Nimodipine and inhibition of alpha adrenergic activation of the isolated canine saphenous vein

The vascular smooth muscle of the canine saphenous vein contains both postjunctional alpha-1 and alpha-2 adrenoceptors. Experiments were designed to elucidate the relationship between alpha adrenoceptor subtypes and sensitivity to calcium entry blockade. Rings of canine saphenous vein were mounted at optimal length for isometric tension recording in organ chambers filled with physiological salt solution. Nimodipine inhibited potassium-induced contractions and depressed contractions to norepinephrine in the presence of prazosin, an alpha-1 adrenoceptor antagonist, but not under control conditions or in the presence of the alpha-2 adrenoceptor antagonist, rauwolscine. Nimodipine depressed the maximal response to B-HT 920, an alpha-2 adrenergic agonist and St-587, a partial alpha-1 adrenergic agonist, but did not affect that to cirazoline, a full alpha-1 adrenergic agonist. However, after treatment with phenoxybenzamine, nimodipine depressed the response to cirazoline. Nimodipine inhibited contractions to tyramine under control conditions or after prazosin but not after rauwolscine. Incubation in calcium-free solution depressed responses to St-587 and B-HT 920 more than those to cirazoline. Incubation in calcium-free solution plus ethylene glycol bis(beta-aminoethyl ether)-N,N-tetraacetic acid abolished responses to all alpha adrenergic agonists. These results suggest that the sensitivity to calcium entry blockade of alpha adrenergic responses is not determined by the subtype of alpha adrenoceptor. Rather, our findings support the concept that it is the efficacy of the agonist-receptor interaction or the efficiency of receptor-response coupling that determines the effect of calcium entry blockade on the adrenergic response.     

Electrophysiological analysis of adrenergic neurotransmission and its modulation by chronic denervation in canine saphenous veins

The present experiments were undertaken to investigate the electrophysiological responses of the canine saphenous vein evoked by perivascular nerve stimulation, norepinephrine or selective alpha adrenergic agonists before and after chronic sympathetic denervation. Unilateral sympathectomy was performed from T12 to L9 in adult female dogs. After 3 to 5 weeks, the denervated saphenous veins were removed. Innervated saphenous veins were obtained from unoperated dogs. In innervated but not in denervated veins, electrical stimulation generated excitatory junction potentials and a slow depolarization. The slow depolarization was inhibited by rauwolscine or phentolamine, but not by prazosin, whereas excitatory junction potentials were not inhibited by alpha adrenergic blockers. Exogenously applied norepinephrine caused a depolarization of the membrane that was inhibited by rauwolscine but not by prazosin. The selective alpha-1 adrenergic agonist, phenylephrine, and the selective alpha-2 adrenergic agonist, UK 14,304, caused depolarization. In denervated veins, the threshold concentrations of norepinephrine or UK 14,304 required to depolarize the smooth muscle cell membrane were reduced. Responses to phenylephrine were not affected by denervation. These results indicate that in the canine saphenous vein norepinephrine, whether added exogenously or released from sympathetic nerves, causes predominant depolarization by activating alpha-2 adrenergic receptors. Denervation augments selectively the electrical response to alpha-2 adrenergic stimulation.     

Migraine therapy: relationship between serotonergic contractile receptors in canine and rabbit saphenous veins to human cerebral and coronary arteries

Canine and rabbit vascular contractile responses to serotonergic agonists have been used to predict antimigraine efficacy for several antimigraine agents, including sumatriptan. The purpose of the present study was to establish the assumed predictive value of contractile responses in canine and rabbit saphenous veins to contractile efficacy for a series of agonists in human cerebral and coronary arteries and to understand better the receptors mediating such responses. The canine and rabbit saphenous veins contracted similarly (both qualitatively and quantitatively) to series of structurally diverse serotonergic agonists, suggesting that the receptors mediating serotonin-induced contractility in these tissues were similar. In addition, the contractile potency (estimated as EC₅₀ values) for these structurally diverse serotonergic agonists in either the rabbit or canine saphenous vein significantly correlated with contractile potency for these agonists in human cerebral arteries. Thus, to the extent that contractile responsiveness of human cerebral arteries may predict antimigraine agents, contractile responses of the rabbit and/or canine saphenous vein may be useful surrogates for antimigraine efficacy. In addition, the contractile potency for this series of serotonergic agonists in the rabbit and/or canine saphenous vein significantly correlated with contractile potency of these agonists in human coronary arteries. These data suggest that the use of the saphenous vein to identify potent vasoconstrictors will also reveal agents capable of contracting human coronary arteries, a liability for using this approach to evaluate promising antimigraine therapies.     

