S. Guimarães

Different receptors for angiotensin II at pre- and postjunctional level of the canine mesenteric and pulmonary arteries

1 This investigation was undertaken to compare pre‐ and postjunctional receptors involved in the responses of the canine mesenteric and pulmonary arteries to angiotensin II. 2 In the mesenteric artery, angiotensin II caused an enhancement of tritium overflow evoked by electrical stimulation (EC30% = 5 nm), the maximal effect representing an increase by about 45%. Postjunctionally, angiotensin II caused concentration‐dependent contractions (pD2 = 8.57). Saralasin antagonized both pre‐ and postjunctional effects of angiotensin II, but it was more potent at post‐ than at prejunctional level (pA2 of 9.51 and 8.15, respectively), while losartan antagonized exclusively the postjunctional effects of angiotensin II (pA2 = 8.15). PD123319 had no antagonist effect either pre‐ or postjunctionally. 3 In the pulmonary artery, angiotensin II also caused an enhancement of the electrically‐evoked tritium overflow (EC30% = 1.54 nm), its maximal effect increasing tritium overflow by about 80%. Postjunctionally, angiotensin II caused contractile responses (pD2 = 8.52). As in the mesenteric artery, saralasin antagonized angiotensin II effects at both pre‐ and postjunctional level and it was more potent postjunctionally (pA2 of 9.58 and 8.10, respectively). Losartan antagonized only the postjunctional effects of angiotensin II (pA2 = 7.96) and PD123319 was ineffective. 4 It is concluded that in both vessels: (1) pre‐ and postjunctional receptors belong to a different subtype, since they are differently antagonized by the same antagonists; (2) postjunctional receptors belong to AT1 subtype, since they are blocked by losartan but not by AT2 antagonists; (3) prejunctional receptors apparently belong to neither AT1 or AT2 subtype since they are blocked by neither AT1 nor AT2 antagonists.

Crosstalk between presynaptic angiotensin receptors, bradykinin receptors and α2‐autoreceptors in sympathetic neurons: a study in α2‐adrenoceptor‐deficient mice

In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release‐enhancing effect when presynaptic α2‐autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with α2 antagonists. We now studied this operational condition in α2‐adrenoceptor‐deficient mice. Release of 3H‐noradrenaline was elicited by electrical stimulation. In tissues from wild‐type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. In tissues from mice lacking the α2A‐adrenoceptor (α2AKO) or the α2B‐adrenoceptor (α2BKO), the concentration–response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the α2C‐adrenoceptor (α2CKO) or both the α2A‐ and the α2C‐adrenoceptor (α2ACKO), the concentration–response curves were shifted to the same extent downwards. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in α2AKO and α2ACKO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven‐fold in WT and α2BKO tissues, three‐fold in α2AKO and α2CKO tissues, and two‐fold in α2ACKO tissues. Results confirm that angiotensin II and bradykinin require ongoing α2‐autoinhibition for the full extent of their release‐enhancing effect. Specifically, they require ongoing α2C‐autoinhibition. The peptide effects that remain in α2C‐autoreceptor‐deficient mice seem to be because of α2B‐autoinhibition. The results hence also suggest that in addition to α2A‐ and α2C‐ mouse postganglionic sympathetic neurons possess α2B‐autoreceptors.

Chloroethylclonidine irreversibly activates postjunctional alpha2-adrenoceptors in the dog saphenous vein

SummaryThis study was aimed at analysing the contractile response of the dog saphenous vein to chloroethylclonidine. At 37°C, chloroethylclonidine (0.1–100 μmol·1−1) caused along-lasting contraction in both proximal and distal segments of the dog saphenous vein, reaching 77.6 and 52.6% of the maximal response to phenylephrine, respectively. At 18°C, and in both segments, the maximal response to chloroethylclonidine was markedly reduced, whereas that to phenylephrine was not changed and that to UK-14,304 was enhanced. The response to chloroethylclonidine was unaffected by pretreatment with cocaine. Warming to 37°C caused contraction of strips which at 18°C had remained unresponsive to chloroethylclonidine, even if these strips were repeatedly washed before warming. At 18°C, chloroethylclonidine (100 μmol·1−1) did not alter the responses to UK-14,304 and phenylephrine.At 37°C, the contractile response to chloroethylclonidine was antagonized by yohimbine, rauwolscine and prazosin, with the potency rank yohimbine = rauwolscine > prazosin. Phenoxybenzamine (30 nmol · 1−1) displaced the concentration-response curve to chloroethylclonidine to the right and depressed its maximum. After phenoxybenzamine, yohimbine continued to be more effective than prazosin, which remained very potent.We concluded that: 1) the contractile response of the canine saphenous vein to chloroethylclonidine (both in the absence and in the presence of phenoxybenzamine) is predominantly alpha2-adrenoceptor-mediated since it is larger at the proximal than at the distal level of the vein and since it is more sensitive to yohimbine and rauwolscine than to prazosin; 2) the response to chloroethylclonidine and UK-14,304 are apparently due to activation of different alpha2-adrenoceptor subtypes, since prazosin was much more effective against chloroethylclonidine than against UK-14,304, and since at 18°C chloroethylclonidine “occupies” receptors without changing the response to UK-14,304; 3) there is a component of alphal-adrenoceptor stimulation in the response to chloroethylclonidine, since 30 nmol·1−1 phenoxybenzamine partly antagonized the effect of chloroethylclonidine; 4) since the responses to UK-14,304 and chloroethylclonidine are differently affected by cooling, there is some step (or steps) in the chain of events between the receptor and the final response, which is different in the two pathways.

