Juan Pablo Huidobro-Toro

ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology.

Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y-Y1 receptors.

Abstract: Neuropeptide Y (NPY) appears to be involved in the sympathetic regulation of vascular tone. To assess the putative role of NPY in mesenteric circulation, the release and biological effect of NPY were examined after electrical stimulation of perimesenteric arterial nerves. Nerve stimulation with trains of 2–30 Hz increased the perfusion pressure of the arterially perfused rat mesenteric bed in a frequency‐ and time‐dependent fashion. Trains of 15–30 Hz significantly displaced to the left, approximately threefold, the noradrenaline (NA)‐induced pressor concentration‐response curve, in addition to increasing significantly its efficacy. Perfusion with 10 nM exogenous NPY mimicked the electrical stimulation effect, causing a threefold leftward shift of the NA concentration‐response curve and increasing the maximal NA response. These effects were antagonized by 100 nM BIBP 3226, indicating the activity of NPY‐Y1 receptors. Electrical stimulation of the perimesenteric nerves released immunoreactive NPY (ir‐NPY) in a frequency‐dependent fashion; the ir‐NPY coelutes with synthetic NPY as confirmed by HPLC. Both the electrically induced pressor response and the calcium‐dependent release of NPY were obliterated in preparations perfused with 1 µM guanethidine or in rats pretreated intravenously for 48 h with 6‐hydroxydopamine, thus revealing the sympathetic origin of these phenomena. Only a small proportion of the total NPY content in the perimesenteric arterial nerves is released after electrical stimulation. Chromatographic studies of the physiological sources of the ir‐NPY support that NPY fragments are generated via peptidase degradation. The present findings demonstrate that NPY is released from the perimesenteric arterial sympathetic nerves and acts, via the activation of NPY‐Y1 receptors, as the mediator responsible for the potentiation of NAs effect on perfusion pressure in the isolated rat mesenteric bed.

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 μm) plus guanethidine (3.4 μm). 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 μm 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 F2α 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.

A human prion protein peptide (PrP59-91) protects against copper neurotoxicity

Human cellular prion protein (PrPC) 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 PrPC (PrP59–91) protects hippocampal neurons against copper neurotoxic effects in vivo. Using a rat bilateral intrahippocampal injection model, we found that PrP59–91 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 PrP59–91 fragments lacking histidine (His) and Trp residues were tested for their capacity to protect from copper toxicity. A PrP59–91 peptide lacking His residue shows as much neuroprotection as the native peptide; however, PrP59–91 without Trp residues only partially protected against copper toxicity. The neuroprotective effect not only occurs with PrP59–91, 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 PrPC protects neurons against copper toxicity by a differential contribution of the binding (His) and reducing (Trp) copper activities of PrP59–91. Our results are consistent with the idea that PrPC function is related to copper homeostasis.

Interactions between morphine and the opioid-like peptides in the rat vas deferens.

1 Morphine, methadone, levorphanol, pethidine, etonitazene and related morphine‐like alkaloids produced an increase in the electrically‐evoked muscular contraction of the rat vas deferens. In contrast, the enkephalins and β‐endorphin caused inhibition of the twitching. 2 The concentration of β‐endorphin required to inhibit by 50% the muscular twitch was about 50 to 100 times less than that of the enkephalins. 3 Pretreatment of the vasa with morphine antagonized the inhibition of the neuromuscular transmission caused by either β‐endorphin or enkephalin. 4 Conversely, pretreatment with β‐endorphin sensitized the vasa to the increase in twitch tension caused by morphine. 5 Morphine did not alter the sensitivity to exogenously administered noradrenaline, dopamine or potassium.

Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.

ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

Wnt-5a increases NO and modulates NMDA receptor in rat hippocampal neurons

Wnt signaling has a crucial role in synaptic function at the central nervous system. Here we evaluate whether Wnts affect nitric oxide (NO) generation in hippocampal neurons. We found that non-canonical Wnt-5a triggers NO production; however, Wnt-3a a canonical ligand did not exert the same effect. Co-administration of Wnt-5a with the soluble Frizzled related protein-2 (sFRP-2) a Wnt antagonist blocked the NO production. Wnt-5a activates the non-canonical Wnt/Ca(2+) signaling through a mechanism that depends on Ca(2+) release from Ryanodine-sensitive internal stores. The increase in NO levels evoked by Wnt-5a promotes the insertion of the GluN2B subunit of the NMDA receptor (NMDAR) into the neuronal cell surface. To the best of our knowledge, this is the first time that Wnt-5a signaling is related to NO production, which in turn increases NMDARs trafficking to the cell surface.

Excitatory neurotensin receptors on the smooth muscle of the rat fundus: Possible implications in gastric motility

1 Picomolar concentrations of neurotensin caused concentration‐dependent contractions of the longitudinal musculature of the fundus of the rat stomach. The EC50 of neurotensin was approximately 1.5 nm. On a molar basis neurotensin was about 5–10 times more potent than 5‐hydroxytryptamine (5‐HT) and approximately 80 times as active as acetylcholine in producing similar contractions. 2 Studies with structurally related peptides indicated that whereas the carboxy terminal portion of neurotensin was essential for biological activity, a substantial part of its amino terminus end could be removed without affecting its potency. The EC50 for the neurotensin fragment 8–13 was identical to that of neurotensin, however its 1–8 or 1–11 fragments were completely inactive. 3 Tetrodotoxin did not modify the potency of neurotensin or structurally related analogues suggesting that the neurotensin receptor is probably located on the smooth muscle membrane. In addition, the potency of neurotensin in contracting the fundus was not modified by pretreatment with atropine, methysergide or diphenhydramine. 4 Fade to the contractile response of neurotensin was followed by the development of tachyphylaxis; desensitization was concentration‐dependent and characterized by a shift in the agonist concentration‐response curve to the right and downwards. Desensitization with a priming concentration of neurotensin (approx. EC50) caused a substantial blockade of its excitability. 5 There was cross‐desensitization between neurotensin and the contractile activity of neurotensin 8–13 or xenopsin, but not with angiotensin II, bradykinin, substance P, acetylcholine, 5‐HT or histamine. 6 Pretreatment of the fundus strip with verapamil 0.3–1 μm antagonized in a concentration‐dependent fashion the neurotensin‐induced contractions but not the muscular contractions caused by acetylcholine. 7 It is concluded that neurotensin activates a specific excitatory receptor probably located on the cell membrane of the smooth muscles of the rat fundus. In addition, we suggest that this receptor is somehow related to a voltage‐dependent calcium channel, sensitive to verapamil.

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.