Involvement of calcium channels in the contractile activity of neurotensin but not acetylcholine: studies with calcium channel blockers and Bay K 8644 on the rat fundus.

The contractile activity of neurotensin and acetylcholine on rat isolated fundus strips was examined in preparations maintained in Tyrode buffer containing 2.5, 1.0 or 0 mM Ca2+. While the neurotensin contractions depended markedly on the external Ca2+ concentration, the acetylcholine-induced muscular responses were not significantly affected by omission of calcium in the superfusion media. Pre-incubation of rat fundus strips with nifedipine (0.03-3.8 microM), diltiazem (0.5-3.5 microM) or methoxyverapamil (0.3-1.3 microM) antagonized in a non-surmountable fashion the contractile activity of neurotensin but not of acetylcholine. Pretreatment with Bay K 8644 potentiated in a concentration-dependent fashion the contractile activity of rat fundus strips to neurotensin without modifying to any significant degree the acetylcholine-induced contractions. Nifedipine blocked in a concentration-dependent manner the Bay K 8644-induced potentiation of the neurotensin contractile responses in the fundus. Results demonstrate the dependence on external calcium of the contractile activity of neurotensin and the resistance of the muscarinic response to external calcium manipulations. The coupling of the neurotensin receptor to calcium channels is discussed.

Gastrointestinal neurotensin receptors: contribution of the aromatic hydroxyl group in position 11 to peptide potency.

Neurotensin structural analogues on tyrosine11 were tested in vitro to determine their ability to contract the fundus or relax the intestine. The rank order of potency was: neurotensin greater than [Phe11]-neurotensin greater than [D-Tyr11]-neurotensin greater than [D-Phe11]-neurotensin. All peptides behaved as full agonists. It is concluded that tyrosine11 is part of the neurotensin pharmacophore; the hydroxyl group increases the affinity not the intrinsic activity of the peptide at the receptor.

Involvement of ETA receptors in the facilitation by endothelin-1 of non-adrenergic non-cholinergic transmission in the rat urinary bladder.

1. Endothelin-1 (ET-1; 3-10 nM) raised the tone of rat bladders bathed in buffer containing atropine (1 microM) plus guanethidine (3.4 microM). In addition, ET-1 potentiated, in a concentration-dependent fashion (1-10 nM), the contractions evoked by both transmural nerve stimulation and applications of exogenous adenosine 5'-triphosphate (ATP). 2. The threshold concentration of ET-1 required to facilitate non-adrenergic non-cholinergic (NANC) transmission and potentiate ATP-induced contractions, was about 10 fold lower than that required to increase the bladder tone (3 nM). 3. The ET-1-induced increase in basal tension reached its maximal effect within 60-90 s. In contrast, the 7.8 microM ATP-induced contractions increased by 50% within the first minute following incubation with 10 nM ET-1 but required about 5 min to develop the maximal effect. 4. The ET-1-induced potentiation of NANC or ATP responses was long-lasting and persisted in spite of extensive washing. The recovery of the bladder excitability depended on the concentration of ET-1. Following the application of 3 nM ET-1, recovery required 30 min; applications of 10 nM ET-1 required at least 60 min for full recovery. 5. The ET-1-induced potentiation of responses was selective for ATP and related structural analogues. ET-1 did not modify the contractions induced by acetylcholine, 5-hydroxytryptamine, prostaglandin F2 alpha or bradykinin. 6. The potency of ET-2 was similar to that of ET-1. ET-3 and ET-C-terminal hexapeptide were inactive up to 100 M. Sarafotoxin S6b was 2 to 3 fold less potent than ET-1 whereas sarafotoxin S6c (100 nM) was inactive. AGETB-9 and AGETB-89, two ETB receptor agonists, were also inactive (up to 100 nM). 7. Removal of one or both disulphide bonds in ET-1 and tryptophan-21 formylation of ET-1, resulted in inactive peptides (up to 100 nM). 8. The ET-1 receptor antagonists, BE-18257B and FR 139317, blocked both the ET-1-induced rise in tone and the potentiation of ATP responses in a concentration-dependent fashion. FR 139317 was at least 30 fold more potent than BE-18257B. Both antagonists blocked at lower concentrations the ET-1 increase in bladder tone as compared to the ATP potentiation. The antagonism was slowly reversible. 9. Results are consistent with the presence of ETA receptors in the rat bladder, which mediate both actions of ET-1. The interaction of ET-1 with purinergic mechanisms is discussed.

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.

Release and functional role of neuropeptide Y as a sympathetic modulator in human saphenous vein biopsies.

