Rat Mesenteric Arterioles Research Articles

Thrombogenic properties of ultra-low-dose of acetylsalicylic acid in a vessel model of laser-induced thrombus formation

Various studies have demonstrated the antithrombotic effect of high dose acetylsalicylic acid (ASA) and more reports show the similar efficiency of the much better tolerated low dose ASA. Recently, in vitro studies hinted the potent thrombotic properties of ultra-low-dose ASA (ULD-ASA, < 1 mg/day) showing a significant decrease in bleeding time( 1,2). This effect was, at least in part, related to the inhibition of endothelial cyclooxygenase -and not to platelet oneleading to a decrease of prostacyclm release (3). To investigate the effect of ASA in vivo, we used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of the microvasculature. Thrombus formation was induced by Argon-laser shot. The instrumental test set-up was completed with a video-system. The kinetic feature of thrombus formation was estimated in terms of number of laser shots required to induce endothelium injury leading to thrombosis, number of emboli removed by blood stream and duration of embolization. The reliability and reproductibility of our laser-induced thrombus model have been previously tested in rat mesenteric arterioles and veinules and we showed that ASA used at current doses (50-200 mgkg) decreased the number of emboli and the duration of embolization (4). The aim of this preliminary study was the investigation of the effects of ULD-ASA in vivo. In so doing, we evaluated ULD-ASA in our experimental laser-induced thrombus model. This might represent a further step in the understanding of the action of ASA according to the range of concentration used.

Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells

Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells

Levcromakalim-induced modulation of membrane potassium currents, intracellular calcium and mechanical activity in rat mesenteric artery.

In freshly-dispersed cells from rat mesenteric artery, levcromakalim (1 and 10 microM) induced a non-inactivating potassium current (IKCO), an event which was associated with increased current noise. IKCO was fully inhibited in the presence of 10 microM glibenclamide. Stationary fluctuation analysis of the current noise associated with IKCO induced by levcromakalim at a holding potential of -10 mV indicated that the unitary conductance of the underlying K-channels was 10.2 pS at 0 mV under the quasi-physiological conditions of the experiment. In isolated arterioles both levcromakalim (10 nM-10 microM) and nifedipine (10 nM-10 microM) each elicited full, concentration-dependent, parallel reductions of the increases in [Ca2+]i (assessed using fura-2) and tension induced by 10 microM noradrenaline. However, the effects of both drugs on KCl-induced increases in tension and in [Ca2+]i, did not follow a simple relationship. Levcromakalim relaxed KCl- and noradrenaline-induced sustained contractions with a similar potency. This was in contrast to nifedipine which was approximately 20 times more potent against KCl-induced contractions. It is concluded that levcromakalim relaxes rat mesenteric arterioles primarily by the opening of a small conductance, glibenclamide-sensitive K-channel. An additional action of levcromakalim is suggested by its relative inability to suppress the increase in [Ca2+]i produced by 30 mM K(+)-PSS.

Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40-200 microns in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of > 98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3-7.5 nM) or angiotensin II (0.001-1000 nM) induced 3H-thymidine incorporation and cell proliferation in a dose-dependent manner (P < 0.01). S-nitroso-N acetylpenicillamine (0.05-0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced 3H-thymidine incorporation (P < 0.05) and cell proliferation (P < 0.01). The antimitogenic effect of S-nitroso-N-acetylpenicillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P < 0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.

Vasodilator nerves

In addition to sympathetic vasoconstrictor nerves, nonadrenergic, noncholinergic vasodilator nerves control the vascular tone. In cerebral arteries of various species of animals, nitric oxide (NO) is a potential candidate for the vasodilator transmitter. In rat mesenteric arterioles, calcitonin gene-related peptide (CGRP) functions as a transmitter. In guinea pig mesenteric arteries, multiple transmitters including NO and CGRP mediate the vasodilator response. The physiological and pathophysiological significance of vasodilator nerves is to be determined.