Denervation augments alpha-2 but not alpha-1 adrenergic responses in canine saphenous veins

Experiments were performed in order to determine the influence of sympathetic denervation on alpha-1 and alpha-2 adrenergic responses in canine saphenous veins. In female dogs anesthetized with sodium pentobarbital, the left lumbar sympathetic chain was excised from L1 to L7. After a 3- to 5-week period, the left (denervated) and right (innervated) saphenous veins were removed, cut into rings and suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution. Denervation reduced significantly the norepinephrine content of the venous rings and the contractile responses evoked by the indirect sympathomimetic amine, tyramine. The contractile responses evoked by exogenous norepinephrine were augmented by denervation under control conditions (16.7-fold shift in concentration-effect curve) and also after inhibition of neuronal and extraneuronal uptake and beta adrenoceptors (3.8-fold shift in curve). Denervation increased the contractile responses evoked by the alpha-2 adrenergic agonist, UK 14,304 (5-fold shift in concentration-effect curve), but not those produced by the alpha-1 adrenergic agonist, phenylephrine. The selective augmentation of alpha-2 adrenergic responses by denervation may reflect the preferential innervation of alpha-2 adrenoceptors in the canine saphenous vein.     

Effect of cooling on alpha-1 and alpha-2 adrenergic responses in canine saphenous and femoral veins

Experiments were designed to determine the effects of cooling on alpha-1 and alpha-2 adrenergic responses in isolated canine veins. Rings of saphenous and femoral veins were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution, gassed with 95% O2 and 5% CO2. Cooling (from 37-24 degrees C) augmented contractions to norepinephrine in saphenous but caused depression in femoral veins. Cooling (to 24 degrees C) had no effect on alpha-1 adrenergic responses evoked by phenylephrine in saphenous veins but caused depression in femoral veins. Alpha-2 adrenergic responses produced by UK 14,304 were augmented by cooling in the saphenous but were virtually abolished by cooling in femoral veins. Cooling decreased the dissociation constant (i.e., increased affinity) of corynanthine for alpha-1 adrenoceptors in saphenous and femoral veins (approximately 3-fold), and the dissociation constant of rauwolscine for alpha-2 adrenoceptors in saphenous veins (approximately 7.5-fold). The influence of cooling on alpha adrenoceptor responsiveness was analyzed using computer-generated receptor-models. The results suggest that the differential sensitivity of cutaneous and deep blood vessels to cooling results from differences in efficiency of alpha-1 and alpha-2 adrenoceptor response coupling. In the saphenous vein, there is a large alpha-1 adrenoceptor reserve which buffers the alpha-1 adrenergic response from the inhibitory influence of cooling. This coupled with a cooling-induced increase in alpha-2 adrenoceptor affinity ensures that cooling augments the response to norepinephrine. In the femoral vein, there is no alpha-1 adrenoceptor reserve and cooling therefore depresses alpha-1 adrenergic responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gallamine and pancuronium inhibit pre- and postjunctional muscarine receptors in canine saphenous veins.

The influence of gallamine, pancuronium, suxamethonium and d-tubocurarine on adrenergic neurotransmission was studied in the isolated saphenous vein of the dog. Pancuronium but not the other muscle relaxants increased significantly the response to sympathetic stimulation and to exogenous norepipephrine; these increases were abolished after blockade of neuronal uptake by cocaine. Pancuronium and gallamine inhibited both the relaxation produced by lower doses of acetylcholine added during sympathetic stimulation (prejunctional effect) and the direct contractions evoked by high doses of the amine (postjunctional effect). In strips previously incubated with [3H]norepinephrine, gallamine had no effect on [3H]norepinephrine efflux in basal conditions and during sympathetic stimulation; it increased markedly the efflux evoked by sympathetic stimulation in the presence of acetylcholine confirming that gallamine inhibits the prejunctional effect of the latter on adrenergic transmission. When extrapolated to the intact organism, the present experiments indicate that gallamine and pancuronium augment the release of norepinephrine in vascular tissue under vagal control, which explains in part the cardiovascular effects of these muscle relaxants.     