Transmural electrical stimulation of the sympathetic nerve endings of human saphenous vein biopsies released two forms of NPY identified chromatographically as native and oxidized peptide. The release process is dependent on extracellular calcium, the frequency, and the duration of the stimuli. While guanethidine reduced the overflow of ir-NPY, phenoxybenzamine did not augment NPY release, but increased that of noradrenaline. Oxidized NPY, like native NPY, potentiated the noradrenaline and adenosine 5'-triphospahate-induced vasoconstriction, an effect blocked by BIBP 3226 and consonant with the RT-PCR detection of the mRNA encoding the NPY Y1 receptor. These results highlight the functional role of NPY in human vascular sympathetic reflexes.

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.

Pharmacological characterization of CGRP1 receptor subtype in the vascular system of the rat: studies with hCGRP fragments and analogs.

In order to examine whether the truncated fragments of hCGRP, hCGRP(8-37) or hCGRP(12-37), behave as competitive CGRP receptor antagonists in the vascular system of the rat, systemic blood pressure was continually monitored in pentobarbital-anesthetized Sprague-Dawley rats. The IV administration of 7.9-527 pmol hCGRP/rat caused dose-related reductions in mean arterial blood pressure that lasted, depending on the dose, about 3-10 min. In contrast, hCGRP fragments 8-37 or 12-37 proved inactive up to 60,000 pmol/rat. Pretreatment with either 10 or 30 nmol hCGRP(8-37) or 20 or 90 nmol hCGRP(12-37)/rat reduced the magnitude of the CGRP-induced hypotensive responses caused by 79 pmol hCGRP/rat; pretreatment with 10 nmol of the hCGRP fragments displaced about 3-fold the hCGRP as well as the [Cys(ACM)2.7]hCGRP dose-response curve to the right in a parallel fashion. The specificity of hCGRP(8-37) as a CGRP receptor antagonist was documented by the finding that it did not antagonize the hypotensive responses induced with bradykinin, histamine or substance P.

Endothelin-1 (ET)-induced mobilization of intracellular Ca2+ stores from the smooth muscle facilitates sympathetic cotransmission by potentiation of adenosine 5'-triphosphate (ATP) motor activity: studies in the rat vas deferens.

Endothelin-1 (ET) enhances nerve-stimulated contractions in epididymal (E) and prostatic (P) halves of the rat vas deferens, in addition to raising the basal tone in E. Whereas the peak increase in basal tone occurs in about 30 s, the maximal enhancement of neurotransmission is observed within 5 min. The latter effect is long lasting and is maintained even after extensive tissue washout. Furthermore, ET potentiates, in a concentration-dependent fashion, the adenosine 5'-triphosphate (ATP) or the adenylylimidodiphosphate (AMP-PNP) but not the noradrenaline (NA)-induced motor activity. The ATP motor response is partially blocked in media without Ca2+ plus 0.1 mM EGTA or following tissue incubation in buffer containing 10-50 nM nifedipine. However, these procedures do not modify significantly the ET-induced potentiation of the ATP contractions. The ET-induced potentiation of the ATP motor response is not modified by tissue preincubation in Ca(2+)-free buffer plus 10-30 microM ryanodine or 5-20 mM caffeine. The ET-induced rise in E basal tension is significantly reduced in the absence of external Ca2+ or by nifedipine; ryanodine does not modify this effect. Surgical denervation of the tissues does not obliterate the ET-induced potentiation of the ATP motor responses nor the ET increase in E basal tension in tissues superfused in Ca(2+)-free media or buffer with 2.5 mM Ca2+. Endothelin-1 does not significantly modify the overflow of 3H-NA, following transmural electrical depolarization of tissue nerve terminals. Hoe 140 did not interfere with the ET activity.

Involvement of postjunctional purinergic mechanisms in the facilitatory action of bradykinin in neurotransmission in the rat vas deferens.

To investigate the nature of the bradykinin-induced potentiation of electrically driven muscle twitches in the isolated vas deferens, bradykinin, noradrenaline and adenosine 5'-triphosphate (ATP) concentration-response curves were made with control, reserpinized and chemically sympathectomized rats. Bradykinin potentiated the ATP- but not the noradrenaline-induced contractions in the epididymal and prostatic segments of the ductus. The epididymal segment of the ductus did not respond to transmural electrical stimulation following reserpine treatment. Bradykinin potentiated the muscular contractions caused by exogenous ATP but not by noradrenaline. In contrast, the transmurally evoked twitches of the prostatic portion of the ductus remained almost unaltered; bradykinin increased the motor effect of ATP without modifying the potency of noradrenaline. All neuronally induced contractile activity was absent in sympathectomized rats; bradykinin potentiated the contractile effect of ATP without altering the noradrenaline-induced contractions. These results suggest that bradykinin potentiates the ATP-evoked contractions by acting postjunctionally.