Decrease of hydrodynamic resistance in rat mesenteric arterioles in response to injection of polyethylene oxide polyox WSR-301

Decrease of hydrodynamic resistance in rat mesenteric arterioles in response to injection of polyethylene oxide polyox WSR-301

"Caged" phenylephrine: development and application to probe the mechanism of alpha-receptor-mediated vasoconstriction.

A "caged" analogue of the alpha-adrenergic receptor agonist phenylephrine (PE) was prepared by exploiting the 2-nitrobenzyl protecting group and using a synthetic procedure developed to permit preferential derivatization at the amino group. On isolated adult rat mesenteric arterioles, caged-PE had no measurable effects at concentrations up to 100 microM; 0.5-ms light flashes in the presence of caged-PE, however, produced marked and dose-dependent vasoconstriction. Flash-induced vasoconstrictions were blocked by the alpha-receptor antagonist phentolamine and were unaffected by the beta-receptor antagonist propranolol, indicating that the light-induced responses reflect the selective activation of alpha-adrenergic receptors. After a single flash, a large transient decrease in vessel diameter was recorded, and in most vessels, this was followed by a smaller, sustained constriction. The sustained component of the contraction was selectively eliminated when Ca2+ was removed from the bath, which suggests that different mechanisms underlie the transient and the sustained responses to PE. The responses to single flashes of varying intensities occurred with a mean latency of 460 ms, which is consistent with the intermediacy of several steps between alpha-receptor activation and contraction. We anticipate that it will be possible to extend this approach to develop caged analogues of other neurotransmitters for mechanistic and kinetic studies.

Effect of long-term aerobic exercise on helium-neon-laser-induced thrombogenesis in rat mesenteric arterioles and platelet aggregation.

We have previously investigated the antithrombotic effect of aerobic exercise in rat arterioles and venules under conditions of variable exercise load. In the present study, rats were subjected to constant exercise of 90% maximal oxygen uptake for 30 min, 5 times a week for 2, 4, 8 or 18 weeks. Thrombotic tendency was assessed by the He-Ne-laser-induced thrombus formation method. Platelet aggregation, platelet adhesion, whole blood clotting time, fibrinogen levels and blood cell count were also measured. The thrombotic tendency in arterioles decreased significantly after 18 weeks training, but not in venules. Thrombotic tendency increased slightly after 2 weeks training though the differences were not significant. A significant decrease in collagen-induced platelet aggregation was observed after 18 weeks training. The results demonstrated that long-term aerobic training decreased the thrombotic tendency in rat arterioles and that this was partly due to decreased platelet aggregability.

Role of Endothelium in Function of Isolated Arterioles of Rat Mesentery and Gracilis Muscle

The role of endothelium in the mediation of dilation evoked by vasoactive agents in the microcirculation of rat mesentery and gracilis muscle has not been assessed directly and the results obtained so far are controversial. The aim of our study, therefore, was to investigate if changes occurred in the dilator responses of small, isolated arterioles (20 to 200 μm) after physical removal of the endothelium. Isolated arterioles were visualized with television microscopy and diameters measured with an image-shearing device. Dilation of three different orders of rat mesenteric (193.5 ± 7.0, 92.5 ± 4.6, and 35.6 ± 4.8 μm respectively) and gracilis (76.9 ± 1.6, 32.3 ± 1.1, and 22.3 ± 1.6μm) muscle arterioles was investigated in response to acetylcholine (ACh; 10−6M) and sodium nitroprusside (SNP; 10−7M or 5 × 10 −8M) at constant intravascular pressures (100, 80 and 80/50 mm Hg, respectively). In the presence of the endothelium, all arterioles dilated (20–100%) in response to ACh and SNP. Endothelium removal was ...

Characteristics and origin of myogenic response in isolated mesenteric arterioles.