The role of the endocardium in the facilitatory effect of bradykinin on electrically-induced release of noradrenaline in rat cardiac ventricle

1 The present investigation was undertaken to study the role of bradykinin in noradrenaline release from the ventricle of the rat induced by electrical stimulation. Slices of the left ventricle of adult Wistar rats with or without endocardium were previously loaded with 0.2 μm [3H]‐noradrenaline and washed out before electrical stimulation was applied. 2 Bradykinin (0.1–100 nM) concentration‐dependently increased tritium release evoked by electrical stimulation (EC50 = 3.5 (1.2–10.2) nM; n= 12). The angiotensin converting enzyme inhibitor, captopril (1 μm), which per se had no effect on tritium release, caused a marked enhancement of the bradykinin facilitatory effect, shifting the concentration‐response curve of bradykinin to the left by about one log unit. The compound Hoe 140, a selective inhibitor of B2‐bradykinin receptors, competitively antagonized the effect of bradykinin, indicating the involvement of these receptors in the action of bradykinin. 3 In endocardium‐free ventricle, bradykinin had no effect either in the absence or in the presence of captopril. 4 These results show that: (1) bradykinin is able to facilitate noradrenaline release evoked by electrical stimulation of the rat ventricle through activation of B2‐bradykinin receptors located on endocardial cells; (2) this action of bradykinin which is markedly potentiated by the inhibition of the angiotensin‐converting enzyme seems to be exerted through the release of some factor which is formed in the endocardium and diffuses into the myocardium where it acts.

The effectiveness of α2-adrenoceptor activation increases from the distal to the proximal part of the veins of canine limbs

1 The effectiveness of α1‐ and α2‐adrenoceptor activation was compared at different levels of the saphenous and cephalic vein of the dog in vitro. 2 Helically cut strips were used to determine concentration‐response curves to phenylephrine, noradrenaline, UK‐14, 304 (5‐bromo‐6‐(imidazoline‐2‐ylamino)‐quinoxaline) and B‐HT 920 (2‐amino‐6‐allyl‐5,6,7,8‐tetra‐hydro‐4H‐(thiazo)‐4,5‐d‐azepine). The effect of prazosin and yohimbine on these curves was also studied. 3 At the distal level, the maximum response to UK‐14,304 amounted to 33 and 50% of those to noradrenaline in the saphenous and cephalic vein, respectively, while at the proximal level the maximum response to UK‐14,304 amounted to 72 and 78% of those to noradrenaline, in the saphenous and cephalic vein, respectively. 4 In both vessels, the results obtained with B‐HT 920 were very similar to those for UK‐14,304. 5 The pD2 values for UK‐14,304 — which were identical at the three levels of both vessels — and the pA2 values for the antagonism exerted by either prazosin or yohimbine against the responses to UK‐14,304 indicate that the α2‐adrenoceptors are identical at the different levels of both vessels. 6 These results show that the effectiveness of α2‐adrenoceptor stimulation increases from the distal to the proximal regions of canine limb veins. Apparently, this is due to a greater density of α2‐adrenoceptors in the proximal regions. 7 Yohimbine is much more potent against phenylephrine distally than proximally in both vessels. However, after 30 nm phenoxybenzamine — a concentration which eliminates the vast majority of α1‐adrenoceptors without affecting α2‐adrenoceptors — yohimbine became equally potent at both levels, suggesting that the difference existing before phenoxybenzamine depended on α1‐adrenoceptors. Hence it is concluded that α1‐adrenoceptors in distal and proximal portions may differ.