Sodium pump stimulation by activation of two alpha adrenergic receptor subtypes in canine blood vessels

The effects of alpha-1 and alpha-2 selective adrenergic agents on sodium pump activity were investigated in intact canine femoral artery and saphenous vein by measuring ouabain-sensitive uptake of 86Rb. In both vessels, the alpha-1-selective agonist, phenylephrine, stimulated 86Rb uptake in a dose-dependent manner. The uptake was blocked by prazosin and yohimbine with the order of potency: prazosin greater than yohimbine. The alpha-2-selective agonist, clonidine, also stimulated 86Rb uptake in the saphenous vein but not in the femoral artery. The stimulation was blocked by prazosin and yohimbine with the order of potency: yohimbine greater than prazosin. The potency of phenylephrine to contract saphenous vein or femoral artery was the same as that for stimulation of ouabain-sensitive 86Rb uptake. Clonidine was 10-fold more potent as a contractile agonist than as a Na+ pump stimulant. It caused only a weak contraction in the femoral artery. Reducing extracellular sodium abolished the stimulation of 86Rb uptake by both phenylephrine and clonidine in saphenous vein. Subsequently it was shown that both agonists increased intracellular sodium levels and these increases were blocked by the alpha receptor antagonists, prazosin and yohimbine, with the same selectivity as was observed in the 86Rb uptake experiments. Sodium pump stimulation produced by both phenylephrine and clonidine was blocked by amiloride. These observations suggest that the activity of the vascular sodium pump can be regulated by both alpha-1 and alpha-2 adrenergic receptors and that the mechanism involves an influx of sodium, most likely through a stimulation of Na+/H+ exchange.     

Cooling and alpha adrenergic responses in the saphenous vein of the rabbit

Experiments were designed to determine the effects of cooling on alpha-1 and alpha-2 adrenergic responses in a cutaneous vein of the rabbit. Rings of saphenous vein were suspended in physiological salt solution for the recording of isometric force. Cooling (from 37-24 degrees C) caused no significant increase in force in quiescent rings. Similarly, the same degree of cooling had no significant effect on the response to exogenous norepinephrine (10(-9)-10(-5) M), whether under control conditions or in the presence of either the alpha-1 adrenergic antagonist prazosin (3 X 10(-7) M) or the alpha-2 adrenergic antagonist rauwolscine (10(-7) M). Contractions evoked by the alpha-1 adrenergic agonist phenylephrine were reduced, but those induced by the alpha-2 adrenergic agonist UK 14,304 (10(-9)-10(-5) M) were unaffected by the same degree of cooling. Cooling augmented the response elicited by electrical field stimulation of the sympathetic nerves, although only under conditions of alpha-1 or combined alpha-1 and alpha-2 adrenergic blockade. Data obtained with the sympathomimetic tyramine suggest that both alpha-1 and alpha-2 adrenoceptors are innervated in this blood vessel. Together, the present data suggest that the effects of acute cooling on the saphenous vein of the rabbit, unlike that of the dog, are not mediated by changes in the affinity of postjunctional alpha-2 adrenoceptors.     

Amplification of alpha adrenergic vasoconstriction in canine isolated mesenteric artery and vein.

