M. Verónica Donoso

Rise in endothelium-derived NO after stimulation of rat perivascular sympathetic mesenteric nerves

To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 +/- 7 mmHg, accompanied by peaks of 23 +/- 3 pmol NE, 445 +/- 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 microM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 microM N(omega)-nitro-L-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 microM guanethidine or alpha1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. alpha2-Adrenoceptor blockade with 1 microM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 ± 7 mmHg, accompanied by peaks of 23 ± 3 pmol NE, 445 ± 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 μM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 μM N ω-nitro-l-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 μM guanethidine or α1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. α2-Adrenoceptor blockade with 1 μM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.

Migraine, but not subarachnoid hemorrhage, is associated with differentially increased NPY-like immunoreactivity in the CSF

To test whether migraine and subarachnoid hemorrhage (SAH) are associated with increased sympathetic tone, we compared the neuropeptide Y-like (NPY-LI) and chromogranin A-like immunoreactivities (LI) of cerebrospinal fluid (CSF) from migraneurs and SAH patients with those from control subjects. Increased sympathetic tone was expected to produce higher co-release of these co-stored peptides and concordant changes in their CSF levels. In addition, we investigated a possible disturbed nitric oxide homeostasis by measuring CSF nitrites (NO). More than 70% of CSF NPY-LI corresponded to the chromatographic peak (HPLC) for the intact molecule in all three groups. Migraneurs had 64% higher CSF NPY-LI, but no significant difference in CSF chromogranin A-LI, as compared to controls. In contrast, SAH patients had 74% less CSF chromogranin A-LI and a trend to lower NPY-LI, as compared to controls. No differences in CSF NO were detected among groups. These results argue against an increased sympathetic tone in patients with either migraine or SAH, and suggest that the higher CSF NPY-LI of migraneurs probably originates from central neurons. Furthermore, our findings in SAH patients argue in favor of a decreased sympathetic tone; this could be a homeostatic response to counterbalance vasoconstriction mediated by other mechanisms.

P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation

The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y1R and P2Y2R revealed a 6‐ to 8‐fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y2R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y1R and P2Y2R were detected by RT‐PCR and the mRNA coding for the P2Y4R, P2Y6R and P2Y11R was also identified. Application of 2‐MeSADP and uridine triphosphate (UTP), preferential P2Y1R and P2Y2R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration‐dependent manner by 10–100 nm indomethacin or 10 nm GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y1R antagonist, reduced the 2‐MeSADP‐ but not the UTP‐evoked contractions. Perfusion of cotyledons with 2‐MeSADP or UTP evoked concentration‐dependent reductions in perfusion pressure mediated by the NO–cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2‐MeSADP but not by UTP. In summary, P2Y1R and P2Y2R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses.

Neuropeptide Y is a vasoconstrictor and adrenergic modulator in the hamster microcirculation by acting on neuropeptide Y1 and Y2 receptors

