Rat Vena Cava Research Articles

Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava.

1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

Formation of endovasal structures and intramural channels in rat vena cava after application of microsurgical suture: prophylactic effect of heparin and trental administration.

The study addresses the histopathology of end-to-end microanastomosis in the rat inferior vena cava. The vessels were harvested in 4 hours and 1, 3, 7, 14, and 30 days after the operation and were evaluated using light, scanning, and transmission electron microscopy. The endothelium was completely restored within 7 days after the surgical procedure. Endovasal structures at the microsuture site and intramural endothelial channels opening into the vena cava lumen developed as a result of endothelialization and parallel substitution of the three-dimensional fibrin framework of the mural microthrombus with connective tissue. By 1 month, aggregates of platelets, fibrin, and adhering leukocytes were observed in the intramural channel mouths. Administration of heparin and Trental for 1 week after the operation reduced the number of intramural channels and prevented the formation of endovasal structures at the site of microvenous anastomosis.

Structural features of dermatan sulfates and their relationship to anticoagulant and antithrombotic activities

Dermatan sulfate is a polydisperse, microheterogeneous sufated copolymer of N- acetyl- d- galactopyranose and idopyranosyluronic acid that is currently under clinical investigation as a new antithrombotic agent. The structure and activity of two pairs of dermatan sulfates, isolated from bovine and porcine mucosa, were studied. One dermatan sulfate from each species demonstrated high in vivo antithrombotic activity in the rat vena cava assay. The in vitro anticoagulant activity of each dermatan sulfate was determined using activated partial thomboplastin time (APTT), thrombin time (TT) (5 units), calcium thrombin time (CaTT ) (5 units), Heptest®, anti-factor Xa and anti-factor IIa antithrombin assays and heparin cofactor II amidolytic assays. The coagulation-based assays gave the best correlation to in vivo antithrombotic activity. The physical and chemical properties of each dermatan sulfate were determined using 1H-NMR and 13C-NMR spectroscopy, molecular weight determination, potentiometric titration, chemical degradative analysis, chondroitin lyase degradative analysis and oligosaccharide mapping. These analyses indicated that the major difference between dermatan sulfates from a particular species having high and low in vivo antithrombotic activity was their iduronic acid content. The relation between increased iduronic acid content and increased in vivo antithrombotic activity may be the result of the conformational flexibility of this residue.

Local inhibition of thrombosis using urokinase linked to a monoclonal antibody which recognises damaged endothelium

A monoclonal antibody which binds to damaged endothelial cells was covalently linked to urokinase in an attempt to produce a thrombolytic agent which could be localised to areas of endothelial damage. The conjugate was shown to be effective in inducing localised thrombolysis in vitro, and in a rat vena cava thrombosis model brief exposure of the intimal surface of the isolated vena cava to the antibody -urokinase conjugate prevented or reduced subsequent stasis-induced thrombosis without inducing a systemic lytic state.

Angiotensin II generation in the rat vena cava: Stimulation of local synthesis by ?-adrenoceptor activation

There is considerable evidence to suggest that isolated tissues have the capacity to generate angiotensin II and that angiotensin II thus generated may enhance noradrenergic neurotransmission. In the present study the possibility that there may be local formation of angiotensin II within the rat vena cava and its consequence to noradrenergic transmission has been investigated. The angiotensin II precursors, angiotensin I and a synthetic tetradecapeptide renin substrate, each enhanced the stimulation-induced efflux of radioactivity from tissues previously incubated with 3H-noradrenaline. The effects of angiotensin I were blocked by the converting enzyme inhibitor captopril and the receptor antagonist saralasin, indicating local conversion to angiotensin II. The effect of the tetradecapeptide was blocked by saralasin, but not by captopril, suggesting either a direct effect of this peptide or conversion to angiotensin II by a pathway not involving angiotensin converting enzyme. The beta-adrenoceptor agonist isoprenaline enhanced noradrenergic transmission in the rat vena cava, relaxed guinea-pig trachea precontracted with carbachol and increased heart rate in rat isolated atria. Captopril and saralasin blocked the effect of isoprenaline in the vena cava, but did not alter its effects in the atria or trachea. This suggests that in the rat vena cava the facilitation of noradrenergic transmission by isoprenaline may involve stimulation of local angiotensin II production.

Heterogeneity of Presynaptic Serotonin Receptors on Sympathetic Neurones in Blood Vessels

Presynaptic serotonin (5-HT) receptors on the postganglionic sympathetic nerves, which mediate inhibition of noradrenaline release in blood vessels of various species and which interact with the presynaptic alpha 2-autoreceptors, are heterogeneous. In the rat vena cava, they are of the 5-HT1B subtype, in the pig coronary artery they belong to a novel, so far unknown class of 5-HT receptors, and in the human saphenous vein they could be classified as 5-HT1D. These results point to marked species differences and the need to carry out experiments in human vascular preparations. Presynaptic 5-HT receptors may be involved in the mechanism of action of the new antimigraine drug sumatriptan.

