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.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

A human prion protein peptide (PrP(59-91)) protects against copper neurotoxicity.

Human cellular prion protein (PrP(C)) is involved in several neurodegenerative disorders; however, its normal function is unknown. We report here that a synthetic peptide corresponding to the four-octarepeat sequence of the PrP(C) (PrP(59-91)) protects hippocampal neurons against copper neurotoxic effects in vivo. Using a rat bilateral intrahippocampal injection model, we found that PrP(59-91) protects against copper-induced neurotoxicity, including a recovery in spatial learning performance and a reduced neuronal cell loss and astrogliosis. Previous studies from our laboratory indicated that a tryptophan (Trp) residue plays a key role in the reduction of copper(II) to copper(I); therefore several PrP(59-91) fragments lacking histidine (His) and Trp residues were tested for their capacity to protect from copper toxicity. A PrP(59-91) peptide lacking His residue shows as much neuroprotection as the native peptide; however, PrP(59-91) without Trp residues only partially protected against copper toxicity. The neuroprotective effect not only occurs with PrP(59-91), in fact a full neuroprotection was also observed using just one octamer of the N-terminal region of prion protein. We conclude that the N-terminal tandem octarepeat of the human PrP(C) protects neurons against copper toxicity by a differential contribution of the binding (His) and reducing (Trp) copper activities of PrP(59-91). Our results are consistent with the idea that PrP(C) function is related to copper homeostasis.

Pharmacological identification of P2X1, P2X4 and P2X7 nucleotide receptors in the smooth muscles of human umbilical cord and chorionic blood vessels.

To ascertain the role of extracellular adenosine 5'-triphosphate (ATP) receptors in human placenta circulation, we identified and pharmacologically characterized the P2X receptor population in its superficial vessels. Total RNA was extracted from segments of chorionic and umbilical arteries and veins of terminal placentae delivered by vaginal or Caesarian births. Polymerase chain reaction (PCR), followed by sequencing of the products, identified the presence of P2X 1, 4, 5, 6, and 7mRNAs in smooth muscle from chorionic and umbilical arteries and veins. Umbilical vessels proximal to the fetus expressed the same population of P2X subtypes, except for the P2X(5), but additionally expressed the P2X(2). Rings of chorionic vessels contracted upon addition of nucleotides and analogs with the following relative rank order of potencies in arteries and veins: alpha,beta-methyleneATP>beta,gamma-methyleneATP>PNP>ATP=diBzATP>2-MeSATP>ADP>AMP; in umbilical vessels alpha,beta-methyleneATP was at least 100-fold more potent than ATP. Nucleotide potency was less than that of PGF(2alpha) or endothelin-2, but had the same magnitude as serotonin. ATP-desensitized receptors evidenced cross desensitization to alpha,beta-methyleneATP, 2-MeSATP and diBzATP, effect not observed when desensitization was elicited by alpha,beta-methyleneATP, confirming the presence of various P2X receptor subtypes in the smooth muscles of these vessels. The vasocontractile efficacy of alpha,beta-methyleneATP was unaltered by endothelium removal, while that of ATP was significantly attenuated and those elicited by 2-MeSATP were blunted, indicating the presence of additional endothelial nucleotide receptors. These results suggest that P2X receptors participate in the humoral regulation of placental blood flow.

Diabetes-induced changes in responsiveness of rat bladder and vas deferens to peptides in vitro: susceptibility to reversal by insulin.