The microvascular effects of neuropeptide Y, and two analogs with preferential affinity for different neuropeptide Y receptor subtypes, were assessed by intravital microscopy on the hamster cheek pouch. The interaction of neuropeptide Y and its analogs with noradrenaline was also studied. Superfusion with 0.1-300 nM neuropeptide Y caused a concentration-dependent reduction in microvascular conductance that was paralleled by reductions in arteriolar and venular diameters. These effects of neuropeptide Y were equipotent with noradrenaline, but slower to develop and longer-lasting than that of noradrenaline. Neuropeptide Y did not affect permeability to macromolecules, as measured by extravasation of fluorescent dextran. The neuropeptide Y Y1 receptor agonist, [Leu31,Pro34]neuropeptide Y, mimicked neuropeptide Y with similar potency but shorter duration, while neuropeptide Y-(13-36), a neuropeptide Y Y2 receptor agonist, was at least 10-fold less potent than neuropeptide Y to induce a delayed and prolonged reduction in microvascular conductance. The joint superfusion of 1 nM neuropeptide Y plus 0.1 mu M noradrenaline did not cause synergism, nor even summation of effects, but reduced the contractile effect of noradrenaline. No synergism was observed after a 10 min priming with 1 nM neuropeptide Y, followed by its joint application with 0.1 mu M noradrenaline, but a significant vasodilation and hyperemia ensued upon stopping noradrenaline application. Priming with 1 nM [Leu31,Pro34]neuropeptide Y prolonged noradrenaline vasoconstriction without evidence of hyperemia. In contrast, priming with 1 nM neuropeptide Y-(13-36) significantly antagonized noradrenaline vasoconstriction. These findings indicate that both neuropeptide Y receptor subtypes are present in arterioles and venules of the hamster, and suggest that their activation with neuropeptide Y induces a rapid (Y1 receptor subtype activation) and a delayed (Y2 receptor subtype activation) vasocontractile response. The interaction with noradrenaline is complex, without evidence for synergism, but neuropeptide Y Y2 receptor activation seems to antagonize noradrenaline and/or to facilitate auto-regulatory vasodilation after the catecholamine-induced vasoconstriction.

The role of adenosine A2A and A3 receptors on the differential modulation of norepinephrine and neuropeptide Y release from peripheral sympathetic nerve terminals

The pre‐synaptic sympathetic modulator role of adenosine was assessed by studying transmitter release following electrical depolarization of nerve endings from the rat mesenteric artery. Mesentery perfusion with exogenous adenosine exclusively inhibited the release of norepinephrine (NA) but did not affect the overflow of neuropeptide Y (NPY), establishing the basis for a differential pre‐synaptic modulator mechanism. Several adenosine structural analogs mimicked adenosines effect on NA release and their relative order of potency was: 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride = 1‐[2‐chloro‐6‐[[(3‐iodophenyl)methyl]amino]‐9H‐purin‐9‐yl]‐1‐deoxy‐N‐methyl‐β‐d‐ribofuranuronamide = 5′‐(N‐ethylcarboxamido)adenosine >> adenosine > N6‐cyclopentyladenosine. The use of selective receptor subtype antagonists confirmed the involvement of A2A and A3 adenosine receptors. The modulator role of adenosine is probably due to the activation of both receptors; co‐application of 1 nm 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride plus 1 nm 1‐[2‐chloro‐6‐[[(3‐iodophenyl)methyl]amino]‐9H‐purin‐9‐yl]‐1‐deoxy‐N‐methyl‐β‐d‐ribofuranuronamide caused additive reductions in NA released. Furthermore, while 1 nm of an A2A or A3 receptor antagonist only partially reduced the inhibitory action of adenosine, the combined co‐application of the two antagonists fully blocked the adenosine‐induced inhibition. Only the simultaneous blockade of the adenosine A2A plus A3 receptors with selective antagonists elicited a significant increase in NA overflow. H 89 reduced the release of both NA and NPY. We conclude that pre‐synaptic A2A and A3 adenosine receptor activation modulates sympathetic co‐transmission by exclusively inhibiting the release of NA without affecting immunoreactive (ir)‐NPY and we suggest separate mechanisms for vesicular release modulation.

P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling.

The nucleotide P2Y1 receptor (P2Y1R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y1R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y1R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y1R and obliterated the P2Y1R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y1R agonist, not only displaced within 4 min the P2Y1R localization out of membrane rafts but also induced its subsequent internalization. 2′-Deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y1R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y1R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y1R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y1R to membrane rafts, highlighting the role of this microdomain in P2Y1R signaling.

α2-Adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals

Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.

