Enrique F. Castillo

Pharmacological profile of the receptors that mediate external carotid vasoconstriction by 5-HT in vagosympathectomized dogs.

1 5‐Hydroxytryptamine (5‐HT) can produce vasodilatation or vasoconstriction of the canine external carotid bed depending upon the degree of carotid sympathetic tone. Hence, external carotid vasodilatation to 5‐HT in dogs with intact sympathetic tone is primarily mediated by prejunctional 5‐HT1‐like receptors similar to the 5‐HT1D subtype, which inhibit the carotid sympathetic outflow. The present investigation is devoted to the pharmacological analysis of the receptors mediating external carotid vasoconstriction by 5‐HT in vagosympathectomized dogs. 2 Intracarotid (i.c.) infusions for 1 min of 5‐HT (0.3, 1, 3, 10, 30 and 100 μg) resulted in dose‐dependent decreases in both external carotid blood flow and the corresponding conductance; both mean arterial blood pressure and heart rate remained unchanged during the infusions of 5‐HT. These responses to 5‐HT were resistant to blockade by antagonists at 5‐HT2 (ritanserin) and 5‐HT3/5‐HT4 (tropisetron) receptors, but were partly blocked by the 5‐HT1‐like and 5‐HT2 receptor antagonist, methiothepin (0.3 mg kg−1); higher doses of methiothepin (1 and 3 mg kg−1) caused little, if any, further blockade. These methiothepin (3 mg kg−1)‐resistant responses to 5‐HT were not significantly antagonized by MDL 72222 (0.3 mg kg−1) or tropisetron (3 mg kg−1). 3 The external carotid vasoconstrictor effects of 5‐HT were mimicked by the selective 5‐HT1‐like receptor agonist, sumatriptan (3, 10, 30 and 100 μg during 1 min, i.c.), which produced dose‐dependent decreases in external carotid blood flow and the corresponding conductance; these effects of sumatriptan were dose‐dependently antagonized by methiothepin (0.3, 1 and 3 mg kg‐1), but not by 5‐HT1D‐like receptor blocking doses of metergoline (0.1 mg kg−1). 4 The above vasoconstrictor effects of 5‐HT remained unaltered after administration of phentolamine, propranolol, atropine, hexamethonium, brompheniramine, cimetidine and haloperidol, thus excluding the involvement of α‐ and β‐adrenoceptors, muscarinic, nicotinic, histamine and dopamine receptors. Likewise, inhibition of either 5‐HT‐uptake (with fluoxetine) or cyclo‐oxygenase (with indomethacin), depletion of biogenic amines (with reserpine) or blockade of calcium channels (with verapamil) did not modify the effects of 5‐HT. 5 Taken together, the above results support our contention that the external carotid vasoconstrictor responses to 5‐HT in vagosympathectomized dogs are mainly mediated by activation of sumatriptan‐sensitive 5‐HT1‐like receptors. It must be emphasized, notwithstanding, that other mechanisms of 5‐HT, including an interaction with a novel 5‐HT receptor (sub)type and/or an indirect action that may lead to the release of a known (or even unknown) neurotransmitter substance cannot be categorically excluded.

Does segmental difference in α1-adrenoceptor subtype explain contractile difference in rat abdominal and thoracic aortae?

The cyclooxygenase inhibitor, indomethacin, depresses adrenergic agonist constriction of endothelium-denuded rat abdominal, but not thoracic, aorta. In order to explain this finding, we explored the possibility of segmental differences in the population of alpha 1-adrenoceptor (AR) subtypes. In endothelium-denuded tissues, phenylephrine elicited concentration-dependent contractions in the thoracic and abdominal aortic rings with potencies and maximal effects that, respectively, did not differ significantly (P > .05). Indomethacin (1 x 10(-5) M) inhibited phenylephrine-induced contractions only in abdominal aorta. The subtype-selective alpha 1D-AR antagonist, BMY 7378, was found to antagonize contractions to phenylephrine competitively in abdominal (pA2 8.44) and thoracic (pA2 8.56) aortic rings. These data are consistent with published alpha 1D-AR functional potency and clonal alpha 1D-AR binding affinity. In addition, cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat abdominal and thoracic aortae by prazosin, 5-methylurapidil and WB 4101, with pA2 values of 9.39 and 9.61, 7.64 and 7.85, and 9.43 and 9.58, respectively. These compounds with varying degrees of subtype selectivity inhibited contractions of the thoracic and abdominal aortae with affinities consistent with those determined at the alpha 1D-AR subtype. The results of this study suggest that the contraction to phenylephrine of the rat abdominal and thoracic aorta is mediated via the same alpha 1D-AR subtype.

