Interference of transmethylation inhibitors with thromboxane synthesis in rat platelets

In order to determine whether a methylation reaction is involved in the platelet metabolism of arachidonic acid (AA), we investigated the effect of the transmethylase inhibitors 3-deazaadenosine (DZA) and L-homocysteine-thiolactone (Hcy) on the production of immunoreactive thromboxane (TX) B2 by rat platelets. Incubation for at least one hour of the plateletrich plasma with DZA and Hcy led to an inhibition of TX synthesis induced by collagen (5 μg.ml^?1). Platelets in plasma were then preincubated for 4 hours with DZA (10^?3M) in association with Hcy(5×10^?4M), washed, resuspended in buffer, and stimulated with 3 different activators. The formation of TXB2 in response to collagen (25 μg.ml^?1) was markedly reduced, whereas no inhibition occurred when AA (5×10^?6M) or the calcium ionophore A 23,187 (5×10^?6M) were used. In addition labelled AA was incorporated into the platelet phospholipids (PL). Its release induced by collagen (25 μg.ml^?1) was inhibited when platelets were preincubated with DZA and Hcy under the same experimental conditions. By contrast, the release of AA induced by A 23187 (10^?6M) was unaffected. This results strongly suggest the association of a methylation reaction with platelet activation, at a calcium-independent step of endogenous AA metabolism, before the cyclo-oxygenase level. Its precise biochemical nature remains to be determined.     

In vitro effects of synthetic antioxidants and vitamin E on arachidonic acid metabolism and thromboxane formation in human platelets and on platelet aggregation

The “in vitro” effects of α-tocopherol, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were studied on aggregation of human platelets induced by collagen and arachidonic acid (AA), on the metabolic conversion of ¹⁴C AA through the cyclooxygenase and lipoxygenase pathways and on the formation of thromboxane B₂ (TXB₂) in washed platelets after stimulation with collagen.Vitamin E completely inhibited AA induced platelet aggregation only at high concentration (mM) and after 10 minutes of preincubation, with limited effects on AA metabolism in platelets and no effect on TXB₂ formation from endogenous substrate. BHA completely inhibited platelet aggregation in the 10⁻⁶M range, gave 50% inhibition of AA metabolism in the 10⁻⁵M range and almost complete inhibition of thromboxane formation in the 10⁻⁴M range. BHT was about 100 times less active on platelet aggregation and AA metabolism. The lipoxygenase and cyclooxygenase pathways were differentially affected at low concentrations of BHA and only at concentrations greater than 5×10⁻⁵M were both pathways depressed.     

Effects of extracellular Ca2+ and the intracellular Ca2+ antagonist 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate on rabbit platelet conversion of arachidonic acid into thromboxane

The regulatory role of Ca²⁺ on the conversion of arachidonic acid (AA) into thromboxane B₂ (TXB₂) was examined in washed rabbit platelets, whose secretoary processes are known to have requirements for extracellular CA²⁺. Varying the extracellular free Ca²⁺ [Ca_f²⁺] concentration from < 10^?8 to 10^?3 M had no significant effect on the synthesis of immunoreactive TXB₂ by rabbit platelets incubated with 1–4 μM AA. On the other hand, 8-(N,N-diethylamino) octyl-3,4,5- trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ movement, inhibited AA-stimulated synthesis of TXB₂ in a concentration dependent manner--an effect which could be partially overcome by increasing the AA concentration. The TMB-8 inhibition could not be reversed by increasing the [Ca²⁺_f]. Studies examining platelet metabolism of ¹⁴C-AA and ¹⁴C-prostaglandin H₂ demonstrated that TMB-8 inhibited platelet cyclooxygenase, but not thromboxane synthetase. These studies demonstrate the absence of a requirement for [Ca²⁺_f] but suggest the presence of a TMB-8 sensitive intracellular Ca²⁺ pool in the rabbit platelet synthesis of TXB₂ from AA.     

Effects of VA-045, a novel apovincaminic acid derivative,on isolated blood vessels: Cerebroarterial selectivity

We investigated the effects of VA-045, an apovincaminic acid derivative, on isolated blood vessels. VA-045 (10⁻⁷−10⁻⁵M) and vinpocetine (10⁻⁷−10⁻⁵M) inhibited the 64 mM KCl-induced and 10⁻⁶M norepinephrine (NE)-induced contraction of rat aortic strips. VA-045 (10⁻⁷−10⁻⁴M) and vinpocetine (10⁻⁷−10⁻⁴M) inhibited the activity of cyclic AMP and cyclic GMP phosphodiesterase in porcine coronary artery. VA-045 (3×10⁻⁹−3×10⁻⁶M) relaxed the 64 mM KCl-induced contraction of the canine basilar artery without affecting the peripheral arteries. These results indicate that VA-045 selectively dilates canine cerebral artery, and that it may be a useful agent for the treatment of cerebrovascular diseases such as stroke.     