Responses to changes in intravascular pressure of isolated rat mesenteric arterioles were investigated under no-flow conditions. First-, second-, third-, and fourth-generation arterioles were isolated and cannulated. Vascular diameters were measured with an image-shearing device and recorded. The arterioles (except for the first-generation vessels) developed spontaneous tone, corresponding to the step increases in intravascular pressure (from 20 to 160 mmHg, by 20-mmHg steps). For example, at 80 mmHg pressure the mean diameters of first-, second-, third-, and fourth-generation vessels were 286.9 +/- 5.0, 203.4 +/- 8.2, 92.5 +/- 4.6, and 35.6 +/- 4.8 microns, respectively; by use of a Ca(2+)-free solution containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (1 mM) and sodium nitroprusside (SNP; 10(-4) M) the passive diameters of these vessels were 295.6 +/- 6.3, 238.4 +/- 11.7, 120.3 +/- 3.7, and 59.4 +/- 3.1 microns, respectively, demonstrating that the degree of pressure-induced constriction increased with the increasing order of generations (3, 14, 24, and 43%, respectively). The vasoactive function of endothelium and vascular smooth muscle was assessed by the responses of arterioles to acetylcholine (ACh; 10(-6) M) and SNP (10(-7) M) before and after removal of the endothelium with air. After removal of the endothelium, dilation to ACh was abolished while dilation to SNP was retained. Removal of the endothelium did not significantly alter the changes in the diameter of arterioles in response to step increases in intravascular pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pyridoxalated hemoglobin polyoxyethylene conjugate on diameter of rat mesenteric arterioles in situ.

The effects of exchange transfusion (ET) with a modified hemoglobin (pyridoxalated hemoglobin polyoxyethylene conjugate, or PHP) on endothelium-dependent relaxation of microvasculature were examined in rat mesenteric arterioles using image-splitter television microscopy. To examine the endothelium-dependent relaxation, we applied acetylcholine (ACh) to arterioles preconstricted with norepinephrine (NE) and measured changes in arteriolar diameter. Topical application of NE (6 x 10(-8) to 6 x 10(-6)M) decreased the diameter of the arterioles dose-dependently to a mean of 45% (SE +/- 4%, n = 6) of the control. Topical application of ACh (7 x 10(-8) to 7 x 10(-6)M) increased the diameter of the arterioles preconstricted with NE in a dose-dependent manner and almost fully reversed the NE-induced decrease in diameter. ET with 6% PHP solution (30 ml/kg) induced an increase in the mean arterial blood pressure (MABP) from 117 +/- 5 to 132 +/- 4 mm Hg (n = 6, p < 0.05), with a concomitant decrease in diameter of arterioles, from 22.6 +/- 2.5 to 19.8 +/- 2.7 microns (p < 0.05). The MABP and arteriolar diameter gradually returned to the control level within 30 min after ET. Arteriolar changes in diameter in response to NE and ACh examined 30 min after ET were similar to those of the control. ET with 6% stroma-free hemoglobin (SFH; 30 ml/kg) did not alter arteriolar changes in diameter in response to NE and ACh. Results suggest that circulating PHP and SFH moieties do not interfere with endothelium-derived relaxing factor(s) induced by ACh in rat mesenteric microvasculatures in situ.

Insulin Attenuates Vasoconstriction by Noradrenaline, Serotonin and Potassium Chloride in Rat Mesenteric Arterioles

We investigated the effect of insulin on the vascular reactivity to noradrenaline, serotonin and potassium chloride in rat mesenteric resistance arterioles in vitro. Mesenteric artery segments were placed in a myograph system. Sensitivity to noradrenaline, serotonin and KCl was tested after an equilibration at 37 degrees C. Thereafter, arteries were incubated with buffer alone or with insulin (40, 100, 250 and 400 mU/ml) for one hour at 37 degrees C. Sensitivity to the three vasoconstrictors was retested. Incubation with noradrenaline, serotonin and KCl resulted in a dose dependent increase in wall force. Exposure with buffer did not change the shape of the dose-response-curve. The same was true for the lowest dose of insulin (40 mU/ml). However, incubation with insulin at concentrations of 100, 250 and 400 mU/ml led to a reduction in wall force by 37-77%. The reduction in the slope of the curve and the maximal response suggest a non-competitive inhibition. Supraphysiological doses of insulin attenuate the vasoconstriction by noradrenaline, serotonin and KCl in rat mesenteric arteries in vitro.