Hypertension and enhanced β‐adrenoceptor‐mediated facilitation of noradrenaline release produced by chronic blockade of adenosine receptors

1 The study was undertaken to compare the β‐adrenoceptor‐mediated facilitation of noradrenaline release in the tail artery of vehicle‐treated rats and of rats rendered hypertensive by chronic administration of 1,3‐dipropyl‐8‐sulphophenylxanthine (DPSPX). Artery rings were loaded with [3H]‐noradrenaline, and five periods of electrical stimulation (1 Hz for 2 min) were applied. To eliminate the influence of prejunctional α2‐adrenoceptors, the tissues were pre‐exposed to 1 μm phenoxybenzamine. 2 Isoprenaline caused a concentration‐dependent increase of tritium overflow elicited by electrical stimulation. It was more effective in arteries from DPSPX‐treated than in those from vehicle‐treated rats; isoprenaline (27.8 nM) increased by 30% tritium overflow in vessels from vehicle‐treated rats whereas isoprenaline (7.0 nM) produced a 30% increase in vessels from DPSPX‐treated animals. Furthermore, the maximal effect of isoprenaline was a 32.6% increase in control rats but a 48.6% increase in DPSPX‐treated rats. 3 These results show that the sympathetic nerve endings of the rat tail artery are endowed with prejunctional β‐adrenoceptors which mediate facilitation of noradrenaline release elicited by electrical stimulation. They also suggest that adenosine receptors and β‐adrenoceptors interact at the prejunctional level and that impairment of this ‘talk’ may lead to the development of a hypertensive state.

Prejunctional α2A-autoreceptors in the human gastric and ileocolic arteries

This study was undertaken to determine the subtype of prejunctional α2-autoreceptors in human blood vessels. Segments of gastric and ileocolic arteries were incubated with [3H]noradrenaline and subsequently perifused with modified Krebs-Henseleit solution containing cocaine (12 µM). Five periods of electrical stimulation (S1–S5) were applied (1 Hz, 1 ms, 50 V for 1 min). Concentration-response curves for the facilitatory effect of eight α-adrenoceptor antagonists [rauwolscine, 2-[2-(2-methoxy-1,4-benzodioxanyl)] imidazoline (RX821002), yohimbine, phentolamine, idazoxan, 2-(2’,6’-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxan (WB4101), spiroxatrine and prazosin] were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, indicating the existence of α2-autoreceptors. The EC30% values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity to the autoreceptors. Correlations between the pEC30% values obtained in the present study and the pKi values of the same antagonists at cloned human α2A-, α2B-, α2C-adrenoceptors expressed in Chinese hamster lung cells and at α2D-adrenoceptors in the rat submaxillary gland or the bovine pineal gland showed that the α2-autoreceptors in the human gastric and ileocolic arteries resemble most closely the α2A-subtype.

Adenosine and the endothelium-dependent modulation of 3H-noradrenaline release in the canine pulmonary artery

This study aimed at characterizing the influence of endothelium on noradrenaline release from the canine pulmonary artery. Tritium overflow from intact or endothelium-free vessels preloaded with 0.2 μmol.1−13H-noradrenaline was evoked by electrical stimulation (1 Hz, during 5 min) or potassium (25–100 mmol. 1−1).The fractional release of tritium evoked by electrical stimulation was increased by removing the endothelium [from 1.7 (1.2; 2.4) to 2.7(2.3; 3.2) × 10−5. pulse−1, n = 10; P < 0.05]. Neither NG-nitro-l-arginine methyl ester (l-NAME) (up to 300 μmol.l−1) nor indomethacin (up to 30 μml.l−1), nor endothelin-1 (up to 30 nmol.l−1), nor suramin (up to 300 μmol.l−1) changed tritium release evolved by electrical stimulation. In contrast, the selective A1-adenosine antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (3.3-33 nmol.l−1) concentration-dependently increased, and the selective A1-adenosine agonist N6-cyclopentyladenosine (CPA) (3.3–100 nmol. l−1) concentration-dependently decreased the evoked release of noradrenaline. Since the effects of DPCPX were observed in endothelium-intact tissues only, it may be concluded that adenosine secreted by the endothelium activates prejunctional release-inhibiting A1-receptors. Tetraethylammonium (TEA) (3.3–33 mmol. l−1) enhanced tritium overflow evoked by electrical stimulation more in endothelium-free than in endothelium-intact vessels, indicating that some K+-channel opener is involved in the inhibitory role of endothelium on noradrenaline release. Since it had been previously shown that A1-adenosine receptors are coupled to K+-channels, it is suggested that adenosine may inhibit noradrenaline release through the opening of K+-channels.In conclusion, the results show that in the canine pulmonary artery, adenosine is a good candidate for the endothelium-dependent inhibitory factor which is responsible for the reduction of noradrenaline release evoked by electrical stimulation.

