Canine Saphenous Vein Research Articles

Effects of Bay K 8644 on the electrically evoked and KCl-evoked norepinephrine release and the corresponding contraction in isolated canine saphenous veins.

Bay K 8644 caused no significant effects on the 3H overflow and contraction evoked by electrical stimulation in the canine saphenous vein preloaded with [3H]norepinephrine. Bay K 8644 significantly enhanced the 3H overflow evoked by 40 mM KCl in a concentration-dependent manner, which was antagonized by nisoldipine. Bay K 8644 failed to significantly augment the KCl-evoked contraction. These results suggest that there is a difference between the modes of action of Bay K 8644 on norepinephrine releases evoked by electrical stimulation and KCl.

Effects of Bay K 8644 on the Electrically Evoked and KCI-Evoked Norepinephrine Release and the Corresponding Contraction in Isolated Canine Saphenous Veins

Bay K 8644 caused no significant effects on the 3H overflow and contraction evoked by electrical stimulation in the canine saphenous vein preloaded with [3H]norepinephrine. Bay K 8644 significantly enhanced the 3H overflow evoked by 40 mM KCl in a concentration-dependent manner, which was antagonized by nisoldipine. Bay K 8644 failed to significantly augment the KCI-evoked contraction. These results suggest that there is a difference between the modes of action of Bay K 8644 on norepinephrine releases evoked by electrical stimulation and KCl.

Differential effects of Ca antagonists on the noradrenaline release and contraction evoked by nerve stimulation in the presence of 4-aminopyridine.

We previously reported that verapamil, nicardipine and diltiazem inhibited both neurotransmitter release and contraction evoked by transmural nerve stimulation (TNS) in the canine saphenous vein. To evaluate whether the three Ca antagonists act on the nerve endings by inhibiting Ca2+ influx, the effects of the three antagonists were studied in the presence of 4-aminopyridine (4-AP) 3 X 10(-4) M on the TNS-evoked tritium overflow and contraction of canine saphenous veins preloaded with [3H]-noradrenaline. 4-AP increased both tritium overflow and contraction evoked by TNS, but did not enhance the contraction induced by exogenous noradrenaline (10 nmol). In the veins pretreated with 4-AP, verapamil (3 X 10(-5) M) and nicardipine (10(-5) M and 3 X 10(-5) M) caused no significant effects on the TNS-evoked tritium overflow, but they still inhibited the contraction. Diltiazem (10(-5) M and 3 X 10(-5) M) significantly inhibited both responses to TNS in the veins pretreated with 4-AP, the effects being nearly equipotent to those in the absence of 4-AP. The (-)-cis isomer of diltiazem (10(-5) M and 3 X 10(-5) M), which is about 100 times less potent than diltiazem in inhibiting Ca2+ influx, inhibited both responses to TNS in the presence of 4-AP to almost the same degree as diltiazem. When 4-AP was added after the Ca antagonists, it reversed the TNS-evoked tritium overflow inhibiting actions of verapamil (3 X 10(-5) M) and nicardipine (3 X 10(-5) M) much more effectively than that of diltiazem (3 X 10(-5) M). Tetracaine (4 X 10(-6) M) significantly inhibited the TNS-evoked tritium overflow and contraction, which were unaffected by 4-AP. Sodium salicylate (10(-2) M) failed to modify the inhibition of TNS-evoked tritium overflow following diltiazem (3 X 10(-5) M), but it enhanced that of tetracaine (4 X 10(-6) M). Verapamil but not diltiazem and nicardipine significantly increased the spontaneous tritium overflow from veins pretreated with 4-AP. The present study together with previous results suggests that diltiazem but not verapamil and nicardipine may inhibit the TNS-evoked neurotransmitter release through an action other than inhibition of Ca2+ influx into the adrenergic nerve endings, allowing an inhibition of the resulting contraction.

Inorganic phosphate inhibits sympathetic neurotransmission in canine saphenous veins.

Inorganic phosphate has been proposed as the initiator of metabolic vasodilatation in active skeletal muscle. The present study was primarily designed to determine if this substance has an inhibitory effect on adrenergic neurotransmission. Rings of canine saphenous veins were suspended for isometric tension recording in organ chambers. A comparison was made of the ability of inorganic phosphate (3 to 14 mM) to relax rings contracted to the same degree by electrical stimulation, exogenous norepinephrine, and prostaglandin F2 alpha. The relaxation during electrical stimulation was significantly greater at all concentrations of phosphate. In strips of saphenous veins previously incubated with [3H]norepinephrine, the depression of the contractile response caused by phosphate during electrical stimulation was accompanied by a significant reduction in the overflow of labeled neurotransmitter. Thus inorganic phosphate inhibits sympathetic neurotransmission and hence may have a key role in the sympatholysis in the active skeletal muscles during exercise. By contrast, in this preparation, it has a modest direct relaxing action on the vascular smooth muscle.