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) on the endothelium-dependent relaxation in rat mesenteric arterioles.

Effects of exchange-transfusion with a modified hemoglobin (pyridoxalated hemoglobin polyoxyethylene conjugate; PHP) on diameter of rat mesenteric arterioles responding to various vasoactive substances were examined using the image-splitter television microscopy method. The mesentery of the anesthetized rat was spread over the stage of a microscope and the microvasculature was visualized by transillumination. Exchange-transfusion with a 6%PHP solution (30 ml/kg) was performed and effects of topical application of norepinephrine (NE) and acetylcholine(ACh) on diameter of mesenteric arterioles were examined. By exchange-transfusion with PHP, NE induced a dose-dependent decrease in diameter, while ACh induced a dose-dependent increase in diameter of the arterioles preconstricted by NE. There was no difference in vascular responsiveness to ACh and NE before and after exchange-transfusion with PHP solution. Results suggest that circulating PHP moiety does not exert any action on function of endothelial cell to release endothelium-derived relaxing factor (EDRF) in rat mesenteric microvasculature in situ.

Pertussis toxin abolishes the effect of neuropeptide Y on rat resistance arteriole contraction

Pertussis toxin (PTX) was used to investigate the possible involvement of a G protein in the mechanism of action of neuropeptide Y (NPY) on rat mesenteric arterioles. ADP ribosylation of membrane protein was assessed by gel electrophoresis followed by autoradiography. The effect of NPY was studied on contractions elicited either by addition of calcium to depolarized vessels or by the calcium agonist BAY K 8644. Under conditions in which PTX ADP-ribosylated a 40-41 kDa protein, the potentiation of contractions induced by NPY in both protocols was abolished. In contrast, the contraction induced by norepinephrine was unaffected by PTX. It is concluded that a G protein mediates the potentiating effect of NPY on external calcium-dependent contractions in rat mesenteric arterioles.

Enhancement by neuropeptide Y (NPY) of the dihydropyridine‐sensitive component of the response to α1‐adrenoceptor stimulation in rat isolated mesenteric arterioles

