José A. Terrón

Protection Against Ischemia and Improvement of Cerebral Blood Flow in Genetically Hypertensive Rats by Chronic Pretreatment With an Angiotensin II AT1 Antagonist

Background and Purpose— Pretreatment with angiotensin II AT1 receptor antagonists protects against cerebral ischemia. We studied whether modulation of cerebral blood flow (CBF) and morphometric changes in brain arteries participated in this protective mechanism. Methods— We pretreated adult spontaneously hypertensive rats with equally antihypertensive doses of candesartan (0.1 or 0.3 mg/kg per day), nicardipine (0.1 mg/kg per day), or captopril (3.0 mg/kg per day) for 3 or 28 days via subcutaneous osmotic minipumps followed by permanent left middle cerebral artery (MCA) occlusion distal to the origin of the lenticulostriate arteries. We measured CBF by autoradiography with 4-iodo-[N-methyl-14C]antipyrine 3 hours after operation and the areas of infarct and tissue swelling 24 hours after operation. Morphometric changes in the MCA were studied after antihypertensive treatment. Results— Twenty-eight days of candesartan pretreatment decreased the infarct area by 31%; reduced the CBF decrease at the peripheral area of ischemia and the cortical volume of severe ischemic lesion, where CBF was <0.50 mL/g per minute; increased the MCA external diameter by 16%; and reduced the media thickness of the MCA by 23%. Captopril pretreatment for 28 days decreased the infarct area by 25%. Pretreatment with candesartan for 3 days or nicardipine for 28 days was ineffective. Conclusions— Angiotensin II system inhibition protects against neuronal injury more effectively than calcium channel blockade. Protection after AT1 receptor blockade is not directly correlated with blood pressure reduction but with normalization of MCA media thickness, leading to increased arterial compliance and reduced CBF decrease during ischemia at the periphery of the lesion.

Pharmacological evidence for the 5-HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries

We investigated in the present study whether 5‐HT is able to exert direct relaxant responses in canine basilar and middle cerebral arteries via the 5‐HT7 receptor. In arterial rings deprived of endothelium and pre‐contracted with prostaglandin F2α (2 μM), 5‐HT, 5‐carboxamidotryptamine (5‐CT), 5‐methoxytryptamine, sumatriptan or α‐methyl‐5‐HT produced further increase in tone and/or slight relaxation. Blockade of 5‐HT1B/1D and 5‐HT2A receptors with GR127935 (1 μM) and ketanserin (0.1 μM), respectively, antagonized the vasoconstrictor component of the response and unmasked a concentration‐dependent relaxation to 5‐HT, 5‐CT and 5‐methoxytryptamine; sumatriptan and α‐methyl‐5‐HT remained inactive as relaxant agonists. The rank order of agonist potency in both arteries was 5‐CT>5‐HT>5‐methoxytryptamine>>sumatriptanα‐methyl‐5‐HT. In dog basilar artery, pre‐incubated with GR127935 (1 μM) and ketanserin (0.1 μM) and pre‐contracted with prostaglandin F2α (2 μM), the 5‐HT7 ligands, clozapine (1 μM), mesulergine (0.3 μM), methiothepin (3 nM), risperidone (3 nM), spiperone (1 μM) and LY215840 (10–100 nM), produced significant rightward shifts of the concentration‐response curves for 5‐HT and 5‐CT. Only methiothepin and risperidone reduced significantly the maximum relaxant response (Emax), whilst the other drugs behaved as competitive antagonists with affinity values (pKB) that significantly correlated with their binding affinity (pKi) at recombinant 5‐HT7 receptors. These data disclosing the involvement of the 5‐HT7 receptor in cerebrovascular relaxation may be strongly relevant in the light of: (1) the involvement of 5‐HT in migraine; (2) the putative linkage between cephalovascular vasodilatation and migraine headache; and (3) the relatively high 5‐HT7 receptor affinity of migraine prophylactic 5‐HT antagonists.

Restraint Stress Modulates Brain, Pituitary and Adrenal Expression of Angiotensin II AT1A, AT1B and AT2 Receptors