Effect of local anesthetics on plasma protein secretion by rat hepatocytes

The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with l-[¹⁴C]leucine for 9 min at 37°C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpomazine, (3 · 10^?5 M), dibucaine (10^?5 M), lidocaine (10^?3 M) and procaine (5 · 10^?5 M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of l-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phopholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, howover, procaine did not cause the retained plasma proteins to accumulate in Goli-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.     

Contractile activities of leukotrienes C4 and D4 on vascular strips from rabbits

Leukotrienes C₄ (LTC₄) and D₄ (LTD₄), major components of slow-reacting substances of anaphylaxis (SRS-A), caused dose-dependent contractions of rabbit coronary arteries in concentrations of 10^?9 to 10^?7 M and 10^?10 to 10^?7 M, respectively. The potency of LTC₄ and LTD₄, when compared with the concentration that elicits half of the contraction induced by 25 mM KCl, was 17 and 76 times, respectively, greater than that of histamine. In contrast, other blood vessels from rabbits were either unresponsive (renal artery and vein, mesenteric artery and thoracic aorta) or only weakly responsive (pulmonary artery and vein and portal vein) to both leukotrienes even at 10^?7 M. The LTD₄-induced coronary contraction was inhibited by FPL 55712 (10^?7 and 10^?6 M), a selective SRS-A inhibitor, in a dose-dependent manner, but not by diphenhydramine (10^?7 M), a histamine H₁-receptor blocker or by indomethacin (10^?5 M), a prostaglandin synthetase inhibitor, suggesting that LTD₄ has a direct effect on the coronary arteries. These results indicate that the leukotrienes may act as potent, selective coronary vasoconstrictors and that SRS-A responsive receptors exist in the rabbit coronary artery.     

Contractile responses to rat urotensin II in resting and depolarized basilar arteries

The effects of human urotensin II (hUII) on the vascular tone of different animal species has been studied extensively. However, little has been reported on the vasoactive effects of rat urotensin (rUII) in murine models. The aim of the present study was to investigate the effects of rUII on vasoreactivity in rat basilar arteries. Basilar arteries from adult male Wistar rats (300–350 g) were isolated, cut in rings, and mounted on a small vessel myograph to measure isometric tension. rUII concentrations were studied in both resting and depolarized state. To remove endothelial nitric oxide effects from the rUII response, we treated selected arterial rings with N_ω-nitro-L-arginine methyl ester (L-NAME). 10 μM rUII produced a potent vasoconstrictor response in rat basilar arteries with intact endothelium, while isometric forces remained unaffected in arterial rings treated with lower rUII concentrations. Although L-NAME did not have a significant effect on 10 μM rUII-evoked contraction, it slightly increased arterial ring contraction elicited by 1 μM rUII. In depolarized arteries, dose-dependent rUII increased depolarization-induced contractions. This effect was suppressed by L-NAME. Our results show that the rat basilar artery has a vasoconstrictor response to rUII. The most potent vasoconstrictor effect was produced by lower doses of rUII (0.1 and 1 μM) in depolarized arteries with intact endothelium. This effect could facilitate arterial vasospasm in vascular pathophysiological processes such as subarachnoid hemorrhage and hypertension, when sustained depolarization and L-type Ca²⁺ channel activation are present.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Effects of prostacyclin on the canine isolated basilar artery

Prostacyclin (PGI₂) produced a biphasic response in canine isolated basilar arteries. In low doses (1 × 10^?8M?1 × 10^?7M) PGI₂ caused a slight but consistent relaxation of resting muscle tone. In low concentrations (1 × 10^?8M?1 × 10^?6M) PGI₂ antagonized muscle contractions caused by serotonin or prostaglandin (PG) F_2α. This relaxant effect with low doses of PGI₂ on the isolated cerebral artery contrasts with findings obtained with other PGs and supports the hypothesis that PGI₂ is a mediator of vasodilatation. However, in 1 × 10^?5M concentrations PGI₂ contracted the arterial muscle and did not antagonize contractions induced by serotonin or PGF_2α.     