Flunarizine and α-adrenergic responses in isolated canine saphenous veins

1. 1. Experiments were designed to determine how flunarizine affects contractions of cutaneous veins to α-adrenergic activation. 2. 2. Rings of canine saphenous vein were mounted at optimal length for isometric tension recording in organ chambers filled with physiological salt solution. 3. 3. At concentrations higher than those needed to inhibit KCl-induced contractions, flunarizine inhibited the contractile responses evoked by α 2-adrenergic agonists (B-HT 920, xylazine), partial (St-587) and full (cirazoline, phenylephrine) α 1-adrenergic agonists and the combined α 1-/ α 2-adrenergic agonist, norepinephrine. 4. 4. The inhibitory effect of flunarizine against α 2-adrenergic responses was similar to that produced by other calcium-antagonists and results presumably from inhibition of the influx of extracellular calcium. 5. 5. The inhibitory effect of flunarizine against α 1-adrenergic responses was greater than expected and appears to result from competitive antagonism of α 1-adrenoceptors (p A 2 = 5.79). 6. 6. Therefore, flunarizine can decrease adrenergic contractile responses by depressing the influx of extracellular calcium and by blocking postjunctional α 1-adrenoceptors.

Effects of Bay K 8644 on the Electrically Evoked and KCI-Evoked Norepinephrine Release and the Corresponding Contraction in Isolated Canine Saphenous Veins

Bay K 8644 caused no significant effects on the 3H overflow and contraction evoked by electrical stimulation in the canine saphenous vein preloaded with [3H]norepinephrine. Bay K 8644 significantly enhanced the 3H overflow evoked by 40 mM KCI in a concentration-dependent manner, which was antagonized by nisoldipine. Bay K 8644 failed to significantly augment the KCI-evoked contraction. These results suggest that there is a difference between the modes of action of Bay K 8644 on norepinephrine releases evoked by electrical stimulation and KCI.

Tetrodotoxin-resistant relaxation to transmural nerve stimulation in canine saphenous veins: a possible neural origin

Transmural nerve stimulation following sympathetic (guanethidine 10(-4) mol/L, phenoxybenzamine 2 X 10(-5) mol/L, propanolol 2 X 10(-6) mol/L) and muscarinic blockade (atropine 5 X 10(-5) mol/L) produces a relaxatory response in canine saphenous veins contracted with prostaglandin F2 alpha. This relaxatory response was shown previously to be resistant to tetrodotoxin. Transmural nerve stimulation (10 V, 1.0 ms) was applied as intermittent trains of stimuli of 30 s duration at frequencies of 1-32 Hz. The veins showed a frequency dependent relaxation (maximum 2.65 +/- 0.20 g). The stimulations were repeated in the presence of lignocaine (10(-3) mol/L), apamin (10(-8) mol/L), ascorbic acid (10(-4) mol/L), or catalase (50 micrograms/mL). The relaxatory response was unaffected by apamin, scorpion toxin, superoxide dismutase, ascorbic acid, and catalase (p greater than 0.05). However, lignocaine (10(-3) mol/L) reduced significantly the relaxatory response to transmural nerve stimulation in this preparation (p less than 0.05). In a separate group of veins, lignocaine (10(-3) mol/L)l abolished the contractile response to transmural nerve stimulation with little effect upon the contractile response to exogenous noradrenaline and the relaxatory responses to isoprenaline and sodium nitrite. These findings support the proposition that the nonadrenergic, noncholinergic tetrodotoxin-resistant relaxatory response observed with transmural nerve stimulation in the canine saphenous vein is mediated by a neural mechanism.