Angiotensin II (Ang II) AT₁ receptors are involved in the regulation of the stress response. In adult male rats, acute restraint increased AT_1A mRNA in paraventricular nucleus. Repeated restraint increased AT_1A mRNA and AT₁ binding in paraventricular nucleus and AT₁ binding in subfornical organ and median eminence. AT_1B and AT₂ receptors were not expressed in brain areas involved in the stress response. Acute restraint increased anterior pituitary AT_1A mRNA and AT₁ binding and decreased AT_1B mRNA. During repeated restraint, the increase in AT_1A mRNA in the anterior pituitary was maintained, but AT_1B mRNA and AT₁ binding returned to normal levels. In adrenal zona glomerulosa, AT_1B mRNA, AT₁ binding, AT₂ mRNA and AT₂ binding decreased during acute restraint. Receptor mRNA and binding returned to normal after repeated stress, with the exception of rebound increase in adrenal zona glomerulosa AT₂ mRNA. In adrenal medulla, AT_1A mRNA increased and AT₂ mRNA decreased during acute restraint. AT_1A mRNA remained increased during repeated restraint, while alterations in AT₂ mRNA were no longer present. Expression of AT_1A, AT_1B and AT₂ receptors in the hypothalamic-pituitary-adrenal axis is tissue specific and is different in acute and repeated stress. Increased brain, pituitary and adrenomedullary AT_1A receptor expression correlates with hypothalamic-pituitary-adrenal axis stimulation, supporting the hypothesis of Ang II, through selective AT_1A receptor stimulation, as an important determinant of the acute and repeated stress response. Decreased adrenal zona glomerulosa and anterior pituitary AT_1B receptors during acute stress can be interpreted as compensatory to increased stimulation by Ang II. There may be additional roles for adrenal AT₂ receptors during acute stress, possibly related to interaction or cross-talk with AT₁ receptors.

Mediation of 5‐HT‐induced external carotid vasodilatation in GR 127935‐pretreated vagosympathectomized dogs by the putative 5‐HT7 receptor

The vasodilator effects of 5‐hydroxytryptamine (5‐HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho‐inhibitory 5‐HT1B/1D receptors and postjunctional 5‐HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5‐HT with the 5‐HT1B/1D receptor antagonist, GR 127935, unmasks a dose‐dependent vasodilator effect of 5‐HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5‐HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg−1, i.v.). One‐minute intracarotid (i.c.) infusions of 5‐HT (0.330 μg min−1), 5‐carboxamidotryptamine (5‐CT; 0.010.3 μg min−1), 5‐methoxytryptamine (1100 μg min−1) and lisuride (31000 μg min−1) resulted in dose‐dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5‐CT>>5‐HT5‐methoxytryptamine>lisuride, whereas cisapride (1001000 μg min−1, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg−1, i.c.), but not cisapride (mean dose of 67±7 μg kg−1, i.c.), specifically abolished the responses induced by 5‐HT, 5‐CT and 5‐methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8‐OH‐DPAT (33000 μg min−1) produced dose‐dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg−1, i.c.) the agonist‐induced vasodilator responses. The external carotid vasodilator responses to 5‐HT, 5‐CT and 5‐methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)‐pindolol (4 mg kg−1) or ritanserin (100 μg kg−1) plus granisetron (300 μg kg−1), but were dose‐dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg−1, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg−1), metergoline (1 and 3 mg kg−1), methysergide (1 and 3 mg kg−1) or clozapine (0.3 and 1 mg kg−1). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5‐ht7 receptor, our results indicate that the 5‐HT receptor mediating external carotid vasodilatation in GR 127935‐pretreated vagosympathectomized dogs is operationally similar to the putative 5‐HT7 receptor mediating relaxation of vascular and non‐vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea‐pig ileum) as well as tachycardia in the cat.

The relaxant 5-HT receptor in the dog coronary artery smooth muscle : pharmacological resemblance to the cloned 5-ht7 receptor subtype

1 The relaxant effect of 5‐hydroxytryptamine (5‐HT) in the dog isolated coronary artery deprived of endothelium is mediated by a receptor unrelated to the 5‐HT1, 5‐HT2, 5‐HT3 or 5‐HT4 types. Based upon the pharmacological characteristics of this relaxant 5‐HT receptor and those reported for the new members of the 5‐HT receptor family, the present study explored the possibility that the relaxant 5‐HT receptor referred to above, corresponds to the cloned 5‐ht7 subtype. Thus, the relaxing and/or blocking effects of several 5‐HT receptor drugs as well as some typical and atypical antipsychotic drugs with high affinity for the cloned 5‐ht7 receptor in precontracted ring segments were analyzed. 2 5‐HT, 5‐carboxamidotryptamine (5‐CT) and 5‐methoxytryptamine, but not 8‐OH‐DPAT or sumatriptan, produced concentration‐dependent relaxations in endothelium‐denuded canine coronary artery rings precontracted with prostaglandin F2α (2 μm). Clozapine (1 μm) produced in some cases a small relaxing effect and antagonized 5‐HT‐ and 5‐CT‐induced relaxation suggesting a partial agonist effect. In the presence of the 5‐HT1D receptor antagonist, GR127935 (100 nM), the rank order of agonist potency was 5‐CT > 5‐HT > clozapine ≥ 5‐methoxytryptamine. 8‐OH‐DPAT and sumatriptan remained inactive as agonists. 3 In GR 127935‐treated preparations, methiothepin (3 nM) and mianserin (1 μm), as well as the antipsychotics, clozapine (1 μm), pimozide (300 nM), risperidone (3 nM) and spiperone (1 μm), failed to induce a significant relaxation in prostaglandin F2α‐precontracted vessels, but produced significant rightward displacements of the concentration‐response curves to 5‐HT and 5‐CT without significantly reducing the Emax. In a final set of experiments with 5‐CT, metergoline (100 nM) and mesulergine (300 nM) behaved as competitive antagonists. In contrast, lisuride (3 nM) noncompetitively antagonized 5‐CT‐induced relaxation. The estimated affinity (apparent pKB values) of the above antagonist drugs for the relaxant 5‐HT receptor significantly correlated with their reported affinity at the cloned 5‐ht7 receptor. 4 Taken together, the above pharmacological data may suggest that the relaxant 5‐HT receptor in the smooth muscle of the canine coronary artery is similar to the cloned 5‐ht7 receptor subtype.