1. The mechanism by which neuropeptide Y (NPY) potentiates the vasoconstriction induced by alpha 1-adrenoceptor agonists was investigated in 3rd generation mesenteric arterioles of the rat. 2. At a maximally active concentration, nitrendipine (10(-6) M) displaced to the right the concentration-response curves to noradrenaline (pD2 decreased from 6.2 +/- 0.06 to 5.7 +/- 0.03) and phenylephrine (pD2 decreased from 5.6 +/- 0.03 to 5.3 +/- 0.03). Diltiazem (10(-5) M) also shifted to the right the concentration-response curve to phenylephrine (pD2 decreased from 6.0 +/- 0.06 to 5.5 +/- 0.04). In addition, the maximal response to phenylephrine was significantly decreased in the presence of either nitrendipine or diltiazem. 3. In the absence of a calcium channel blocking agent, NPY (100 nM) produced a leftward shift of the concentration-response curves to noradrenaline (pD2 increased from 6.2 +/- 0.06 to 6.5 +/- 0.05) and phenylephrine (pD2 increased from 5.6 +/- 0.03 to 6.0 +/- 0.06 and from 6.0 +/- 0.06 to 6.3 +/- 0.11). In the presence of either nitrendipine (10(-6) M) or diltiazem (10(-5) M), NPY (100 nM) did not alter the concentration-response curves to either noradrenaline or phenylephrine. 4. NPY was added to arterioles brought to the same level of tension (40% of the maximal contraction) either by phenylephrine alone (1.5 x 10(-6) M) or by a higher concentration of phenylephrine (3 x 10(-6) M) followed by the addition of prazosin (1.3 x 10(-9) M; a concentration at which it partially blocks alpha 1-adrenoceptors). In these conditions, the response to phenylephrine was completely abolished by nitrendipine (10-6 M) or by diltiazem (10-5M). Furthermore, NPY (10-1" to 10-7M) increased the arteriolar tension up to the maximal contractile capacity of the vessels with pD2 values of 8.6 + 0.02 and 8.7 + 0.01, in the absence and presence of prazosin, respectively. 5. Prazosin was replaced in the above protocol by other vasodilator agents acting through different mechanisms. Whether in the presence of 2 x 10-7M forskolin, 6 x 10-7M sodium nitroprusside (which stimulate adenylate cyclase or guanylate cyclase, respectively) or 2 x 10- 7M diltiazem (a concentration at which calcium entry is partially blocked), NPY enhanced phenylephrine-induced contraction to the maximum level with an identical potency (pD2 values of the peptide ranged from 8.3 to 8.7). 6. The results show that, in rat mesenteric arterioles, NPY potentiates only the calcium entry blockersensitive component of contraction induced by stimulation of alpha,-adrenoceptors. In addition, they provide evidence that the peptide counteracts with an equal potency the inhibitory effect of partial block of alpha,-adrenoceptors and of relaxing agents acting through different mechanisms. It is suggested that NPY enhances calcium entry induced by stimulation of alpha l-adrenoceptors in this tissue.

Dilator response of rat mesenteric arcading arterioles to increased blood flow velocity.

Arcading arterioles (average diam 68 microns ID) connecting adjacent triangular vascular sectors in the rat mesentery were examined in vivo for the presence of flow-dependent vasodilation. When a feed artery to one of these sectors was occluded, the affected sector was supplied by collateral flow through the arcading arteriole, and red cell velocity in the arteriole increased by 10-66 mm/s. The velocity increase was followed (with an average delay of 7.7 s) by dilation of the arcading arteriole, which averaged 68%. The dilation was closely correlated with red cell velocity (r = 0.96), volume flow (r = 0.96), and wall shear rate (r = 0.89). The dilation was sustained for the duration of increased velocity (1-10 min) and was not affected when direction of flow in the arteriole was reversed. The flow-induced dilation was equal to the maximal dilation attained with topically applied papaverine. Dilation of the arcading arteriole could be almost completely abolished if the arteriole was also occluded during occlusion of a feed artery. These observations indicate that a potent flow-dependent dilator mechanism is present in arcading arterioles of rat mesentery and may play an important role in local regulation.

Effect of antibiotics on laser-induced thrombus formation in rat mesenteric arterioles.

Antibiotics were tested in a thrombosis model, in which thrombi are produced in small rat mesenteric arterioles. An interference contrast system based on a Leitz Orthoplan microscope, was used to visualize thrombus formation. Vascular lesions were produced with a Coherent CR-2 supergraphite ion laser (argon laser) in arterioles of 25-35 microns diameter. Cefmenoxim, cefotaxim (i.v.) and cephalexin orally at doses of 20 and 40 mg/kg and gentamycin 10 and 20 mg/kg (i.v.) had a marked and significant antithrombotic effect. Even more effective were cefoperazon and lamoxactam 20 and 40 mg/kg (i.v.) and tobramycin 10 and 20 mg/kg (i.v.). Azlocillin, cefoxitin, mezlocillin or spectinomycin (20 mg/kg i.v.) and penicillin or piperacillin (50 mg/kg i.v.) also showed a significant antithrombotic effect, which, however, disappeared on doubling of the applied dose.