Changes in responsiveness of the vas deferens and urinary bladder to bradykinin (BK) receptor agonists (Tyr8-BK and des-Arg9-BK), substance P (SP), and endothelin-1 (ET-1) were assessed 8 weeks after streptozotocin (STZ)-induced diabetes. Preparations from control or STZ-treated (60 mg/kg i.p.) male rats were tested for contractile and neurogenic twitch potentiating (TP, in VD only) effects of all four agonists (1 nM to 0.3 or 3 microM). In diabetic VD, contractile effects of Tyr8-BK, des-Arg9-BK, and SP were enhanced, but ET-1 effects were unchanged. In contrast, TP by des-Arg9-BK was unaffected, that by Tyr8-BK was decreased, and those by SP and ET-1 were increased. In diabetic UB, only contractions to des-Arg9-BK and SP were enhanced. Following insulin replacement (human, 1-3 U/day s.c.), starting 1 week after STZ, TP induced by Tyr8-BK and des-Arg9-BK in VD were further inhibited, but all other changes in both preparations were reversed at least partially. Insulin treatment of nondiabetic rats, however, also affected VD (but not UB) responsiveness, such that contractions to Tyr8-BK and TP by ET-1 were increased, but TP by Tyr8-BK was decreased. Thus, STZ-induced type I diabetes causes important alterations in responsiveness of non-vascular smooth muscle tissues of the rat to BK, SP, and ET-1. Long term insulin replacement, at doses normalising glycaemia, effectively reversed most changes in VD or UB responsiveness, but it is unclear if this is truly due to blocking of STZ-induced changes, since the treatment also affected responsiveness of nondiabetic tissues.

Basal tonic release of nitric oxide coupled to cGMP production regulates the vascular reactivity of the mesenteric bed.

To reveal a basal production of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) in the rat arterial mesenteric bed, mesenteries were perfused in the absence and in the presence of selective blockers of the L-arginine cascade. Endothelium removal or inhibition of NO synthase significantly reduced the release of NO and tissue cGMP. A significant correlation between these messengers was shown. Blockade of soluble guanylyl cyclase with 0.3-10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) only reduced basal cGMP production; 1-100 nM sildenafil (Sild), an inhibitor of phosphodiesterase V, increased basal tissue cGMP without modifying the release of NO. Acetylcholine (0.01-10 microM) caused a concentration-dependent rise in NO and cGMP evoking a proportional vasodilatation, demonstrating the interdependence between these messengers and vascular reactivity. Endothelium removal or NO synthase blockade reduced the acetylcholine-induced increase of messengers and the vasodilatation. ODQ attenuated only the increase in cGMP and the vasodilatation, while sildenafil increased cGMP without significantly altering luminal NO release. The present results highlight a tonic release of NO and its involvement in endothelial-smooth muscle signaling; NO and cGMP are determinants of vascular reactivity.

Clonidine-induced nitric oxide-dependent vasorelaxation mediated by endothelial alpha(2)-adrenoceptor activation.

1. To assess the involvement of endothelial alpha(2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2. In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 microM L(omega)-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3. In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+/-13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L(omega)-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten microM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 - 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4. alpha(2)-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential alpha(2D)-adrenoceptor activation, the K(B)s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5. Rat pretreatment with 100 mg kg(-1) 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+/-15.5 pmol NO. Perfusion with 1 microM guanethidine or 1 microM guanethidine plus 1 microM atropine did not modify the NO surge evoked by 100 nM clonidine. 6. Clonidine and congeners activate endothelial alpha(2D)-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.

[Spontaneous rhythmic contractions of human placental vessels: is it an evidence for a physiological pacemaker in blood vessels?]. / Actividad rítmica espontánea en vasos de placenta humana: un marcapaso fisiológico en los vasos sanguíneos?