Manipulation of chloride flux affects histamine-induced contraction in rabbit basilar artery

Cl(-) efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl(-) flux in histamine-induced contraction in the rabbit basilar artery. Male New Zealand White rabbits (n = 16) weighing 1.8-2.5 kg were euthanized by an overdose of pentobarbital sodium. The basilar arteries were removed for isometric tension recording. Histamine produced a concentration-dependent contraction that was attenuated by the H(1) receptor antagonist chlorpheniramine (10(-8) M) but not by the H(2) receptor antagonist cimetidine (3 x 10(-6) M) in normal Cl(-) Krebs-Henseleit bicarbonate solution (123 mM Cl(-)). The histamine-induced contraction was reduced by the following manipulations: 1) inhibition of Na(+)-K(+)-2Cl(-) cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free HEPES solution to disable Cl(-)/HCO exchanger, and 3) blockade of Cl(-) channels with the use of niflumic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, and indoleacetic acid 94 R-(+)-methylindazone. In addition, substitution of extracellular Cl(-) (10 mM) with methanesulfonate acid (113 mM) transiently enhanced histamine-induced contraction. Manipulation of Cl(-) flux affects histamine-induced contraction in the rabbit basilar artery.     

Optimization of an assay for studying the effects of agents on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in washed human platelets

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (10(8) platelets/0.5 ml) incubated for various times with 2 microCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10(-7) to 10(-4) M AA and found that the cyclooxygenase was saturated with 10(-5) M AA whereas the lipoxygenase was not saturated with 10(-4) M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10(-5) M AA, while lipoxygenase products predominated at 3 x 10(-5)-10(-4) M AA. Using 3 microM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 microM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10(-4) M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Cl- current in endothelin-1-induced contraction in rabbit basilar artery

Cl- efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl- channels in endothelin-1 (ET-1)-induced contraction in rabbit basilar artery. Male New Zealand White rabbits (n = 26), weighing 1.8-2.5 kg, were euthanized by an overdose of pentobarbital. The basilar arteries were removed for isometric tension recording. ET-1 produced a concentration-dependent contraction of the rabbit basilar artery in the normal Cl- Krebs-Henseleit bicarbonate buffer (123 mM Cl-). The ET-1-induced contraction was reduced by the following manipulations: 1) inhibition of Na+-K+-2Cl- cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free solution to disable Cl-/HCO exchanger, and 3) preincubation of rings with the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and indanyloxyacetic acid 94. The ET-1-induced contraction was enhanced by substitution of extracellular Cl- (10 mM) with methanesulfonic acid (113 mM). Cl- channels are involved in ET-1-induced contraction in the rabbit basilar artery.     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β-estradiol on isolated canine basilar and internal carotid arteries

Progesterone and 17β-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17β-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17β-estradiol on KCl- and/or PGF_2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl₂ was also determined. In both arteries progesterone (5.6–180 μM) and 17β-estradiol (1.8–180 μM): (1) produced concentration-dependent relaxations of KCl- or PGF_2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM), potassium channel blockers and ICI 182,780 (only for 17β-estradiol). In the basilar artery the vasorelaxation induced by 17β-estradiol was slightly blocked by tetraethylammonium (10 mM) and glibenclamide (K_ATP; 10 μM). In both arteries, progesterone (10–100 μM), 17β-estradiol (3.1–31 μM) and nifedipine (0.01–1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM–10 mM). These results suggest that progesterone and 17β-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca²⁺ channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17β-estradiol in the internal carotid artery; meanwhile K_ATP channels play a minor role on the effect of 17β-estradiol in the basilar artery.     

Adrenergic response of splanchnic arteries from cirrhotic patients: role of nitric oxide, prostanoids, and reactive oxygen species

Peripheral and splanchnic vasodilatation in cirrhotic patients has been related to hyporesponsiveness to vasoconstrictors, but studies to examine the vascular adrenergic response provide contradictory results. Hepatic arteries from cirrhotic patients undergoing liver transplantation and mesenteric arteries from liver donors were obtained. Segments 3 mm long from these arteries were mounted in organ baths for testing isometric adrenergic response. The concentration-dependent contraction to noradrenaline (10(-8) to 10(-4) M) was similar in hepatic and mesenteric arteries, and prazosin (alpha 1-adrenergic antagonist, 10(-6) M), but not yohimbine (alpha 2-adrenergic antagonist, 10(-6) M), produced a rightward parallel displacement of this contraction in both types of arteries. Phenylephrine (alpha 1-adrenergic agonist, 10(-8) to 10(-4) M) and clonidine (alpha 2-adrenergic agonist, 10(-8) to 10(-4) M) also produced concentration-dependent contractions that were comparable in hepatic and mesenteric arteries. The inhibitor of cyclooxygenase meclofenamate (10(-5) M), but not the inhibitor of nitric oxide synthesis N(w)-nitro-l-arginine methyl ester (l-NAME, 10(-4) M), potentiated the response to noradrenaline in hepatic arteries; neither inhibitor affected the response to noradrenaline in mesenteric arteries. Diphenyleneiodonium (DPI; 5 x 10(-6) M), but neither catalase (1000 U/ml) nor tiron (10(-4) M), decreased the maximal contraction for noradrenaline similarly in hepatic and mesenteric arteries. Therefore, it is suggested that, in splanchnic arteries from cirrhotic patients, the adrenergic response and the relative contribution of alpha 1- and alpha 2-adrenoceptors in this response is preserved, and prostanoids, but not nitric oxide, may blunt that response. Products dependent on NAD(P)H oxidase might contribute to the adrenergic response in splanchnic arteries from control and cirrhotic patients.     