The interactions between UK-14304 and other vasoconstrictor agents were investigated using isolated canine mesenteric vascular rings mounted in tissue baths for the measurement of isometric contraction. In the mesenteric artery, exposure to UK-14304 caused a small contraction, producing 8% of the KCl maximal response. In the presence of either 20 mM KCl or 10(-9) M endothelin-1, which had small contractile effects, UK-14304 produced a biphasic concentration-response curve; 10(-7) M rauwolscine inhibited the responses to low concentrations of UK-14304 and 10(-7) M prazosin blocked the responses to high concentrations of UK-14304. In the presence of 10(-8) M Bay K 8644, UK-14304 elicited a monophasic concentration-dependent contraction that was antagonized by 10(-7) M prazosin, not by 10(-7) M rauwolscine. In the mesenteric vein, UK-14304 elicited concentration-dependent contractions, producing 63% of the KCl maximal response. The lower part of the biphasic concentration-response curve was inhibited by 10(-7) M rauwolscine and the upper part of the curve was antagonized by 10(-7) M prazosin. The presence in the medium of 20 mM KCl, 10(-11) M endothelin-1 or 10(-9) M Bay K 8644, which increased resting tension less than 10% of the KCl maximal response, markedly enhanced the responses to UK-14304 primarily at concentrations lower than 10(-6) M. The enhancement of responses were prazosin (10(-7) M)-resistant and rauwolscine (10(-7) M)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between alpha adrenergic receptor occupation and contractile response: radioligand and physiologic studies in canine aorta

The relation between occupation of alpha-1 adrenergic receptors by an agonist and the resulting force was examined in the canine aorta in vitro. Methods of preparing membrane preparations for radioligand studies were improved to maximize the yield of receptors from strips of aortic wall. [3H]Prazosin was a reliable ligand, binding to a single class of receptors with a dissociation constant of 97 (+/- 10) pM. Agonists and antagonists displaced [3H]prazosin in a manner consistent with binding to a single class of sites, with Hill coefficients near unity. The rank order of drug potencies determined by competition with [3H]prazosin was characteristic of alpha adrenergic receptors of the alpha-1 subtype. Conventional measurements of maximum isometric force and dose-response relations for l-phenylephrine in strips of ascending aorta in vitro were followed by radioligand studies on homogenates made from the same strips. The number of receptors was 38.8 (+/- 2.9) fmol/mg of protein in the homogenate, corresponding to 580 (+/- 41) fmol/g of aortic wall, a value at least twice as high as any reported previously. Maximum active stress was 440 (+/- 23) g/cm2. Concentration-response curves to l-phenylephrine were relatively shallow, with a Hill coefficient of 0.63 and an ED50 of 1.1 (+/- 0.24) microM. The curve relating receptor occupation to response (force development) in the ascending aorta was consequently nonlinear; 50% of the maximum response occurred when only 10% of the receptors were occupied. Occupation of virtually all receptors (greater than or equal to 97%) was required to produce maximum response, suggesting an absence of "spare" receptors.     

Alpha-1 and alpha-2 adrenoceptor: response coupling in canine saphenous and femoral veins

The aim of the present study was to analyze alpha-1 and alpha-2 adrenoceptor response coupling in isolated canine blood vessels. Rings of saphenous and femoral veins and of femoral arteries were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution, gassed with 95% O2-5% CO2 and maintained at 37 degrees C. Dissociation constants for the alpha-1 adrenergic agonists, phenylephrine and cirazoline, and the alpha-2 adrenergic agonist, UK 14,304, were determined by analysis of concentration-effect curves to the agonists under control conditions and after partial inactivation of alpha adrenoceptors by phenoxybenzamine. The dissociation constant of phenylephrine for alpha-1 adrenoceptors in saphenous veins was approximately 10-fold higher than that obtained for the agonist in femoral arteries or femoral veins. Similarly the dissociation constant for cirazoline in the saphenous vein was higher than that obtained in other alpha-1 adrenergic systems. Dissociation constants were used to determine alpha adrenoceptor occupancy-response relationships. The alpha-1 adrenergic responses evoked by high intrinsic-efficacy agonists (cirazoline and phenylephrine) were associated with a very large receptor-reserve in the saphenous vein, but no, or only a limited receptor-reserve in the femoral vein. The dissociation constant for UK 14,304 in saphenous veins was significantly lower than that obtained for alpha-2 adrenergic stimulation by norepinephrine. There was no alpha-2 adrenoceptor reserve in the saphenous vein for these putative high intrinsic-efficacy agonists. The differences in receptor-reserve between alpha-1 adrenoceptors in canine saphenous and femoral veins and between alpha-1 and alpha-2 adrenoceptors in saphenous veins may help to explain the differential modulation of adrenergic responses in these blood vessels.     