Role of 5-HT1A and 5-HT7 receptors in the facilitatory response induced by 8-OH-DPAT on learning consolidation

The present study further explored the mechanisms involved in the facilitatory effect induced by (+/-)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on learning consolidation. For this purpose, we analyzed in parallel the effects of LY215840 and ritanserin, two 5-HT(2) receptor antagonists with high affinity for the 5-HT(7) receptor, and WAY100635, a selective 5-HT(1A) receptor antagonist, on the facilitatory effect induced by 8-OH-DPAT on learning consolidation. We also determined whether LY215840 and/or ritanserin could be beneficial in restoring a deficient learning condition. Using the model of autoshaping task, post-training injection of LY215840 or WAY100635 had no effect on learning consolidation. However, both drugs abolished the enhancing effect of 8-OH-DPAT, with LY215840 being slightly more effective than WAY100635 in this respect. Ritanserin produced an increase in performance by itself and also abolished the effect of 8-OH-DPAT. Remarkably, selective blockade of 5-HT(2A) and 5-HT(2B/2C) receptors with MDL100907 and SB200646, respectively, failed to alter the 8-OH-DPAT effect. LY215840 and ritanserin, at the doses that inhibited the 8-OH-DPAT-induced response, reversed the learning deficits induced by scopolamine and dizocilpine. The present results suggest that the enhancing effect produced by 8-OH-DPAT on learning consolidation involves activation of 5-HT(1A) receptors and an additional mechanism, probably related to the 5-HT(7) receptor. Blockade of 5-HT(2) receptors, and perhaps of 5-HT(7) receptors as well, may provide some benefit in reversing learning deficits associated with decreased cholinergic and/or glutamatergic neurotransmission.

Role of 5‐ht7 receptors in the long‐lasting hypotensive response induced by 5‐hydroxytryptamine in the rat

The receptor mediating the long‐lasting hypotensive effect of intravenous (i.v.) 5‐hydroxytryptamine (5‐HT) in the rat was originally classified as 5‐HT1‐like. Since some pharmacological properties of this receptor are closely similar to those for the cloned 5‐ht7 receptor, the present study investigated the effects of several 5‐HT receptor agonists and antagonists showing high affinity for the cloned 5‐ht7 receptor in pithed rats with artificially raised blood pressure. I.v. bolus administration of 5‐HT, 5‐carboxamidotryptamine (5‐CT), 5‐methoxytryptamine, lisuride and sumatriptan to bilaterally vagotomized pithed rats pretreated with ketanserin (0.18 μmol kg−1, i.v.), the diastolic blood pressure of which had been raised by a continuous i.v. infusion of methoxamine (60–80 nmol kg−1 min−1), produced dose‐dependent hypotensive responses; only 5‐HT and 5‐CT displayed similar maximum effects. In addition to mimicking the hypotensive action of 5‐HT with a lower maximum effect, lisuride strongly antagonized the 5‐CT‐induced hypotensive responses thus suggesting a partial agonist effect. The rank order of hypotensive agonist potency was 5‐CT >> 5‐HT 5‐methoxytryptamine lisuride >> sumatriptan. In experiments with antagonists, i.v. treatment with metergoline (2.48 μmol kg−1), mesulergine (2.76 μmol kg−1), methysergide (2.13 μmol kg−1), lisuride (0.22 μmol kg−1), methiothepin (0.68 μmol kg−1), mianserin (10.6 μmol kg−1), or the atypical antipsychotic drugs, clozapine (11.0 μmol kg−1) or risperidone (78.0 nmol kg−1), produced significant rightward displacements of the dose‐response curve for 5‐CT in methoxamine‐infused pithed animals pretreated with ketanserin (0.18 μmol kg−1, i.v.); lisuride, methiothepin and risperidone behaved as non‐competitive antagonists as they elicited a significant reduction of the maximum effect to 5‐CT. In contrast, blockade of 5‐HT1, 5‐HT3 and 5‐HT4 receptors with i.v. propranolol (3.38 μmol kg−1), MDL‐72222 (1.59 μmol kg−1) and GR125487 (1.91 μmol kg−1), respectively, did not alter 5‐CT‐induced hypotensive responses; ketanserin (0.18 μmol kg−1, i.v.) failed to modify the dose‐response curve for 5‐CT in saline‐pretreated animals. Lastly, inhibition of the prostaglandin‐forming cyclo‐oxygenase and nitric oxide synthase with indomethacin (14 μmol kg−1, i.v.) and NG‐nitro‐L‐arginine methyl ester (L‐NAME, 120 μmol kg−1, i.v.), respectively, had no significant effects on 5‐CT‐induced hypotensive effects. Taken together, the present pharmacological data suggest that the long‐lasting vasodepressor action of 5‐HT in the rat involves activation of receptors closely similar to the cloned 5‐ht7 subtype. Since no evidence for an indirect mechanism could be obtained, these receptors may be primarily located in the vascular smooth muscle of the systemic resistance vessels. These findings represent further evidence favouring the functional role of the 5‐ht7 receptor.