BACKGROUND: Placental vessels are not innervated. Therefore the vasomotor activity and vascular tone is not regulated by the nervous system. AIM: To assess the existence of pacemaker mechanisms related to rhythmic motor activity of blood vessels. MATERIAL AND METHODS: Isometric contractions of rings from umbilical and chorionic vessels of term human placentas were monitored. RESULTS: Recordings of the circular layer of chorionic and umbilical vessels revealed rhythmic spontaneous contractions with a frequency of 1.4 +/- 0.05 cycles/min, the duration of each cycle was 42.8 +/- 0.24 s (n = 12). The amplitude of contractions was larger in veins than in arteries, predominating in umbilical vein biopsies, proximal to the fetus. Both the frequency and the amplitude of contractions were relatively constant during the first 30 min. However, after an hour, the frequency declined while the amplitude increased. The absence of the endothelium neither modified the frequency nor the amplitude of the rhythmic activity. Blockage of voltage dependent sodium channels or calcium channels did not alter the frequency of spontaneous contractions, although their magnitude was reduced. Glibenclamide, an ATP-dependent K+ channel blocker or the blockade of gap junctions ablated the frequency and amplitude of spontaneous contractions. CONCLUSIONS: We propose that rhythmic contractions are triggered by pacemaker cells located in the circular layer of the smooth muscle of blood vessels and spread via gap junctions; they likely contribute to the control of blood flow.

[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.

Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat.

The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N(omega)-nitro-L-arginine methyl ester, L-NAME (1-100 microM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 microM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca(2+) ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study.

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.

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.

Synergism between neuropeptide Y and norepinephrine highlights sympathetic cotransmission: studies in rat arterial mesenteric bed with neuropeptide Y, analogs, and BIBP 3226.

Although abundant literature supports the notion that neuropeptide Y (NPY) synergizes in vivo and in vitro, the vasomotor activity elicited by norepinephrine (NE), the converse interaction (i.e., the adrenergic modulation of the NPY vasomotor response) has been less characterized. To assess whether NE synergizes the vasomotor effect of NPY, the rat arterial mesenteric bed was chosen as a model experimental system. Mesenteries were precontracted with NE and few minutes later were perfused with exogenous NPY. Under these conditions, NPY contracted the arterial mesenteric bed with an EC50 value of 0.72 +/- 0.06 nM. NPY was unable to contract this vascular territory without an agonist-induced precontraction. Other agonists, such as endothelin-1, a synthetic analog of prostaglandin F2alpha, or 5-hydroxytryptamine, also were effective primers because in their presence, NPY was a potent vasoconstrictor. In contrast, mesenteries precontracted with KCl failed to evidence the NPY-induced rise in perfusion pressure. Two structural analogs of NPY, PYY and [Leu31, Pro34]NPY, mimicked the activity of NPY. The NPY fragment 13-36 did not elicit such a response. All NPY analogs exhibited less efficacy and potency relative to NPY. The NPY- and related structural analog-induced vasoconstriction was competitively and reversibly antagonized by BIBP 3226; the pA2 of the NPY interaction was 7.0. The application of 0.1 to 1 microM BIBP 3226 or 0.1 to 10 nM prazosin at the peak of the NPY vasomotor response elicited a gradual blockade of the vasoconstriction. Although BIBP 3226 blocked the increase in perfusion pressure elicited by NPY, leaving unaffected the NE-induced tone, 10 nM prazosin blocked the full response, including the NE-induced component. Tissue preincubation with 200 nM nifedipine abolished the NPY-induced vasoconstriction; likewise, the acute application of 10 to 100 nM nifedipine blocked gradually the maximal NPY-induced contraction. Removal of the mesenteric endothelial layer increased the potency of NPY by 2-fold; it also slightly potentiated the antagonist activity of BIBP 3226. The synergism between NPY and NE backs the principle of sympathetic cotransmission.

Central B2 receptor involvement in the antinociceptive effect of bradykinin in rats.