Effects of CRL 41034 on the adrenergic neuroeffector interaction in the canine saphenous vein

Experiments were designed to determine the effect of CRL 41034, a buflomedil analogue, on the adrenergic responsiveness of canine veins. Rings of saphenous vein (without endothelium) were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution at 37 degrees C. CRL 41034 produced a concentration-dependent inhibition of the contractions evoked by the alpha adrenergic agonists norepinephrine, phenylephrine and UK 14304 which was insensitive to the blockade of neuronal uptake by cocaine. CRL 41034 was more potent in inhibiting the concentration-dependent contractions evoked by UK 14304 than those by phenylephrine and the antagonism it caused against the response to UK 14304 fulfilled the criteria for competitivity. CRL 41034, at 10(-5) M significantly depressed, and at 10(-4) M abolished the contractions induced by electrical stimulation of the adrenergic nerves and those evoked by the indirect sympathomimetic amine tyramine. Strips of canine saphenous vein were superfused after incubation with [3H] norepinephrine. During sympathetic nerve activation, CRL 41304 increased the stimulation-evoked overflow of [3H] norepinephrine and 3-methoxy-4-dihydroxyphenylglycol; in the presence of rauwolscine the compound only increased the stimulation-evoked overflow of 3,4-dihydroxyphenylglycol. These experiments suggest that the major vascular effects of CRL 41034 in canine veins are blockade of alpha 2-adrenoceptors on vascular smooth muscle, and inhibition of prejunctional alpha 2-adrenoceptors on adrenergic nerve endings.     

Subtypes of muscarinic receptors on adrenergic nerves and vascular smooth muscle of the canine saphenous vein

Experiments were designed to characterize pre- and postjunctional muscarinic receptors in the canine saphenous vein. In rings contracted by 2-Hz electrical stimulation, acetylcholine produced concentration-dependent relaxations that result from prejunctional inhibition of norepinephrine release from adrenergic nerve endings (prejunctional effect). In quiescent preparations, acetylcholine caused concentration-dependent contractions (postjunctional effect). Both responses to acetylcholine were inhibited in a competitive manner by atropine, pirenzepine and gallamine. The affinity of atropine for pre- and postjunctional muscarinic receptors was similar (pKB = 8.7 and 9.2, respectively). However, gallamine displayed higher affinity for the prejunctional than for the postjunctional receptor (pKB = 6.1 and 4.7, respectively), whereas the converse was true for pirenzepine (pKB = 6.5 and 8.1). The data support the presence in the canine saphenous vein of two muscarinic receptor subtypes, M1 postjunctionally and M2 prejunctionally, which are distinguishable by selective antagonists.     

Cholinergic and alpha adrenergic augmentation of histamine-induced contraction of canine airway smooth muscle

The effect of i.v. methacholine (MC) and norepinephrine (NE) in causing synergistic contraction of airways with histamine was studied in 31 anesthetized dogs. Dose-response studies were performed for individual agonists and then with agonists administered together for the same animal. The additive airway contractile response to i.v. MC and histamine and i.v. NE and histamine was less than that obtained for the same doses of these agonists administered by i.v. injection together in the same syringe. Synergism was observed for 0.005 microgram/kg to 5.0 microgram/kg i.v. of MC and 0.0025 microgram/kg i.v. of histamine, as measured by change in dynamic compliance (P less than 0.1), airway resistance (P less than .001), isometric tracheal smooth muscle tension (P less than .01) and duration of the tracheal contractile response (P less than 0.005). Synergism was also observed for 0.002 microgram/kg to 2.0 microgram/kg of NE and 0.0025 microgram/kg to 2.5 microgram/kg of histamine i.v. for airway resistance and dynamic compliance (P less than .025), isometric tracheal contraction (P less than .05) and duration of tracheal contraction (P less than .01). No augmentation was observed for seven animals receiving NE and MC at any dose nor was the duration of tracheal contraction to these combined agonists greater than additive. The addition of histamine to cholinergic or alpha adrenergic stimulation results in synergistic contraction of airways and augmentation of the duration of contractile response. This effect is specific for histamine, since no augmentation is seen when MC and NE are administered together.     