Different types of relationship between β-adrenergic relaxation and activation of cyclic AMP-dependent protein kinase in canine saphenous and portal veins

We examined the relationships between the relaxation mediated by β-adrenoceptor and either the associated cyclic AMP production or the activation of cyclic AMP-dependent protein kinase (A-PK) in canine saphenous and portal veins. In the saphenous vein constricted with methoxamine, isoproterenol caused concentration-dependent relaxation (maximum relaxation 92.7%), and concomitant increases in cyclic AMP and A-PK activity ratio from (52.8 to 73.5%). The portal vein was only slightly relaxed by isoproterenol (14.7% in the longitudinal strips) after constriction with methoxamine, while cyclic AMP and A-PK activation increased significantly. Isoproterenol markedly activated A-PK of the portal vein after KCl construction (from 52.6 to 74.6%) but the maximum relaxation was only slight (13.3%). The portal vein also showed a smaller relaxation response to either forskolin or dibutyryl cyclic AMP than the saphenous vein. These results indicated that the relationship between the relaxation response to isoproterenol and either cyclic AMP production or A-PK activation was different in the saphenous and portal veins.

Cyclo-oxygenase blockers influence the effects of 15-lipoxygenase metabolites of arachidonic acid in isolated canine blood vessels

In canine saphenous veins both the 15-hydroxy- and 15-hydroperoxy derivatives of arachidonic acid, 15HETE and 15HPETE, caused endothelium-independent contractions which were not affected by a variety of classical receptor antagonists. These contractions were markedly augmented by cyclooxygenase blockers; nifedipine, which did not influence the contractions induced by lipoxygenase products, inhibited the potentiating effect of indomethacin. In the veins, 15HETE and 15HPETE also induced spotaneous rhytmic contractions which persisted after several washings but could be blocked by inhibitors of clyclooxygenase. In coronary, splenic, renal and femoral arteries, 15HETE and 15HPETE caused contractions which were also augmented by indomethacin and were dependent on the influx of extracellular calcium as they were inhibited by verapamil. Both 15-lipoxygenase metabolites evoked relaxations during contractions induced by prostaglandin F 2α or the thromboxane-mimetic U46619. These relaxations were not endothelium-dependent but were inhibited by indomethacin; they did not occur when the initial contractions were caused by K +, norepinephrine or 5-HT. Our results illustrate multiple vascular actions of 15HETE and 15HPETE in dog blood vessels.

Norepinephrine Uptake in Canine Saphenous Veins in the Presence and Absence of Halothane

Studies were done to examine neuronal and extraneuronal uptakes of norepinephrine in canine saphenous veins and to ascertain whether these uptakes were altered by halothane. Minced vein was incubated for 1 min in Krebs-Ringer solution containing L-[ring-2,5,6-3H]-norepinephrine (1, 2.5, 6, or 12 microM). After incubation, radioactivity in tissue was measured by liquid scintillation counting. Neuronal uptake at each norepinephrine concentration was determined by the difference in norepinephrine uptake between tissues in which both neuronal and extraneuronal uptakes were operative and in tissues in which only extraneuronal uptake was operative. In time studies with 1 microM norepinephrine, neuronal uptake was still dominant at 10 min; with 12 microM norepinephrine, extraneuronal uptake predominated at all incubation periods tested. In other studies, tissues were incubated in the absence or presence of halothane (2.5%). The Km values for neuronal uptake in control and halothane-treated tissues were 2.49 and 2.32 microM, respectively. Corresponding Vmax values were 0.049 and 0.060 nMol X min-1 X 100 mg-1, respectively. No significant effect of halothane on neuronal or extraneuronal uptake could be detected.

Effect of calcium antagonists on adrenergic mechanisms in canine saphenous veins.

Experiments were designed to investigate the effect of two calcium antagonists, diltiazem and nicardipine (concentration range: 10(-7)-10(-4) M), on the contractile responses to transmural nerve stimulation, exogenous noradrenaline and tyramine in isolated canine saphenous vein rings. Both diltiazem and nicardipine inhibited the contractile response to transmural nerve stimulation in a non-competitive, concentration-dependent manner. At a concentration of 10(-4) M, diltiazem and nicardipine inhibited the maximum contractile response to transmural nerve stimulation to 0.8 +/- 0.8% and 20 +/- 10% of control respectively. Effects of diltiazem and nicardipine (up to 10(-4)M) on the contractile response to exogenous noradrenaline were minimal. The only significant difference observed was a 30% depression of the maximum contractile response with a shift in ED50 at high concentrations of nicardipine. Diltiazem (up to 10(-4) M) had no significant effect on concentration-effect curves for tyramine. Nicardipine inhibited the response to tyramine in a non-competitive manner with the maximum response depressed to 46% of control at 10(-4) M-nicardipine. Release of [3H]noradrenaline during transmural nerve stimulation was reduced by both calcium antagonists in a concentration-dependent manner. However, release of [3H]noradrenaline produced by the indirect sympathomimetic agent tyramine was not significantly inhibited by nicardipine. These experiments suggest that the calcium antagonists diltiazem and nicardipine inhibit the contractile response to transmural nerve stimulation in the canine saphenous vein predominantly by inhibiting the release of endogenous noradrenaline. However, nicardipine appears to have an additional post-synaptic inhibitory effect on the responses to exogenous as well as endogenous noradrenaline.