1. The effect of intracerebroventricular (i.c.v.) injection of bradykinin (BK) and related peptides was tested on the dental pulp electrical stimulation threshold (DPEST) in rats. 2. BK (4, 8 and 16 nmol) induced a dose-dependent increase of DPEST, indicative of an antinociceptive effect. 3. I.c.v. injection of equimolar doses of BK-related peptides, Lys-BK and Met-Lys-BK, also induced an increase of DPEST, but the magnitude of the effect was not as intensive as that induced by BK, when the maximum increase of DPEST was considered. The peptide T-kinin induced a short lasting and weak antinociceptive effect. 4. The B1 agonist, des-Arg9-BK (8 nmol) induced a significant antinociceptive effect, but this was not as intensive as that induced by BK. 5. The B2 antagonist D-Arg0-Hyp3-Thi5,8-D-Phe7-BK (D-Arg0) competitively antagonized the BK-induced antinociception. Likewise, Hyp3-Thi5,8-D-Phe7-BK (Hyp) also antagonized BK effect. However, the compound Thi5,8-D-Phe7-BK (Thi), initially considered a pure BK antagonist, induced an antinociceptive effect, supporting previous observations that this peptide can also act as a partial agonist. 6. It is concluded that the dose-dependent antinociceptive effect induced by i.c.v. injection of BK is mediated by the stimulation of brain B2 receptors.

Neuropeptide Y inhibits 3[H]noradrenaline release in the rat vas deferens independently of cAMP levels.

The purpose of the present investigation was to ascertain the functional significance of the reduction in cyclic AMP (cAMP) levels in the inhibitory action of neuropeptide Y (NPY) on [3H]noradrenaline ([3H]NA) release, as well as to further characterize the subtype(s) of NPY receptors involved in the peptide's actions in the rat vas deferens. We studied the effects of NPY, carboxyterminal fragments of this peptide and the NPY analog (Leu31,Pro34)-NPY on three functional responses, namely, the release of [3H]NA and the associated muscle contractions evoked by electrical stimulation, and the accumulation of cAMP stimulated by forskolin. NPY, a known inhibitor of the electrically-evoked [3H]NA release and neurogenic contractions is also a potent inhibitor of the forskolin-stimulated cAMP synthesis in the prostatic portion of the rat vas deferens. However, the ability of NPY to inhibit cAMP accumulation is lost upon tissue denervation, suggesting that this is likely to be a prejunctional effect. Elevation of cAMP levels by the use of the cell permeant analog of cAMP, 8-(p-chlorophenylthio)-cAMP (8pCPTcAMP) increases the electrically-evoked release of [3H]NA. However, the inhibition of [3H]NA release by NPY is not prevented by 8pCPTcAMP. Structure-activity relationship studies reveal that NPY and related peptides inhibit the release of [3H]NA, the muscle contractions and the synthesis of cAMP with a similar pharmacological profile. NPY is the most potent inhibitory agent, whereas [Leu31,Pro34]-NPY and NPY13-36, the respective Y1 and Y2 selective agonists, display similar potencies to inhibit the three responses. It is concluded that NPY inhibits neurotransmission in the rat vas deferens through the activation of a peptide receptor different from the known NPY-Y1 or NPY-Y2 receptor subtypes. NPY receptor activation in the vas deferens is negatively coupled to adenylyl cyclase activity. This intracellular signalling pathway is, however, not likely to mediate the peptide effects on the prejunctional regulation of noradrenaline release.

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.

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.

[Opioid receptors in the era of molecular biology: they account for more than 6,000 years of empirical pharmacology]. / Receptores opioides a la luz de la biología molecular: explican mas de 6.000 años de farmacología empírica.

The medicinal use of opium and of morphine in different cultures and ancient civilizations is described. Research within the past 40 years have demonstrated the existence of brain opiate receptors. Morphine and related opioid analgetic interact at these sites in the nervous system to produce the characteristic pharmacological effects of these drugs. The opiate receptors have structural homologies with a variety of other cell membrane receptors; they activate second messenger-based chemical transduction systems in the cell membrane and are endowed with several regulation mechanisms. These opiate receptors are presumably activated under specific physiological conditions by endogenous ligands (opiopeptins). It is currently thought that morphine mimicks the opiopeptins by interacting with these receptors either at different molecular subsites or with a different mode of action.

Characterization of kinin receptors modulating neurogenic contractions of the mouse isolated vas deferens.