Pertussis toxin reduces endothelium-dependent and independent responses to alpha-2- adrenergic stimulation in systemic canine arteries and veins.

A pertussis toxin-sensitive guanine nucleotide regulatory protein (G-protein) is involved in the signal transduction of certain endothelium-dependent responses in mammalian arteries. To determine whether a similar mechanism mediates endothelium-dependent responses in mammalian veins, rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers. In femoral arteries, incubation of the rings with pertussis toxin (from Bordetella pertussis, 100 ng/ml for 2 hr) in the presence of indomethacin and propranolol did not reduce significantly endothelium-dependent relaxations to acetylcholine and adenosine diphosphate, thrombin or the calcium ionophore A23187. However, endothelium-dependent relaxations evoked by the alpha-2 adrenergic agonist UK 14,304 were blocked by the pertussis toxin. In venous rings, endothelium-dependent relaxations to acetylcholine were reduced by the toxin, whereas the endothelium-dependent relaxations evoked by adenosine diphosphate, thrombin and A23187 were not affected. UK 14,304 contracted the veins; these contractions were augmented by removal of the endothelium. Pertussis toxin inhibited contractions to UK 14,304 in venous rings without but not with endothelium. Relaxations of arterial and venous smooth muscle to nitric oxide were unaffected by the toxin. Contractions to phenylephrine were not altered by either removal of the endothelium or the toxin in the arteries or veins. These results suggest that the release of endothelium-derived relaxing factor in response to stimulation of purine and thrombin receptors probably does not involve a pertussis toxin-sensitive G-protein in canine femoral arteries or veins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two subtypes of alpha adrenergic receptors in canine airway smooth muscle

The contractile response of canine tracheal muscle to i.a. phenylephrine, clonidine and norepinephrine was studied isometrically in situ in 32 dogs after beta adrenergic and ganglionic blockade. Intra-arterial phenylephrine caused dose-related tracheal contraction which was not altered by yohimbine (5 microgram/kg i.a.). Prazosin (4 mg/kg i.v.) caused a 77 +/- 3% decrease in tracheal response to i.a. phenylephrine. Clonidine also caused dose-related tracheal contraction, which was not altered by prazosin (4 mg/kg i.v.) but was 95 +/- 2% blocked by 5 microgram/kg i.a. of yohimbine. Norepinephrine caused tracheal muscle contraction which was greater than both phenylephrine (P less than .05) and yohimbine (P less than .001). Prazosin (4 mg/kg i.v.) caused 53 +/- 6% blockade and yohimbine (5 microgram/kg i.a.) caused 76 +/- 2% blockade of the response to i.a. norepinephrine; prazosin plus yohimbine caused greater than 98% blockade of the response to i.a. norepinephrine. The dose-response curve to i.a. acetylcholine was not altered by treatment with prazosin (4 mg/kg i.v.) plus yohimbine (5 microgram/kg i.a.). These results demonstrate that tracheal contraction induced by sympathomimetic amines is mediated by two subtypes of alpha adrenergic receptors on tracheal muscle, alpha-1 for phenylephrine, alpha-2 for clonidine and both alpha-1 and alpha-2 for norepinephrine.     

Alpha adrenergic and serotonergic agents in the treatment of spastic hypertonia.

In the treatment of patients with problematic spasticity, it is important to consider the following steps: 1. Establish the functional impact of the spasticity. 2. Identify the functional goal to be achieved by treatment. 3. Eliminate any remediable spasticity aggravating factors. 4. Evaluate the effects of previous antispasticity treatments. 5. Consider nonpharmacologic and pharmacologic treatments. 6. Initiate therapy with a low dosage, and titrate judiciously. 7. Stop the titration when functional goal is achieved. 8. If goal is not achieved or if side effects are intolerable, consider a second medication.