α1D‐Adrenoceptors contribute to the neurogenic vasopressor response in pithed rats

Summary— The aim of the present study was to assess the role of vascular α1D‐adrenoceptors in the sympathetic vasopressor response in vivo. Specifically, we evaluated the effect of a selective α1D‐adrenoceptor antagonist, BMY 7378 (8‐(2‐(4‐(2‐methoxyphenyl)‐1‐piperazinyl)ethyl)‐8‐azaspiro(4,5)decane‐7,9‐dione 2HCI), on the vasopressor response induced by preganglionic (T7‐T9) sympathetic stimulation in the pithed rat. The vasopressor response was dose‐dependently sensitive to inhibition by intravenous BMY 7378 (0.1, 0.31, 1 and 3.1 mg/kg), doses of 1 and 3.1 mg/kg being equally effective. Like BMY 7378, 5‐methylurapidil (0.1, 0.31, 1 and 3.1 mg/kg) antagonized the vasopressor response to spinal stimulation; doses of 1 and 3.1 mg/kg were also equally effective. In combination experiments, BMY 7378 (1 mg/kg, iv) and the α1A‐adrenoceptor antagonist, 5‐methylurapidil (1 mg/kg, iv), showed an additive effect. The present results demonstrate that the α1D‐adrenoceptor subtype plays an important role in the pressor response to sympathetic nerve stimulation in the pithed rat, and confirm the participation of the α1A‐adrenoceptor subtype in the same response.

Participation of K+ Channels in the Endothelium-Dependent and Endothelium-Independent Components of the Relaxant Effect of Rosuvastatin in Rat Aortic Rings

Rosuvastatin was tested on rat aortic rings in the presence and absence of K+ channel blockers, mevalonic acid, and inhibitors of nitric oxide, prostaglandins, or endothelial-derived hyperpolarizing factor synthesis. The direct vascular effects of rosuvastatin were then evaluated by obtaining dose—response curves. Rosuvastatin relaxed aortic rings with and, to a lesser degree, without endothelium. Under both these conditions this effect was partially inhibited by L-NAME, tetraethylammonium, apamin + charybdotoxin (only administered together), or mevalonic acid. The combination of L-NAME with any of the other 3 treatments completely inhibited the effect of rosuvastatin, but indomethacin, clotrimazol, glibenclamide, charybdotoxin, or apamin alone had no effect. Therefore, the relaxation induced by rosuvastatin, even in the absence of endothelium, is partially related to 2 different mechanisms, one that is isoprenoid dependent and NO mediated and the other that is tied to the opening of Ca 2+-dependent K+ channels of the slow subfamily.

Evidence against α2-adrenoceptors mediating relaxation in rat thoracic aortae : α2-agonists relaxation depends on interaction with α1-adrenoceptors

In rat aorta, the presence of functional α2‐adrenoceptors (α2‐AR) was investigated in ring preparations preconstricted with α1‐adrenergic and non‐ α1‐adrenergic agonists. Particularly, the hypothetical interference of α2‐AR agonists with α1‐AR‐mediated vasoconstriction was evaluated. Relaxant and contractile responses to α2‐AR agonists were obtained. In endothelium‐intact and endothelium‐denuded aortic rings preconstricted with phenylephrine (1 × 10−6 m), the imidazoline derivatives, clonidine and UK14304, induced relaxations with similar order of potencies (−log EC50) and maxima relaxant effects respectively. Pretreatment with the NO synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME) had no effect on the relaxant responses to clonidine and UK14304. In phenylephrine‐constricted rings with endothelium, relaxations to clonidine and UK 14304 were not antagonized by the selective α2‐AR antagonist, rauwolscine (≤1 × 10−6 m). Clonidine and UK 14304 induced only contractions on endothelium‐intact and endothelium‐denuded aortic rings contracted with prostaglandin F2α (3 × 10−7 m). Moreover, clonidine and UK 14304‐induced relaxation of endothelium‐denuded arteries precontracted with methoxamine but not with serotonin. Finally, the concentration–contraction curves to clonidine and UK 14304 in endothelium‐denuded aortic rings were significantly shifted to the right by the α1D‐AR selective antagonist, BMY 7378, and rauwolscine. The pA2 and pKB values for BMY 7378 and rauwolscine, respectively, against endothelium‐independent actions of clonidine and UK 14304 were characteristic of an effect on the α1D‐AR. The other selective α2‐AR agonist tested BHT 933 (an azepine derivative), lacks considerable relaxant and contractile effects in rat aorta. The results provide no evidence for the presence of functional α2‐AR in rat aorta. Respectively, the relaxant and contractile effects of the imidazoline derivatives, clonidine and UK 14304, may be due to an adjustable (in relation to the agonist‐dependent active state of the α1‐AR), inhibitory and excitatory, interaction with α1‐ARs.