Pharmacological studies on frozen stored canine saphenous veins and basilar arteries.

Canine saphenous veins were either placed in Krebs-Henseleit solution and stored for 24 h at +4 degrees C, or immersed in FCS (fetal calf serum) containing 1.8 mol/l DMSO (dimethyl sulfoxide), slowly frozen to -70 degrees C and stored for 4 weeks at -70 degrees C or -190 degrees C. Canine basilar arteries were either stored in Krebs-Henseleit solution for 24 h at +4 degrees C or slowly frozen and stored for 3 months in FCS plus 1.8 mol/l DMSO at -70 degrees C. Subsequent pharmacological investigations revealed a considerable attenuation of the contractile force of frozen-stored vessels but the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and uptake1 mechanisms in veins stored at -190 degrees C and there is an excellent correlation of the pD2 values for various tryptamine derivatives on canine basilar arteries stored for 3 months at -70 degrees C with those calculated on fresh preparations. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.

O-184 - Differential effects of diltiazem and verapamil on Ca2+-dependent and Ca2+-independent norepinephrine releases and the corresponding contractions in isolated canine saphenous veins

O-184 - Differential effects of diltiazem and verapamil on Ca2+-dependent and Ca2+-independent norepinephrine releases and the corresponding contractions in isolated canine saphenous veins

Moderate cooling depresses the accumulation and the release of newly synthesized catecholamines in isolated canine saphenous veins.

Moderate cooling (from 37 degrees to 24 degrees C) depressed the formation of 3H-dopamine and 3H-norepinephrine from 3H-tyrosine by isolated canine saphenous veins. Cooling reduced the evoked release of newly synthesized catecholamine to the same extent as that of stored norepinephrine. Hence the augmentation by cold of the contractile response to sympathetic nerve stimulation observed in earlier work is not accompanied by an augmented release of newly synthesized norepinephrine.

Prejunctional adrenergic inhibition by aggregating platelets in canine blood vessels.

Experiments were performed to determine the effect of aggregating platelets on adrenergic neurotransmission. Rings of canine saphenous veins and left circumflex coronary arteries were incubated with [3H]norepinephrine and suspended for superfusion. Aggregating platelets and exogenous 5-hydroxytryptamine decreased the overflow of [3H]norepinephrine evoked by electrical stimulation of the adrenergic nerve endings. The reduction of transmitter overflow caused by 5-hydroxytryptamine was prevented by the serotonergic antagonist methiothepin in a concentration that did not significantly affect the release of 5-hydroxytryptamine or thromboxane B2 from the aggregating platelets. Methiothepin decreased but did not abolish the inhibitory effect of aggregating platelets on neurotransmitter overflow. These experiments demonstrate that 5-hydroxytryptamine and other substances released from aggregating platelets can exert prejunctional inhibition of adrenergic neurotransmission in isolated blood vessels.

Relationship between α2-adrenoceptor occupancy and response for B-HT 933 in canine saphenous vein

The relationship between α 2-adrenoceptor occupancy and contractile response for B-HT 933 was investigated in canine saphenous vein. B-HT 933 produced a concentration-dependent, α 2-adrenoceptor mediated contractile response in canine saphenous vein with an ED 50 of 0.65 μM. The dissociation constant (K A) of B-HT 933 was calculated to be 5 μM. The relationship between α 2-adrenoceptor occupancy and contractile response for B-HT 933 in canine saphenous vein was hyperbolic, typical of full agonists or agonists with high intrinsic efficacies. B-HT 933 produced a half-maximal response in canine saphenous vein at only 11% α 2-adrenoceptor occupancy, with a maximal response being obtained with 40–60% α 2-adrenoceptor occupancy. We conclude that the selective α 2-adrenoceptor agonist, B-HT 933, has high efficacy at α 2-adrenoceptors, such that a significant α 2-adrenoceptor reserve, or spare α 2-adrenoceptors, exists for this compound in canine saphenous vein.