Effect of temperature on α2‐ and α1‐adrenoceptor‐mediated responses in the pithed rat and in the rat vena cava

1. The influence of body temperature on the alpha 1- and alpha 2-adrenoceptor-mediated pressor responses has been investigated in the pithed rat. 2. The pressor responses to noradrenaline, to the full alpha 1-adrenoceptor agonists phenylephrine and cirazoline and to tyramine were not influenced by lowering the temperature from 36-37 degrees C to 27-29 degrees C. In contrast, the dose-response curves for the pressor effects of the partial alpha 1-adrenoceptor agonist ST 587 and of the alpha 2-adrenoceptor agonist B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine), B-HT 933 (2-amino-6-ethyl-5,6,7,8-tetrahydro-4H-[4,5-d]azepine), clonidine, moxonidine and M-7 (2-dimethylamino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide) were markedly depressed (without change in the ED50 values), when the body temperature was lowered from 36-37 degrees C to 27-29 degrees C. 3. After i.v. administration of yohimbine, there was a rightward shift of the dose-response curve for B-HT 920, and the degree of this shift was the same at all temperatures investigated. 4. In the rat vena cava preincubated with [3H]-noradrenaline, the B-HT 920-induced alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium overflow was also reduced at lower temperature. 5. These results are compatible with the suggestion that cooling decreases the alpha 2-adrenoceptor-mediated pre- and postsynaptic responses in the rat vena cava and pithed rat respectively, leaving the pressor effect induced by full, but not partial alpha 1-adrenoceptor agonists in the pithed rat unaffected. 6. These differences may partly be related to differences in receptor reserve for alpha 1- and alpha 2-adrenoceptors in the pithed rat preparation.

The 5‐HT1B Receptors

The 5-HT1B receptors have been identified by radioligand binding techniques predominantly in the basal ganglia of the rat and mouse brain. A number of 5-HT receptor agonists have been shown to display high affinity but limited selectivity for the 5-HT1B recognition site. These include 5-CT, 5-HT, RU 24969, TFMPP, MCPP, and CGS 12066B. Antagonists at the 5-HT1B site include the drugs metitepin, metergoline, cyanopindolol, isamoltane, and propranolol but none of these drugs are selective for this receptor. Functional correlates of 5-HT1B receptor activation have been most closely defined in vitro. These include inhibition of transmitter release, inhibition of forskolin-stimulated adenylate cyclase and actions on the mouse urinary bladder strip and the rat vena cava. Many functional correlates of 5-HT1B receptor activation in vivo have been proposed, but convincing evidence from antagonist studies is generally lacking. The development of selective 5-HT1B receptor agonists and antagonists will be a key step in defining the physiological role of this receptor site in the brain and periphery of the mouse and rat although it must be realized that these compounds, if they are developed, are unlikely to have functional effects in man since the 5-HT1B recognition site is absent in the human CNS. Nevertheless many of these studies on the 5-HT1B receptor may aid the development of drugs acting at the 5-HT1D site since this receptor has been identified as being the equivalent of the 5-HT1B site in species other than the rat and mouse.

Mutual interaction between presynaptic ?2-adrenoceptors and 5-HT1B receptors on the sympathetic nerve terminals of the rat inferior vena cava

Segments of the rat vena cava preincubated with [3H]noradrenaline and superfused with [3H]noradrenaline-free solution containing desipramine and corticosterone were stimulated electrically (standard parameters: 150 mA, 0.3 ms, 0.66 Hz; duration 6 min). In some experiments the stimulation parameters were modified in order to obtain similar absolute release values despite the presence of an alpha-adrenoceptor agonist or antagonist or of 5-hydroxytryptamine (5-HT). In a first set of experiments, the vascular segments were first exposed to an alpha-adrenoceptor ligand, which was kept present throughout the remainder of superfusion, and then to 5-HT. The release-inhibiting effect of 5-HT was attenuated by the alpha 2-adrenoceptor agonists clonidine and B-HT 920 whereas it was enhanced by the alpha-adrenoceptor antagonists phentolamine and idazoxan. The alpha 1-adrenoceptor antagonist prazosin did not change the 5-HT-induced inhibition of noradrenaline (NA) release. In a second set of experiments, 5-HT was administered first and kept present in the superfusion fluid for the remainder of the experiment. In the presence of 5-HT, the overflow-inhibiting effects of B-HT 920 and clonidine and the overflow-enhancing effect of idazoxan were reduced. The results demonstrate that activation of one kind of receptor decreased the inhibition of noradrenaline release produced by activation of the other. These effects were not the consequence of the change of release per se induced by the interacting drugs, since they also occurred when the release was adjusted to similar levels by modification of the stimulation parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonistic properties of RU 24969, a preferential 5-HT1 receptor agonist, at presynaptic alpha 2-adrenoceptors of central and peripheral neurones.