Aging differentially modifies arterial sensitivity to endothelin-1 and 5-hydroxytryptamine: Studies in dog coronary arteries and rat arterial mesenteric bed

The influence of age on vascular reactivity to endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) was studied in coronary artery rings from dogs of 9 years of age or younger, and dogs older than 9 years. ET-1 caused concentration-dependent contractions that developed about 100% of the 70 mM KCl-induced tension in the younger dogs; those from older dogs did not generate more than 20%. In contrast, 5-HT developed only about 20% of the KCl-induced tension in rings from young dogs, whereas in the older animals, it developed up to 120% of the KCl tension. No significant difference in the tension developed by 70 mM KCl was noted between both groups of dogs. Mechanical denudation of the endothelial cell layer caused a modest, yet significant, leftward shift of the ET-1 and 5-HT concentration-response curves only in the younger dogs. N omega-Nitro-L-arginine (15 microM) shifted the ET-1 concentration-response curves to the left in rings from both groups of dogs. Rings precontracted with 20 mM KCl relaxed in a concentration-dependent fashion with acetylcholine; its sensitivity was about threefold less in the older group of dogs. To validate the changes in vascular reactivity with age, a parallel study was performed perfusing the arterial mesenteric bed of rats of 3, 7, and 30 weeks of age. In this experimental model, the efficacy of ET-1 significantly decreased with age and that of 5-HT was significantly increased. The vasomotor reactivity of noradrenaline was modestly affected by aging, whereas the acetylcholine-induced vasorelaxation was significantly reduced with age.

UTP controls cell surface distribution and vasomotor activity of the human P2Y2 receptor through an Epidermal growth factor receptor-transregulated mechanism

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y2 receptor (P2Y2R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y2R agonist, displaced the P2Y2R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y2R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y2R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y2R in intact human blood vessels.

Pharmacological Characterization of the ETA Receptor in the Vascular Smooth Muscle Comparing its Analogous Distribution in the Rat Mesenteric Artery and in the Arterial Mesenteric Bed

The potency of ET-1, ET-2, and ET-3 to contract the isolated perfused rat arterial mesenteric bed was 2.73 +/- 0.57, 1.63 +/- 0.32, and 144 +/- 30 nM, respectively. The vasomotor effect of the ETs was slow in onset, persistent but reversible. Sarafotoxin S6b mimicked the ETs with a potency twofold lower than ET-1; sarafotoxin S6c and the C-terminal hexapeptide of ET-1 was inactive. ETH agonists such as IRL-1620 and AGETB-89 were inactive as vasoconstrictors within the range of concentrations examined. Minor chemical modifications of ET-1 amino acids residues in position 7 or 21 decreased significantly the peptide potency; ET-1 analogues with one or none of the disulfide bonds resulted inactive. The vasomotor effect of ETs was blocked in a competitive, reversible, and selective manner by FR 139317 and BQ-123, the latter being about threefold less potent than the former antagonist. The potency of FR 139317 was 20-fold higher to antagonize ET-3 than ET-1, and threefold higher to block ET-2 than ET-1. In strict analogy to FR 139317, BQ-123 was 12-fold more potent to antagonize ET-3 than ET-1, and fourfold more potent to antagonize ET-2 than ET-1. Upon removal of the endothelial cell layer, the vasomotor potency of ET-1 or the antagonist potency of FR 139317 remained unaltered, suggesting that the vasomotor receptors are localized in the arterial smooth muscles. The ET-1-induced vasomotor responses desensitized, an effect not crossed to noradrenaline (NA); perfusion with 10 microM indomethacin did not alter the vasomotor potency of ET-1, excluding the participation of eicosanoids in the arteriolar effects of ET-1. In isolated rings of the rat mesenteric artery, set to record isometric contractions of the circular muscular layer, the potency of the ETs and their structural analogues was as follows; ET-2 = ET-1 = sarafotoxin S6b > ET-3 > sarafotoxin S6c. The C-terminal hexapeptide of ET-1 and [Ala 1,3,11,15]ET-1 were inactive. The ET-1-induced vasoconstriction was antagonized in a concentration-dependent fashion by FR 139317. These results allow to conclude that the ETA receptors present in the arterial mesenteric circulation are localized in the vascular smooth muscle of the large-sized arteries as well as the smaller arterioles and precapillary vessels of the rat arterial mesenteric bed.