that would complicate the analysis of the results. In control animals (glycemia 7.7 +/- 0.4 mmol/L), application of insulin (10 to 500 microU/mL/min) evoked vasodilatation in a dose-dependent fashion in arterioles (4.9 +/- 3.2% to 50.9 +/- 6.5%, smallest and largest concentration, respectively, values expressed in percent of the initial diameter as mean +/- SE) and venules (-2.1 +/- 3.1% to 14.3 +/- 5.1%), decreased and finally abolished the spontaneous vasomotion frequency (from 9.5 +/- 0.3 cycles per minute [cpm] to 0.0 +/- 0.0 cpm) and amplitude (from 8.6 +/- 0.3 to 0.0 +/- 0.0 microm). Addition of metformin, 0.2 mg/mL/min, did not significantly change either the observed vasodilatation in arterioles and venules or the vasomotion frequency and amplitude curves. Two types of diabetic hamsters were studied: severely diabetic, induced with three intraperitoneal injections of streptozotocin, diluted in physiological saline, 50 mg/kg/dose, given in three consecutive days, and mildly diabetic, induced by a single dose of streptozotocin. All diabetic animals were studied four weeks after the onset of diabetes and no specific treatment for diabetes was given. In severely diabetic hamsters (glycemia 18.0 +/- 2.2 mmol/L), application of insulin, in the same concentration range, evoked a significantly reduced vasodilatation in arterioles as compared with control animals (5.9 +/- 1.3% to 18.9 +/- 3.5%) and did not change the vasodilatation observed in the venules (5.9 +/- 1.4% to 21.3 +/- 2.5%). In these preparations no spontaneous arteriolar vasomotion could be detected. Addition of metformin did not significantly improve the impaired vasodilatation. In mildly diabetic hamsters (glycemia 12.1 +/- 0.8 mmol/L), application of insulin, in the same concentration range, evoked vasodilatation, in a dose-dependent fashion, equivalent to the one observed in control animals, in arterioles (3.1 +/- 2.5% to 53.4 +/- 10.0%) and venules (7.1 +/- 3.0% to 29.9 +/- 4.8%) and also reduced the vasomotion frequency (from 10.1 +/- 0.3 to 0.1 +/- 0.1 cpm) and amplitude (from 9.2 +/- 0.6 to 0.2 +/- 0.2 microm). Addition of metformin tended to increase the observed arteriolar dilatation (6.6 +/- 3.0% to 67.8 +/- 5.5%), did not change the venular dilatation (6.7 +/- 4.8% to 28.0 +/- 3.3%), and tended to preserve vasomotion frequency and amplitude. These experiments show that (1) insulin has a direct dilatatory effect on arterioles and venules; (2) the vasodilatation evoked by insulin is impaired in severe diabetes, and (3) no significant abnormality could be detected on microvascular reactivity in mild diabetes. Further addition of metformin helped to maintain the spontaneous arteriolar vasomotion even during moderate vasodilatation and tended to augment the arteriolar dilatation evoked by insulin in mildly diabetic animals.

Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischaemia/reperfusion in the hamster cheek pouch.

1. The effects of a purified micronized flavonoid fraction (S5682) on mean internal diameter and blood flow of arterioles and venules, as well as the functional capillary density (FCD) were evaluated in the hamster cheek pouch microvasculature before and after 90 min of total ischaemia. 2. Male hamsters were treated for ten days, twice a day, with oral doses of S5682 (5, 20, 80 and 160 mg kg-1 day-1) or placebo (10% lactose solution). The cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. Local ischaemia was obtained by a cuff mounted around the neck of the everted pouch where it leaves the mouth of the hamster. 3. Measurements were performed before ischaemia, at the onset of reperfusion and 10, 20, 30, 45 and 60 min thereafter. Diameters were measured by means of an image shearing device. Red blood cell (RBC) velocity was analysed by use of the dual-slit photometric technique. Blood flow was calculated from diameters and RBC velocities. FCD, defined as the number of capillaries with flowing blood per field of observation, was also assessed. 4. During reperfusion, placebo-treated animals showed a significant vasodilatation, a decrease in blood flow and FCD and S5682-treated animals showed a clear trend, dose-dependent, towards maintaining these parameters closer to the value found before ischaemia. 5. In conclusion, our results indicate that S5682 improves the microvascular reactivity and FCD after ischaemia/reperfusion. These data suggest that S5682 could function as an antioxidant, which may explain its beneficial therapeutic effect in chronic venous insufficiency where oxidative stress is involved in the pathological mechanism.

Effects of Cyclo 3 Fort on microvascular reactivity and the venoarteriolar reflex in diabetic hamsters.

Cyclo 3 Fort is used in the treatment of chronic venous insufficiency. In this study, the effects of Cyclo 3 Fort, 2, 10 and 50 mg/kg, were studied in cheek pouch preparations from diabetic hamsters. The mean arteriolar diameter in animals receiving Cyclo 3 Fort, 10 and 50 mg/kg, was significantly greater than in control animals (p < 0.05), and the mean venule diameter was significantly lower in animals receiving the 50 mg/kg dose than in the control group. This suggests that Cyclo 3 Fort, 50 mg/kg/day, has a venotonic effect in diabetic animals. The venoarteriolar reflex was studied by measuring the internal diameter of arterioles during venular occlusion. The reflex was impaired in animals treated with either placebo or 2 mg/kg, but this was reversed by treatment with Cyclo 3 Fort, 10 and 50 mg/kg.

Influence of aldose reductase inhibition on the microvascular reactivity in experimental diabetes.

1. To verify if tolrestat, an aldose reductase inhibitor, corrects the impaired responses of microvessels to histamine and bradykinin in alloxan-diabetic rats, the mesenteric microcirculation was studied in vivo in anaesthetised animals. 2. The impaired responses were corrected by tolrestat 5 mg/kg/day for 7 days p.o. Similar responses to acetylcholine and sodium nitroprusside were obtained in preparations of diabetic and control rats and were not altered by tolrestat treatment. 3. As in diabetes, galctosemia induced impaired responses to histamine and bradykinin; these altered responses were corrected by tolrestat treatment. 4. These data allow us to suggest that the polyol pathway activity might be involved in the altered responses of microvessels observed in diabetic rats. It is possible that polyol activation may play an important role in the development of vascular dysfunction in diabetes mellitus.

Effect of endothelin-1 on arterioles and venules in vivo.

The vasoconstrictor effects of endothelin-1 (ET-1) and norepinephrine (NE) were compared in the exteriorized rat mesentery in situ. ET-1 was 1,000 times more potent than NE in constricting both arterioles and venules. Vasoconstrictions induced by ET-1 lasted much longer than those caused by the catecholamine. At nonrelaxant doses, acetylcholine and sodium nitroprusside antagonized the constrictive response of microvessels to ET-1 and NE. However, larger doses of acetylcholine were necessary to inhibit the response to ET-1 than that to NE.