Extracellular calcium dependence of the neurotensin-induced relaxation of intestinal smooth muscles: studies with calcium channel blockers and BAY K-8644.

To investigate whether the neurotensin-induced relaxation of the rat duodenum and ileum is dependent on the external concentration of calcium, the effect of the neuropeptide was studied in isolated intestinal segments superfused with Tyrode solution containing no calcium, 1 or 2.5 mM Ca2+. The neurotensin-induced intestinal relaxation was reduced when the extracellular calcium concentration was lowered. In addition, the inhibitory effect of neurotensin was cancelled when the tissues were incubated in the presence of diltiazem, methoxyverapamil or nifedipine. BAY K-8644, a structural analog of nifedipine that functions as an agonist of the calcium channel, potentiated the neurotensin-induced smooth muscle relaxation. The facilitatory effect of BAY K-8644 was antagonized by nifedipine, indicating competition between the 2 dihydropyridines. Apamin, the K+ channel blocker, antagonized the neurotensin-induced visceral relaxation, displacing the concentration-response curve of the peptide to the right. Furthermore, apamin also blocked the effect of neurotensin when the neuropeptide was assayed in the presence of BAY K-8644. It is concluded that the smooth muscle relaxation induced by neurotensin is dependent on external calcium, suggesting that the activation of the neuropeptide receptor causes an influx of calcium which leads to the opening of K+ channels before smooth muscle relaxation is triggered.

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.

D-myo-inositol 1,2,6-trisphosphate blocks neuropeptide Y-induced facilitation of noradrenaline-evoked vasoconstriction of the mesenteric bed.

Perfusion of the rat mesenteric bed with 0.1 or 10 nM neuropeptide Y potentiated the noradrenaline-induced increase in mesenteric pressure; the peptide did not modify basal perfusion pressure. While perfusion with 0.1 nM neuropeptide Y significantly increased the maximal noradrenaline-evoked vasoconstriction without modifying its EC50, 10 nM neuropeptide Y potentiated the maximal noradrenaline effect and significantly shifted its concentration-response curve to the left. Perfusion with 1-10 microM D-myo-inositol 1,2,6-trisphosphate (alpha-trinositol) reduced, in a concentration-dependent fashion, the neuropeptide Y-induced potentiation of the noradrenaline-evoked vasoconstriction without altering the potency or maximal response evoked by the catecholamine alone. Perfusion with 0.1 nM neuropeptide Y plus 1 microM alpha-trinositol completely abolished the neuropeptide Y-induced facilitation of the noradrenaline effect. alpha-Trinositol 1 microM in the presence of 10 nM neuropeptide Y caused a nonparallel rightward shift of the noradrenaline concentration-response curve as compared to that obtained in the presence of 10 nM neuropeptide Y alone. The alpha-trinositol blockade of the facilitatory action of neuropeptide Y was reversible.

Neuropeptide Y Y1 receptors are involved in the vasoconstriction caused by human sympathetic nerve stimulation.

Neuropeptide Y, a novel neurotransmitter, interacts with selective membrane receptors to cause vasoconstriction. Frequency- and concentration-dependent isometric contractions were observed in human inferior mesenteric artery and vein mounted rings that were stimulated with either electrical pulses (70 V, 0.5 ms, 2.5-20 Hz) or noradrenaline. The antagonism elicited by 100 nM tetrodotoxin and 1 microM guanethidine confirmed the neuronal and sympathetic origins of the vasomotor response. Incubation with BIBP 3226 ((R)-N2-(di-phenacetyl)-N-(4-hydroxyphenyl)-methyl-D-arginineam ide), a selective neuropeptide Y Y1 receptor antagonist, significantly reduced the vasoconstriction. The incomplete antagonist activity of BIBP 3226 tends to support the hypothesis of sympathetic co-transmission involving neuropeptide Y, adenosine 5'-triphosphate and noradrenaline. These findings were confirmed in parallel studies using rat superior mesenteric artery and vein ring preparations.

Adenosine 5'-triphosphate (ATP), the neurotransmitter in the prostatic portion of the longitudinal muscle layer of the rat vas deferens.