Long-term administration of 1,3-dipropyl-8-sulphophenylxanthine (DPSPX) alters α2-adrenoceptor-mediated effects at the pre- but not at the postjunctional level

The present investigation was undertaken to see whether a long-term inhibition of adenosine receptors —leading to hypertension — interferes with α2-adrenoceptor-mediated modulation of noradrenaline release. Rat tail arteries were removed from normal and from hypertensive animals obtained by chronic treatment with intraperitoneally infused DPSPX (1,3,-dipropyl-8-sulphophenylxanthine) or orally administered L-NAME (NG-Nitro-L-arginine methyl ester). To study prejunctional effects, the influence of UK-14,304 (5-bromo-6(imidazoline-2-ylamino)-quinoxaline) and yohimbine on the overflow of tritium evoked by electrical stimulation (100 V; 1 Hz; 2 ms; 5 min) from tissues preloaded with 3H-noradrenaline was analysed. To study postjunctional effects, concentration-response curves to UK-14,304 were determined. In DPSPX-treated rats there was an enhancement of the prejunctional effects of UK-14,304: its EC30% was reduced from 381 (250; 579) to 85 (73; 99) nmol.l−1 (n = 5; P<0.05) and its maximal effect — expressed as percent reduction of tritium overflow-increased from 45 ± 5% to 61 ± 5% (n = 6; P < 0.05). In L-NAME-treated rats there was no change in either of these two parameters. At the postjunctional level, there was no change in the sensitivity to UK-14,304 in tissues from either DPSPX- or L-NAME-treated rats. Yohimbine (10–1000 nmol.l−1) caused a concentration-dependent increase of tritium overflow evoked by electrical stimulation in both control and hypertensive animals (either DPSPX- or L-NAME-treated). The EC50%-pre-antagonist values (concentration of the antagonist that increases the evoked overflow by 50%) were not significantly different in the three situations. We conclude that long-term administration of DPSPX increases the sensitivity to the prejunctional effects of UK-14,304 without changing that to its postjunctional effects, showing a specific interaction between α-adrenoceptors and adenosine receptors at a prejunctional level. The question arises whether there is any link between that alteration and the development of the hypertensive state.

Release and disposition of 3H-noradrenaline in the saphenous vein of neonate and adult dogs

SummaryRelease of 3H-noradrenaline and formation of 3H-metabolites were studied in the saphenous vein of newborn (mean age, 18 h) and adult dogs. Vein strips were incubated with 0.23 μmol/l of 3H-noradrenaline during 1 h and washed out for 110 min; thereafter, the perifusion fluid was collected in 5-min samples. Electrical stimulation was applied at 120 min (1 Hz, 2 ms, 100 V, for 5 min). In some experiments the tissues were preincubated with 1 mmol/l pargyline (to inhibit monoamine oxidase). In these experiments, 12 μmol/l cocaine (to inhibit uptake1), 41 μmol/l hydrocortisone (to reduce uptake2) and 50 μmol/l U-0521 (to inhibit COMT) were present during the perifusion. 3H-noradrenaline, 3H-DOPEG, 3H-NMN, 3H-DOMA and 3H-OMDA were separated by column chromatography. The noradrenaline content of the tissue was estimated by HPLC followed by electrochemical detection. A morphological study was also carried out by light and electron microscopy.The endogenous noradrenaline content of the saphenous vein was 4.3 times higher in adults than in neonates. The number of varicosities was similar in adults and newborns but the number of vesicles per varicosity profile was 5 times higher in adults. Hence, the endogenous noradrenaline content per vesicle was about the same in adults and newborns. The accumulation of 3H-noradrenaline per vesicle was about 5 times higher in newborns than in adults. On the other hand, the vein wall media of neonates was about 3 times thinner than that of adults. The evoked fractional release of tritium was about 10 times higher in neonates than in adults, whether the inactivation pathways were blocked or not. This difference in the evoked fractional release therefore cannot be ascribed to any difference in the efficacy of the inactivation pathways between neonates and adults. On the other hand, the difference also cannot be ascribed to a different alpha2-adrenoceptor-mediated inhibition of 3H-noradrenaline release since, as previously shown, this mechanism is as effective in newborns as in adults.It is concluded that the evoked fractional release of 3H-noradrenaline is higher in neonates than in adults because in neonates the majority of varicosities (and vesicles) is situated closer to the surface of the tissue such that both uptake from the surrounding media into the stores and release from the stores into the surrounding media become easier in the neonates.