Comparison of the Calcium Antagonistic Activity of Nifedipine and FR34235 in the Canine Saphenous Vein

The effects of FR34235 (FR) on contractions and 45Ca2+ uptake stimulated by depolarization (80 mM KCl) and activation of postsynaptic alpha 1-adrenoceptors in the canine saphenous vein (CSV) were studied and compared with those of nifedipine. The 45Ca2+ uptake and contractions evoked by KCl were inhibited in a dose-dependent fashion by FR [concentration producing 50% inhibition [(IC50 = 2.0 +/- 0.5 and 1.2 +/- 0.3 X 10(-8) M, respectively)] and nifedipine (IC50 = 6.0 +/- 1.0 and 4.0 +/- 0.9 X 10(-8) M, respectively). FR was a potent inhibitor of the 45Ca2+ uptake and contractions of CSV induced by the selective alpha 1-agonist, SK&F l-89748 (l-[1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine] ) (IC50 = 7.2 +/- 0.7 and 7.0 +/- 1.8 X 10(-8) M, respectively). In contrast, the contractions and 45Ca2+ uptake elicited by SK&F l-89748 were much less sensitive to nifedipine (IC50 = 6.0 +/- 1.4 and 6.6 +/- 0.4 X 10(-6) M, respectively). The results suggest the following: (a) both FR and nifedipine are potent antagonists of the voltage-dependent Ca2+ channels in CSV; and (b) FR is a more effective antagonist of receptor-operated Ca2+ entry in the saphenous vein than nifedipine.

Further studies on the effects of epimers of thromboxane A 2 antagonists on platelets and veins

The C-15 hydroxy epimers of three TXA 2/PGH 2 antagonists were resolved by HPLC and their pharmacology evaluated in canine saphenous veins and human platelets. For all three compounds, the epimers were equipotent in their ability to antagonize U46619 (1 μM) induced platelet aggregation. In contrast, one of the epimers for all three of the antagonists was significantly more potent than the other in antagonizing U46619 (10 nM) induced saphenous vein contractions. The data support the notion that the TXA 2/PGH 2 receptors in veins may be different from those in platelets.

N-[2-hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide. A potent agonist which releases intracellular calcium by activation of alpha 1-adrenoceptors.

N-[2-Hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide (SK&F 102652) has been prepared and characterized pharmacologically. It is a potent agonist with an EC50 of 25 nM at alpha 1-adrenoceptors as determined in the isolated perfused rabbit ear artery. On the presynaptic alpha 2-adrenoceptors of the guinea pig atrium it was considerably weaker, demonstrating an EC50 for inhibition of neurotransmission of 1200 nM and thus an overall alpha 1/alpha 2 selectivity ratio of greater than or equal to 48. In the vascular smooth muscle of the canine saphenous vein an EC100 concentration of this agonist in the presence of zero external Ca2+ induced 37.9 +/- 1.4% of the maximal contractile response due to this agent while the endogenous ligand norepinephrine evoked only 14.5 +/- 0.4% of the maximum. In the presence of low (1 microM) external calcium, this agent produced 78.3 +/- 5.3% of maximum while norepinephrine gave 45.3 +/- 7.4%. This agent produces alpha 1-adrenoceptor-mediated contraction primarily by release of intracellular Ca2+ and should provide a useful tool for characterizing alpha 1-receptor subtypes.

Effects of clorgyline and (?)deprenyl on the deamination of normetanephrine and noradrenaline in strips and homogenates of the canine saphenous vein

The influence of specific inhibitors of MAO A (clorgyline) and MAO B [(-)deprenyl] on the metabolism of normetanephrine (NMN) in strips of canine saphenous vein was studied, both in the absence and in the presence of inhibitors of neuronal (cocaine) and extraneuronal (hydrocortisone) uptake. Moreover, the formation of metabolites of noradrenaline and of NMN by saphenous vein homogenates and the influence of clorgyline or (-)deprenyl on this formation are described. Clorgyline reduced to the same degree (by about 70%) the formation of methoxy-hydroxy-phenylglycol (MOPEG) and of vanillylmandelic acid (VMA) in strips incubated with NMN, whereas (-)deprenyl reduced by about 50% the formation of MOPEG and had no effect on VMA production. Hydrocortisone had effects very similar to those of (-)deprenyl. Saphenous vein homogenates (O-methylation inhibited), deaminated both noradrenaline and NMN; clorgyline and (-)deprenyl reduced the formation of metabolites of both noradrenaline and NMN. It is concluded that both MAO A and B are able to deaminate noradrenaline and NMN, but that in the intact tissue the former has no access to MAO B. Even in intact tissues MAO B may play a role in the metabolism (but not in the inactivation) of noradrenaline by deaminating the NMN formed from noradrenaline and giving preferentially origin to MOPEG.