Suramin (1-100 microM) and alpha, beta-methylene adenosine 5'-triphosphate (AMPCPP, 39 microM), antagonized the motor activity induced by exogenous adenosine 5'-triphosphate (ATP) but not exogenous noradrenaline (NA) in the longitudinal musculature of prostatic (P) and epididymal (E) segments of the rat vas deferens. Likewise, application of these drugs reduced the fast component of the nerve-stimulated contraction in response to a single transmural electrical pulse in E and P. Suramin also blocked in a concentration-dependent fashion, the contractile responses to trains of 1.5, 5, 15 or 30 Hz transmural electrical pulses in P, while it did not affect those in E. AMPCPP obliterated responses to trains of 1.5, 5, and 15 Hz in P, while reducing these responses in E to a significantly lesser extent. Present results strongly support that ATP is the motor transmitter in P, while in E, ATP and NA are likely the co-transmitters responsible for the motor tone.

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.

Gastrointestinal neurotensin receptors: lack of modulation by thyrotropin releasing hormone.

To examine whether thyrotropin releasing hormone (TRH) antagonized gastrointestinal neurotensin receptors in isolated segments of the rat fundus, duodenum and ileum, tissues were superfused, mounted and the isometric tension recorded. Picomoles of neurotensin caused concentration-dependent contractions of the fundus and relaxation of the smooth muscles of the small intestine. Preincubation with 1-10 microM TRH failed to antagonize the activity of neurotensin but potentiated neurotensin-induced relaxation of the ileum. Pretreatment of the tissues with 0.6 microM of neuropeptide fragment 1-11, also failed to block the neurotensin-induced effects but produced a significant potentiation of the relaxant action of neurotensin.

Reciprocal sympatho-sensory control: functional role of nucleotides and calcitonin gene-related peptide in a peripheral neuroeffector junction.

The rat vas deferens has scattered sensory afferens plus a dense network of sympathetic motor efferens; these fibers are not known to interact functionally. We ascertained whether sensory fibers modulate the release of sympathetic transmitters through the release of calcitonin gene-related peptide (CGRP) and reciprocally assessed whether sympathetic transmitters modulate the overflow of ir-CGRP from sensory fibers. The tissue overflow of electrically evoked sympathetic co-transmitters (ATP/metabolites, noradrenaline (NA), and immunoreactive neuropeptide tyrosine (ir-NPY)) and the motor responses elicited were quantified following either exogenous CGRP or capsaicin application to elicit peptide release. Conversely, the outflow of ir-CGRP was examined in the presence of sympathetic transmitters. Exogenous CGRP reduced in a concentration-dependent manner the electrically evoked outflow of ATP/metabolites, NA, and ir-NPY with EC(50) values of 1.3, 0.18, and 1.9 nM, respectively. CGRP also reduced the basal NA overflow. The CGRP-evoked modulation was blocked by CGRP8-37 or H-89. Release of endogenous CGRP by capsaicin significantly reduced the basal overflow of NA, ir-NPY, and the electrically evoked sympathetic transmitter release. ADP, 2-methylthioadenosine-5'-O-diphosphate (2-MeSADP), or UTP decreased the electrically evoked ir-CGRP overflow, whereas clonidine, α,ß-methyleneadenosine 5'-triphosphate (α,ß-mATP), or adenosine (ADO) were inactive. CGRP acting postjunctionally also reduced the motor responses elicited by exogenous NA, ATP, or electrically evoked contractions. We conclude that CGRP exerts a presynaptic modulator role on sympathetic nerve endings and reciprocally ATP or related nucleotides influence the release of ir-CGRP from sensory fibers, highlighting a dynamic sympatho-sensory control between sensory fibers and sympathetic nerve ending. Postjunctional CGRP receptors further contribute to reduce the tissue sympathetic motor tone implying a pre and postjunctional role of CGRP as a sympathetic tone modulator.

BIBP 3226, suramin and prazosin identify neuropeptide Y, adenosine 5'-triphosphate and noradrenaline as sympathetic cotransmitters in the rat arterial mesenteric bed.