The effect of RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1 H-indole) on the electrically evoked 3H overflow was studied in superfused rat brain cortex slices preincubated with 3H-noradrenaline or 3H-serotonin and in superfused segments of the rat vena cava preincubated with 3H-noradrenaline. In cortex slices preincubated with 3H-noradrenaline, RU 24969 facilitated the electrically (3 Hz) evoked 3H overflow. This effect was abolished by phentolamine but was not affected by desipramine or the 5-HT3 receptor antagonist ICS 205-930. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow (determined in the presence of desipramine) was shifted to the right by RU 24969 32 and 100 mumol/l. In this respect, RU 24969 was about 500 times less potent than phentolamine. In cortex slices preincubated with 3H-serotonin, the inhibitory effect of 3.2 mumol/l RU 24969 on the electrically evoked 3H overflow was increased by phentolamine. In segments of the vena cava, RU 24969 inhibited the electrically (0.66 Hz) evoked 3H overflow. The concentration-response curve of RU 24969 was U-shaped, since at concentrations higher than 0.1 mumol/l the extent of inhibition decreased with increasing concentrations of RU 24969. In the presence of phentolamine, the concentration-dependent attenuation of the RU 24969-induced inhibition of overflow was no longer detectable. The present results suggest that RU 24969 is a weak antagonist at presynaptic alpha 2-adrenoceptors (by more than 2.5 log units less potent than as an agonist at presynaptic 5-HT1B auto- and heteroreceptors).

Receptor-mediated effects of serotonin and 5-methoxytryptamine on noradrenaline release in the rat vena cava and in the heart of the pithed rat.

In segments of the rat inferior vena cava preincubated with 3H-noradrenaline, it was examined whether presynaptic serotonin (5-HT) receptors exist on the postganglionic sympathetic nerves of the circulatory system; for this purpose the effects of 5-HT receptor agonists and antagonists on the electrically evoked 3H overflow were studied. Furthermore, vagotomized pithed rats (treated with atropine and captopril) were used to investigate the effects of these drugs on heart rate and on the tachycardia induced by electrical stimulation of the preganglionic sympathetic nerves (C7--T1) via the pithing rod; these experiments were carried out to provide evidence that the presynaptic 5-HT receptors are operative in vivo. 5-HT and 5-methoxytryptamine (5-OCH3-T) concentration-dependently inhibited the electrically evoked 3H overflow from the vena cava. The inhibitory effect was more pronounced at 0.66 Hz than at 2 Hz. 8-Hydroxy-2-(di-n-propylamino)tetralin did not alter the evoked overflow. The inhibitory effect of 5-HT or 5-OCH3-T was antagonized by metitepin but not affected by ketanserin or rauwolscine. In pithed rats 5-HT and 5-OCH3-T by themselves dose-dependently increased heart rate. The positive chronotropic effect of 5-HT 10 mumol/kg, which was not affected by ketanserin, was considerably decreased by desipramine, indicating that 5-HT at least at this high dose acts predominantly by a tyramine-like indirect sympathomimetic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Innervation pattern of inferior vena cava in mouse and rat.

The innervation pattern of rat and mouse inferior vena cava was studied using catecholamine fluorescence and cholinesterase histochemical methods. Adrenergic nerve fibers innervate only abdominal portions of the inferior vena cava, while cholinergic nerves are chiefly distributed to the thoracic inferior vena cava and show a gradual decrease in the abdomen. Chemical sympathectomy performed with the neurotoxin 6-hydroxydopamine does not alter the pattern of cholinergic innervation of the inferior vena cava, suggesting the parasympathetic nature of cholinergic nerves.

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type.

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha-like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.

Failure of surface potential to influence thrombus formation.

Platinum wire was inserted in rat vena cava for 1 hour, and deposited thrombus was weighed. The wires were charged in 100 mV steps through the range ±400 mV NHE with respect to the blood. Eight preparations were used at each step. Observations were also made using aluminium wire in the range −400 to −1000 mV NHE. Inserted for various times, platinum wires charged at +400 mV NHE gathered thrombus at a constant rate over 60 minutes. Wires with a negative charge of 400 mV gathered thrombus at a similar rate.

Histochemical investigations on the nature of large blood vessel endothelial and medial argyrophilic lines and on the mechanism of silver staining

A study of the mechanisms involved in silver staining of blood vessels has been performed on the rabbit and rat aorta and vena cava, both in fixed and unfixed states. Pretreatment with cationic detergents, organic solvents, and solutions containing free iodide ions inhibited the silver staining. Anionic or neutral detergents, oxidizing agents, binders of such ions as Ca++, Mg++ and SO4-failed to inhibit the staining. Staining of the intercellular gaps between endothelial cells and between smooth muscle cells could also be obtained if vessels were treated with a cationic detergent and bromocresol green, or by a modified Hale's colloidal iron technique. Silver lines could be returned to dechlorinated vessels, if treated with sodium chloride before silver nitrate staining, but not vice versa; by an extended treatment with dilute silver nitrate or with gold chloride following normal silver nitrate staining; and by treatment with heparin prior to silver staining. Dark chamber experiments have demonstrated that a photographic developer can take the place of light in the silver staining procedure and that a photographic fixer has the same effect on vessel silver staining as dechlorination.