Effects of the 5-HT receptor antagonists GR127935 (5-HT1B/1D) and MDL100907 (5-HT2A) in the consolidation of learning

We have previously reported that 5-HT1B/1D and 5-HT2A/2B/2C receptors play a role in learning and memory. The present investigation was devoted to analyze further in the autoshaping learning task: (1) the effects of the 5-HT1A/1B/1D receptor agonist, GR46611, the 5-HT1B/1D receptor antagonist, GR127935, and the selective 5-HT2A receptor antagonist, MDL100907. Consistent with a role of 5-HT1B/1D receptors in learning, the post-training injection of GR46611 (1-10 mg/kg) decreased the consolidation of learning whereas GR127935 (10 mg/kg) increased it; the effects of both drugs were reversed by PCA pretreatment. GR127935 abolished the decrease induced by GR46611, TFMPP and mCPP, whereas MDL100907 (0.1-3.0 mg/kg) had no effect by itself but abolished the effects of DOI, ketanserin and TFMPP and moderately inhibited the effects elicited by mCPP, 1-NP and mesulergine. Neither did GR127935 nor MDL100907 significantly modify the increase in the consolidation of learning induced by 8-OH-DPAT. Thus, the present findings suggest that stimulation of presynaptic 5-HT1B/1D receptors impairs the consolidation of learning whilst stimulation of 5-HT2A/2C receptors enhances it; the blockade of 5-HT2A receptors has no effects. In addition, 5-HT2 receptors seem to modulate this cognitive stage.

Increased angiotensin II AT1 receptor expression in paraventricular nucleus and hypothalamic-pituitary-adrenal axis stimulation in AT2 receptor gene disrupted mice

Angiotensin II AT2 receptor gene-disrupted mice have increased blood pressure and response to angiotensin II, behavioral alterations, greater response to stress, and increased adrenal AT1 receptors. We studied hypothalamic AT1 receptor binding and mRNA by receptor autoradiography and in situ hybridization, adrenal catecholamines by HPLC, adrenal tyrosine hydroxylase mRNA by in situ hybridization and pituitary and adrenal hormones by RIA in AT2 receptor-gene disrupted mice and wild-type controls.To confirm the role of adrenal AT1 receptors, we treated wild-type C57 BL/6J mice with the AT1 antagonist candesartan for 2 weeks, and measured adrenal hormones, catecholamines and tyrosine hydroxylase mRNA. In the absence of AT2 receptor transcription, we found increased AT1 receptor binding in brain areas involved in the regulation of the hypothalamic-pituitary-adrenal axis, the hypothalamic paraventricular nucleus and the median eminence, and increased adrenal catecholamine synthesis as shown by higher adrenomedullary tyrosine hydroxylase mRNA and higher adrenal dopamine, norepinephrine and epinephrine levels when compared to wild-type mice. In addition, in AT2 receptor gene-disrupted mice there were higher plasma adrenocorticotropin (ACTH) and corticosterone levels and lower adrenal aldosterone content when compared to wild-type controls. Conversely, AT1 receptor inhibition in CB57 BL/6J mice reduced adrenal tyrosine hydroxylase mRNA and catecholamine content and increased adrenal aldosterone content. These results can help to explain the enhanced response of AT2 receptor gene-disrupted mice to exogenous angiotensin II, support the hypothesis of cross-talk between AT1 and AT2 receptors, indicate that the activity of the hypothalamic-pituitary-adrenal axis parallels the AT1 receptor expression, and suggest that expression of AT1 receptors can be dependent on AT2 receptor expression. Our results provide an explanation for the increased sensitivity to stress in this model.

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.