The physiological role of neuropeptide Y (NPY) and extracellular adenosine 5'-triphosphate (ATP) in sympathetic neurotransmission is becoming increasingly clear. To assess whether NPY and ATP act as cotransmitters together with noradrenaline (NA) in the sympathetic nerves of the superior mesenteric artery, the changes in perfusion pressure of the arterial mesenteric bed caused by nerve stimulation were recorded. Depolarization of the perivascular superior mesenteric arterial nerves caused frequency- and time-dependent increases in the perfusion pressure that were abolished by guanethidine, which implied the sympathetic origin of these responses. Independent perfusion with either 500 nM BIBP 3226, an NPY Y1 antagonist; 3 microM suramin, a competitive purinoceptor antagonist; or 0.1 nM prazosin, a competitive alpha-1 adrenoceptor antagonist, evoked approximately a 30% reduction in the rise in perfusion pressure caused by the 20- to 30-Hz electrical depolarization of the perimesenteric arterial nerves. Prazosin (0.1 nM) blocked the increases in perfusion pressure caused by electrical stimulation of the perimesenteric nerves but did not significantly reduce the vasomotor effect of exogenous NA. Likewise, 5-methyl urapidil and chloroethylclonidine, alpha-1 adrenoceptor antagonists with selectivity for the alpha-1A and alpha-1B receptor subtypes, respectively, concentration-dependently decreased the increase in perfusion pressure elicited by electrical stimulation of the perimesenteric nerves at concentrations lower than that required to block the vasoconstriction elicited by exogenous NA. The combined perfusion of 3 microM suramin plus 0.1 nM prazosin did not result in a complete inhibition of the physiological response. Only upon the simultaneous application of BIBP plus suramin plus prazosin was the rise in perfusion pressure abolished. These results support the working hypothesis that the sympathetic nerves of the rat mesenteric bed release NPY, ATP and NA that act as postjunctional cotransmitters in this neuroeffector junction.

Neuropeptide Y is released from human mammary and radial vascular biopsies and is a functional modulator of sympathetic cotransmission.

The role of neuropeptide Y (NPY) as a modulator of the vasomotor responses mediated by sympathetic cotransmitters was examined by electrically evoking its release from the perivascular nerve terminals of second- to third-order human blood vessel biopsies and by studying the peptide-induced potentiation of the vasomotor responses evoked by exogenous adenosine 5' triphosphate (ATP) and noradrenaline (NA). Electrical depolarization of nerve terminals in mammary vessels and radial artery biopsies elicited a rise in superfusate immunoreactive NPY (ir-NPY), which was chromatographically identical to a standard of human NPY (hNPY); a second peak was identified as oxidized hNPY. The amount released corresponds to 4-6% of the total NPY content in these vessels. Tissue extracts also revealed two peaks; hNPY accounted for 68-85% of the ir-NPY, while oxidized hNPY corresponded to 7-15%. The release process depended on extracellular calcium and on the frequency and duration of the electrical stimuli; guanethidine blocked the release, confirming the peptide's sympathetic origin. Assessment of the functional activity of the oxidized product demonstrated that while it did not change basal tension, the NA-evoked contractions were potentiated to the same extent as with native hNPY. Moreover, NPY potentiated both the vasomotor action of ATP or NA alone and the vasoconstriction elicited by the simultaneous application of both cotransmitters. RT-PCR detected the mRNA coding for the NPY Y(1) receptor. In summary, the release of hNPY or its oxidized species, elicited by nerve terminal depolarization, coupled to the potentiation of the sympathetic cotransmitter vasomotor responses, highlights the modulator role of NPY in both arteries and veins, strongly suggesting its involvement in human vascular sympathetic reflexes.

[Neuropeptide Y contribution and the physiology of human sympathetic co-transmission. Studies in saphenous vein biopsies]. / Contribución del neuropéptido y a la fisiología de la co-transmisión simpática humana. Estudios en biopsias de vena safena.

BACKGROUND: It is known that the sympathetic varicosities co-store and co-release norepinephrine (NE) together with adenosine S-triphosphate (ATP) and neuropeptide Y (NPY). AIM: To describe the chemical characterization of stored and released NPY from the varicosities of sympathetic nerve terminals surrounding segments of the human saphenous vein, and the vasomotor activity of rings electrically depolarized or contracted by the exogenous application of the co-transmitters. MATERIAL AND METHODS: Saphenous vein tissues were obtained from patients undergoing elective cardiac revascularization surgery. RESULTS: The chromatographic profile of NPY extracted from biopsies is identical to a chemical standard of human NPY. Upon electrical depolarisation of the perivascular sympathetic nerve terminals, we demonstrated the release of NPY to the superfusion media, which did not exceed a 1% of its stored content. The release of the peptide is sensitive to guanethidine, and to extracellular calcium, suggesting that the mechanism of its release is exocytotic in nature. The electrically evoked release of NPY is dependent on the frequency and duration of the electrical pulses. Phenoxybenzamine reduces the electrically evoked release of NPY. Exogenous application of NE and ATP contract saphenous vein rings; the simultaneous application of NE plus ATP causes a synergic response, effect which is further potentiated by the joint co-application of 10 nM NPY. CONCLUSIONS: Present results highlight the role of NPY as a sympathetic co-transmitter in the regulation of human vascular tone.