1. This study analyses the receptors mediating the effects of bradykinin (BK) and analogues on neurogenic twitch contractions of the mouse isolated vas deferens evoked, in the presence of captopril (3 microM), by electrical field stimulation with trains of 4 rectangular 0.5 ms pulses of supramaximal strength, delivered at a frequency of 10 Hz every 20 s. 2. BK (0.1-300 nM) induced a graded potentiation of twitches, with an EC50 (geometric mean and 95% confidence limits) of 4.5 nM (1.7-11.6) and an Emax of 315 +/- 19 mg per 10 mg of wet tissue (n = 6). Similar results were obtained in tissues challenged with Lys-BK, [Hyp3]-BK, Met,Lys-BK and the selective B2 receptor agonist [Tyr(Me)8]-BK (0.1-300 nM). 3. The selective B2 receptor antagonists, Hoe 140 (1-10 nM) and NPC 17731 (3-30 nM), caused graded rightward shifts of the curve to BK-induced twitch potentiation, yielding apparent pA2 values of 9.65 +/- 0.09 and 9.08 +/- 0.13, respectively, and Schild plot slopes not different from 1. Both antagonists (100 nM) failed to modify similar twitch potentiations induced by substance P (3 nM) or endothelin-1 (1 nM). Preincubation with the selective B1 receptor antagonist, [Leu8,des-Arg9]-BK (1 microM), increased the potentiating effect of BK on twitches at 30-300 nM. 4. In contrast to BK, the selective B1 receptor agonist, [des-Arg9]-BK (0.3-1000 nM) reduced the amplitude of twitches in a graded fashion, with an IC50 of 13.7 nM (10.4-16.1) and an Imax of 175 +/- 11 mg (n = 4). The twitch depression induced by [des-Arg9]-BK (300 nM) was not affected by Hoe140 (30nM) or NPC 17731 (100nM), but was abolished by the selective B1 receptor antagonist,[Leu8,des-Arg9]-BK (1 microM), which did not modify the twitch inhibitory effect of clonidine (1 nM) or morphine (300 nM).5. In non-stimulated preparations, BK (100 nM) also potentiated, in a Hoe 140-sensitive (10 nM)manner, the contractions induced by ATP (100 microM), but not by noradrenaline (10 microM), whereas[des-Arg9]-BK (300 nM) did not modify the contractions induced by either agonist.6. It is concluded that the mouse vas deferens expresses both B1 and B2 receptors, which modulate sympathetic neurotransmission in opposing ways. Neurogenic contractions are inhibited by stimulation of possibly prejunctional B, receptors, whereas activation of B2 receptors increases twitch contractions,in part by amplifying the responsiveness of the smooth muscle cells to the sympathetic co-transmitter ATP.

[Reactivity of internal mammary artery and saphenous vein used in human myocardial revascularization: studies with endogenous vasoconstrictors and vasodilators]. / Reactividad de la arteria mamaria interna y vena safena utilizadas en revascularización miocárdica humana: estudios con vasoconstrictores y vasodilatadores endógenos.

Biopsies of human internal mammary artery and saphenous vein were examined to ascertain the functional integrity of these vessels employed in myocardial revascularization. Studies were performed in vascular rings derived from 28 patients without previous consideration of age, sex, underlying additional pathology or drug treatments previous to and during surgery. Isometric muscle contraction of the circular muscle layer was monitored. Endothelin-1 (ET) is equipotent as a vasoconstrictor in arteries and veins, with a potency at least 10 to 100-fold that of noradrenaline (NA) or serotonin (5-HT). The potency of ET, NA or 5-HT is unaltered by mechanical removal of the endothelial cell layer. Arterial rings precontracted with NA relaxed in a concentration-dependent fashion in the presence of acetylcholine and sodium nitroprusside. Whereas the potency of nitroprusside was unaltered by removal of the endothelium, the efficacy of acetylcholine was greatly reduced. Saphenous vein rings were refractory to acetylcholine but not nitroprusside. Results open new perspectives to explain the larger patency of internal mammary artery grafts as compared to that of saphenous vein grafts in human myocardial revascularization.