Impairment of smooth muscle function of rat thoracic aorta in an endothelium‐independent manner by long‐term administration of NG‐nitro‐l‐arginine methyl ester

In this study, we aimed to elucidate whether the daily hypertensive dose of long‐term NG‐nitro‐l‐arginine methyl ester (l‐NAME) treatment, could make a difference between endothelial and smooth muscle functions in rat thoracic aorta. We test the hypothesis that high‐dose, long‐term l‐NAME treatment has a depressive effect on vascular smooth muscle contractile activity which is not related with nitric oxide (NO) synthesis inhibition. After 14 days of treatment, isometric tension and 45Ca2+ influx were measured in aortic tissues isolated from l‐NAME10 and l‐NAME100 hypertensive (10 and 100 mg/kg/day, systolic blood pressures 167 ± 7 and 172 ± 10 mmHg, respectively) and control normotensive rats (132 ± 7 mmHg). In l‐NAME10‐ and l‐NAME100‐treated rats, acetylcholine‐induced relaxation in aortic rings was suppressed with no significant difference between the treatments. l‐NAME100 (but not l‐NAME10) treatment, significantly inhibited contractile responses to phenylephrine, angiotensin II, and K+ (80 mm) in endothelium‐intact tissues. The effect of l‐NAME100 on phenylephrine‐induced contractile responses was not observed after 3 days of treatment. In endothelium‐denuded aortic tissues of l‐NAME100 (but not l‐NAME10)‐treated rats, phenylephrine (1 × 10−6 m)‐ and K+ (80 mm)‐induced contractions and 45Ca2+ influxes were significantly reduced. In Ca2+‐free medium (0.1 mm EDTA), on the contrary, the transient contractions obtained by either phenylephrine (1 × 10−6 m) or caffeine (1 × 10−2 m), or the sustained contractions induced by 12‐o‐tetradecanoylphorbol‐13‐acetate (1 × 10−6 m; a protein kinase C activator) in endothelium‐denuded aortic rings, were not modified by both l‐NAME treatments. These results indicate that in aortic rings from l‐NAME hypertensive rats, low and high doses, long‐term l‐NAME administration may be associated with equivalent inhibition in NO‐dependent vasodilator tone (corresponding to equivalent hypertension values); whereas only high‐dose, long‐term l‐NAME administration produces an endothelium‐independent decrease in vasocontrictor activity, at least partly explained by a reduction in extracellular Ca2+ influx.

Acute intravenous injection and short‐term oral administration of NG‐nitro‐l‐arginine methyl ester to the rat provoke increased pressor responses to agonists and hypertension, but not inhibition of acetylcholine‐induced hypotensive responses

In experiments in vivo, we studied whether the endothelial dysfunction induced by nitric oxide (NO) synthesis inhibition is simultaneously or sequentially manifested as a reduced level of endothelium‐dependent agonist‐induced vasodilatation, an increased responsiveness to vasoconstrictors, and hypertension. Vascular responses to acetylcholine, phenylephrine, and angiotensin II were measured in pithed rats after acute intravenous injection (100 mg/kg) and short‐term oral administration of NG‐nitro‐l‐arginine methyl ester (l‐NAME; 60 mg/kg per day) for 1 and 3 days (l‐NAME1d and l‐NAME3d, respectively). Pithed rats were chosen because drug‐induced cardiovascular responses reflect only peripheral effects. Parallel experiments examined mean arterial pressure (MAP) values in anesthetized rats. After short‐term l‐NAME1d and l‐NAME3d treatments, the MAP was significantly elevated in anesthetized but not pithed rats. Acute intravenous administration of l‐NAME elevated MAP in pithed rats. Intravenous infusion of phenylephrine was used to compensate for the pressor response induced by l‐NAME in pithed animals. The maximum decrease and duration of the hypotensive responses to acetylcholine were unaltered by the acute and both short‐term l‐NAME treatments in pithed rats. These treatments, on the other hand, increased phenylephrine‐ and angiotensin II‐induced pressor responses in pithed animals. In isolated aortic rings prepared from pithed rats treated acutely and short‐term with l‐NAME, acetylcholine‐induced relaxations were inhibited. Thus, the inhibition of NO‐dependent vasodilator tone after acute intravenous injection and short‐term oral l‐NAME administration may be associated with vascular smooth muscle hyper‐responsiveness to pressor agonists and hypertension, whereas the hypotensive responses to acetylcholine could not be associated with the l‐NAME‐induced endothelial dysfunction in pithed rats.