Surprising effects of the sequential administration of pentoxifylline and low dose acetylsalicylic acid on thrombus formation's

The effect of the combined oral administration of pentoxifyl-line (pof) and low dose acetylsalicylic acid (ASA) was evaluated with the help of the laser-induced thrombosis in rat mesenteric arterioles. Laser-induced thrombosis is inhibited in a dose-dependent way by both drugs. The administration of ASA, either simulataneously with or 1 hour prior to pof, does not show any effects in the laser model. On the contrary, the administration of pof followed 1 h later by ASA not only exhibited a significant effect but also produced a supraadditive inhibition of the laser-induced thrombus formation. Specific investigations concerning the time interval between the administration of both drugs determined that a significant effect can be achieved only after an interval of 30 to 90 minutes (principle of HWA 5112). The striking results could also be shown in diseased animals after sequential chronic administration of pof 1 h prior to ASA. HWA 5112 exhibits significant effects on laser-induced thrombus formation in the following chronic animal models: 1. adjuvant arthritic rats, 10→1 mg/kg for 21 days; 2. spontaneously hypertensive stroke-prone rats, 10→1 mg/kg three times within 24 h; 3. cholesterol-induced atherosclerosis in rabbits, 10→1 mg/kg for 14 days. The reported data clearly demonstrate that the sequential drug administration of first pentoxifylline followed 30 to 90 min later by ASA exhibits a supraadditive antithrombotic effect.

Inlfuence of ADP, blood flow velocity, and vessel diameter on the laser-induced thrombus formation

The laser-induced thrombus model in rat mesenteric arterioles and venules represents a reliable and reproducible in vivo method. It is suitable for basic investigations concerning factors involved in thrombus formation as well as for testing antithrombotic effects of drugs. The laser-induced thrombus formation depends significantly on the presence of ADP, as ADP-utilizing enzymes inhibit thrombosis in the animal model. The instrumental test set-up consists of a 4 W Argon laser (Spectra Physics, Mountain View, CA, USA), a ray adaptation and adjusting device (BTG, Munich, FRG), a microscope (ICM 405, Zeiss, Oberkochen, FRG), and a video system (Sony, Japan). RBC velocity data were recorded with the help of a modified dual-slit technique (acc. to Wayland and Johnson). Results were expressed as number of laser injuries necessary to produce a defined thrombus (minimum size: 1/4 of the vessel diameter) under constant conditions (effective capacity: 30 mW, exposure time: 1/5 sec). The number of laser lesions necessary to induce a defined thrombus decreased with an increase in arteriole diameter (10 to 20 μm) but increased again in larger arterioles and small arteries (>25μm). On the arteriolar side there are significant correlation coefficients between vessel diameter and RBC velocity (r = 0.69), vessel diameter and No. of laser injuries (r = 0.70), and RBC velocity and No. of laser injuries (r = 0.71). Due to relative low flow conditions in the venules, the number of laser injuries required to induce a defined thrombus does not significantly depend on the vessel diameter.

Effects of propentofylline (HWA 285) on laser-induced thrombus formation in healthy and diseased rat mesenteric arterioles.

The antithrombotic effects of the xanthine derivative propentofylline (HWA 285) were investigated in the laser-induced thrombus model in healthy and diseased rat mesenteric arterioles. Laser-induced platelet thrombi were documented by the help of an intravital microscope and a video system. The statistical comparison of drug-treated and untreated animals, in respect to the number of laser shots necessary to induce a defined thrombus, served as a quantitative measure for a potential antithrombotic drug effect. Propentofylline and acetylsalicylic acid, in single dosages of 10 and 30 mg/kg orally, significantly increased the number of laser shots required to produce a defined thrombus. Moreover, the drug decreased the laser-induced thrombus formation in a chronic experiment in adjuvant arthritic rats. After oral administration of 30 mg/kg for 21 days, propentofylline and pentoxifylline reduced significantly the thrombus formation, whereas acetylsalicylic acid exerted no effect.