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells

The effects of tumor necrosis factor alpha (TNF-α) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [³H]AA. TNF-α differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-α (10⁻⁸ M) significantly increased the cyclooxygenase pathway, with prostaglandin E₂ (PGE₂) being a major product. However, at the lowest concentration (10⁻¹⁰ M) of TNF-α, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-α was increased to 10⁻⁹ M leukotriene B₄ (LTB₄), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10⁻⁶ M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-α (10⁻⁸ M) and A23187 (10⁻⁶ M) caused an inhibitory effect on each agent-induced PGE₂ or 15-HETE production.     

Effects of diabetes on the vascular response to nitric oxide and constrictor prostanoids: gender and regional differences

To analyze the effects of diabetes mellitus on the vascular responsiveness to nitric oxide and thromboxane receptor stimulation, 2 mm long segments of basilar, coronary, renal and tail arteries from male and female, control (normoglycemic) and streptozotocin-induced diabetic rats, were prepared for isometric tension recording. In the segments at basal resting tension, the thromboxane analog U46619 (10(-9)-10(-5) M) produced concentration-dependent contraction, which was similar in arteries from male and female rats, and was reduced by diabetes in coronary arteries from male and in tail arteries from female rats. In the vascular segments precontracted with endothelin-1 (10(-9) M), acetylcholine (10(-9)-3 x 10(-5) M) produced concentration-dependent relaxation which was similar in all arteries from normoglycemic male and female rats, and was increased by diabetes in tail arteries from female, but not in those from male rats. In precontracted segments the nitric oxide donor sodium nitroprusside (10(-10)-10(-5) M) also produced concentration-dependent relaxation, which was higher in basilar arteries from normoglycemic females compared with males, and was increased by diabetes in tail arteries from female but not from male rats. These results suggest that diabetes may increase the relaxation to nitric oxide in tail arteries, and may reduce the contraction to thromboxane receptor activation in coronary and tail arteries in a gender-dependent way. These changes in vascular reactivity may be adaptative to the vascular alterations produced by diabetes.     

Inhibition of cyclooxygenase-2 and EP1 receptor antagonism reduces human colonic longitudinal muscle contractility in vitro

We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP₁ receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10^?5 M) or diclofenac (10^?6 M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP₁ receptor antagonist SC19220 (10^?5 M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10^?6 M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE₂ acting at EP₁ receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.     

A novel response to propranolol: Contractile response in the isolated rabbit ear artery

Propranolol caused a contractile response in the isolated rabbit ear artery (EA). The concentration of propranolol causing a threshold contraction was 1.76 × 10^?6M while that causing a maximal contraction of 2.2 ± 0.18 g was 3 × 10^?5M. Higher concentrations caused tissue relaxation. Phentolamine, 10^?7 and 10^?6M reduced the propranolol-induced contractions by 50% and 90%, respectively while prazosin, 10^?8, 10^?7 and 10^?6M caused reductions of 54, 74 and 88%, respectively. Reserpinization of the rabbit with 5 mg/kg 24 hours before use eliminated the EA contractile response to tyramine but had no effect on that to propranolol. Desmethylimipramine plus deoxycorticosterone acetate enhanced the submaximal contraction of the EA to propranolol. $\text{In vitro}$ denervation with 6-hydroxydopamine (6-OHDA) decreased the response of the EA to tyramine and propranolol by 96% and 85% respectively but increased that to norepinephrine (NE) by 11%. Rabbit thoracic aorta (TA) did not respond to propranolol. In EA contracted with vasopressin o or 30 mM potassium, propranolol 10^?4 and 3 × 10^?4M caused a 20% and 100% relaxation, respectively. It is concluded that propranolol elicits a contractile response in the EA, at least in part, by direct activation of postsynaptic alpha adrenoceptors.     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.