α-methylnoradrenaline-induced contractions in rat aorta are mediated via α1D-adrenoceptors

Summary— The subtype(s) of α‐adrenoceptor‐mediating contractions to α‐methynoradrenaline in the rat aorta has been investigated by using α‐adrenoceptor‐selective competitive antagonists and the α1‐adrenoceptor selective agonist, phenylephrine, for comparison. α‐Methylnoradrenaline and phenylephrine elicited concentration‐dependent contractions in the endothelium‐denuded and endothelium‐intact aortic rings with similar potencies and maximal effects. α‐Methylnoradrenaline‐ and phenylephrine‐induced contractions in endothelium‐denuded aortic rings were competitively antagonized by prazosin (pA2 = 9.38 and 9.13; respectively) and rauwolscine (pA2 = 7.19 and 6.60, respectively). This confirms that there is an α1‐ and a non α2‐adrenoceptor response to α‐methylnoradrenaline in the rat aorta. The subtype selective α1D‐adrenoceptor antagonist, BMY 7378, was found to antagonize contractions to α‐methylnoradrenaline and phenylephrine competitively in endothelium‐denuded and endothelium‐intact aortic rings. The pA2 values of BMY 7378 against α‐methylnoradrenaline (8.39 and 8.41; endothelium‐intact and endothelium‐denuded, respectively) and phenylephrine (8.64 and 8.76; endothelium‐intact and endothelium‐denuded, respectively), are consistent with its published functional potency and clonal α1d‐adrenoceptor binding affinity. In addition, contractions to α‐methylnoradrenaline and phenylephrine in endothelium‐denuded aortic rings, were potently inhibited by WB 4101 with pA2 values of 9.75 and 9.25, respectively. The high pA2 values for WB 4101 indicate that the α1B‐adrenoceptor subtype does not seem to participate in α‐methylnoradrenaline (and phenylephrine) induced contractions in this artery. These results suggest that the α1D‐subtype plays a determining role in rat aorta contractions induced by α‐methylnoradrenaline.

Lack of heterologous receptor desensitization induced by angiotensin II type 1 receptor activation in isolated normal rat thoracic aorta

We tested whether heterologous receptor desensitization induced by activation of AT1 receptors may explain the purported relaxation produced by angiotensin II in normal rat aorta. Also, the role for AT2 receptors in the promotion of vasodilation was studied. In endothelium-intact and endothelium-denuded aortic rings, angiotensin II elicited biphasic contractions, which were significantly depressed when repeated in each tissue. Angiotensin II produced biphasic responses on phenylephrine preconstricted endothelium-intact and endothelium-denuded tissues, without reducing precontractile tone. These responses were abolished in the presence of the AT1 receptor antagonist losartan, but no relaxing responses to angiotensin II were uncovered. PD123319 did not influence angiotensin II responses in endothelium-intact tissues precontracted with phenylephrine; thus, under AT2 receptors blockade the contractile effects of angiotensin II were not overexposed. In conclusion, angiotensin II-induced biphasic responses can be attributed to AT1 receptors activation and rapid desensitization with time. Desensitization proved to be homologous in nature, since precontractile tone induced by phenylephrine was not depressed by angiotensin II (i.e., angiotensin II did not induce heterologous α1-adrenergic receptors desensitization). We found no functional evidence of the participation of AT2 receptors in angiotensin II elicited biphasic contractions. Angiotensin II does not exert relaxant effects in normal rat aorta.

Isometric contraction increases endothelial nitric oxide synthase activity via a calmodulin antagonist-sensitive pathway in rat aorta.

In this work, the possibility that isometric contraction activates endothelial nitric oxide synthase (eNOS) in a calcium/calmodulin (Ca2+/CaM)-dependent manner was examined in rat thoracic aorta. Step-wise stable contractile responses (precontractions) to phenylephrine were obtained in endothelium-intact and endothelium-denuded aortic rings. The subsequent addition of the NO synthase inhibitor, N(G)nitro-l-arginine methyl ester (l-NAME), or the soluble guanylyl cyclase inhibitor, ODQ, further augmented precontractions in a concentration-dependent manner. The amplitude of l-NAME- and ODQ-induced increases in tone were dependent on the level of precontraction; the maximal increments for l-NAME and ODQ were observed in arteries precontracted with phenylephrine at 67% of its maximal effect. Likewise, in endothelium-intact non-contracted arteries, l-NAME and ODQ induced small but significant increases in tone. Neither l-NAME nor ODQ had any effect in endothelium-denuded preparations. In endothelium-intact aortic rings precontracted with high K+ solutions, l-NAME also elicited supplementary contractions dependent on precontraction level. The CaM antagonist, calmidazolium, inhibited in a concentration-dependent, noncompetitive, manner the effects of l-NAME on the tone of endothelium-intact phenylephrine-precontracted aortic rings. These results suggest that isometric contraction increases the activity of eNOS by means of the Ca2+/CaM complex in rat aorta.