J. Pablo Huidobro-Toro

Dissecting the Facilitator and Inhibitor Allosteric Metal Sites of the P2X4 Receptor Channel CRITICAL ROLES OF CYS132 FOR ZINC POTENTIATION AND ASP138 FOR COPPER INHIBITION

Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and structurally related phenylethylamines are potent 5-HT2A receptor antagonists in Xenopus laevis oocytes

We recently described that several 2‐(2,5‐dimethoxy‐4‐substituted phenyl)ethylamines (PEAs), including 4‐I=2C‐I, 4‐Br=2C‐B, and 4‐CH3=2C‐D analogs, are partial agonists at 5‐HT2C receptors, and show low or even negligible intrinsic efficacy at 5‐HT2A receptors. These results raised the proposal that these drugs may act as 5‐HT2 antagonists. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5‐HT2A or 5‐HT2C receptors. The above‐mentioned PEAs and its 4‐H analog (2C‐H) blocked the 5‐HT‐induced currents at 5‐HT2A, but not at the 5‐HT2C receptor, revealing 5‐HT2 receptor subtype selectivity. The 5‐HT2A receptor antagonism required a 2‐min preincubation to attain maximum inhibition. All PEAs tested shifted the 5‐HT concentration–response curves to the right and downward. Their potencies varied with the nature of the C(4) substituent; the relative rank order of their 5‐HT2A receptor antagonist potency was 2C‐I>2C‐B>2C‐D>2C‐H. The present results demonstrate that in X. laevis oocytes, a series of 2,5‐dimethoxy‐4‐substituted PEAs blocked the 5‐HT2A but not the 5‐HT2C receptor‐mediated responses. As an alternative hypothesis, we suggest that the psychostimulant activity of the PEAs may not be exclusively associated with partial or full 5‐HT2A receptor agonism.

Potency of three opiate antagonists to reverse the inhibitory activity of dynorphin, enkephalins and opioid-like alkaloids on the guinea pig ileum

To test the hypothesis that dynorphin is a K-opiate agonist acting on the myenteric plexus, the potency of two benzomorphan antagonists (Win 44, 441 and Mr 2266) to block the inhibitory action of dynorphin, enkephalins and opioid alkaloids was determined on the longitudinal muscle preparation of the guinea pig ileum. The effectiveness of these antagonists was compared to that of naloxone. Antagonistic potency was established by calculating the apparent antagonist dissociation constant, Ke, as derived from Schild plots. Win 44, 441 and Mr 2266 were about 7-8 times more potent than naloxone against dynorphin, dynorphin-(1-13) or ethylketocyclazocine. Although the Ke obtained with Win 44, 441 or Mr 2266 against dynorphin or ethylketocyclazocine were significantly lower than those of naloxone, the values obtained for these antagonists did not differ significantly in the case of each of these agonists. With respect to the antagonism of the enkephalins or normorphine, Win 44, 441 was the most potent antagonist. Its Ke value for the enkephalins was 2.5-3 times lower than those for dynorphin or ethylketocyclazocine and in comparison to naloxone, Win 44, 441 was about 5 times more potent. Although Mr 2266 was a potent antagonist of dynorphin, ethylketocyclazocine, the enkephalins or normorphine, it showed no selectivity of action. The fact that the 3 opiate antagonists evidenced similar Ke values for dynorphin and ethylketocyclazocine, but different ones for the enkephalins or normorphine supports the conclusion that dynorphin activates preferentially K- but not mu-opiate receptors in the myenteric plexus.

Pharmacological characterization of the inhibitory effects of neurotensin on the rabbit ileum myenteric plexus preparation

1 Neurotensin in picomolar concentrations caused a concentration‐related inhibition of the spontaneous contractile activity of the longitudinal muscle from the rabbit isolated ileum. Neurotensin was approximately 100 times more potent than adrenaline and about 10000 times as active as adenosine triphosphate (ATP) in producing similar relaxations. 2 The neurotensin‐induced inhibitory effect did not follow activation of adrenoceptors or P1‐purinoceptors since the effect of the neuropeptide was not antagonized by a combination of phentolamine plus (−)‐propranolol, nor by pretreatment with theophylline. 3 Tetrodotoxin did not reduce the potency of neurotensin in relaxing the rabbit ileum, suggesting that the neurotensin‐induced inhibition is not neuronally mediated. The inhibitory responses of neurotensin were blocked non‐competitively by apamin. 4 The inhibitory effect of neurotensin was short‐lived with the subsequent development of tachyphylaxis, which was not crossed to the inhibitory action of adrenaline or ATP. Similarly, when tachyphylaxis to adrenaline or to ATP was established, the inhibitory action of neurotensin was unaffected. Desensitization was characterized by a gradual shift of the neuropeptide concentration‐response curve to the right and downwards. 5 Preincubation of rabbit ileum strips with 10 nm dynorphin (1–13) significantly increased the inhibitory action of neurotensin.

Neurotensin receptors on the ileum of the guinea-pig: Evidence for the coexistence of inhibitory and excitatory receptors

Neurotensin caused a complex muscular response of the longitudinal muscle-myenteric plexus preparation of guinea-pig ileum: picomoles of neurotensin produced inhibition while larger concentrations caused an inhibitory effect followed by a delayed dose-dependent contraction. The inhibitory phase of the neurotensin-induced muscular activity was not modified by tetrodotoxin but was potently antagonized in a non-competitive manner by apamin, a bee venom toxin. The contractile component was blocked by tetrodotoxin but not by apamin. These toxins were used to dissect the neurotensin muscular response into an inhibitory phase and an excitatory component. It was possible to further characterize the two neurotensin muscular components by their kinetics of desensitization. The inhibitory neurotensin response showed a fast rate of desensitization and presented a relatively low spare receptor capacity. In contrast, desensitization to the excitatory action of neurotensin was much slower, the excitatory receptors apparently having a larger spare receptor capacity. Desensitization to the action of neurotensin was selective for the neuropeptide not altering the contractile activity of substance P, angiotensin II, bradykinin, histamine or acetylcholine. These results strongly suggest the presence of two subsets of neurotensin receptors in the ileum: the inhibitory set probably localized at the postsynaptic effector level and excitatory neurotensin receptors probably of neuronal origin whose function is probably to modulate the release of neurotransmitters. The physiological implications of these two subtypes of neurotensin receptors in the control of gastrointestinal motility are discussed.

Dual agonist-antagonist effects of 5-hydroxytryptamine (5-HT) in the guinea pig ileum: Evidence for a selective receptor desensitization effect

The application of 5-HT to strips of whole ileum or the longitudinal muscle-myenteric plexus preparation from guinea pigs caused dose-dependent muscle contractions that were followed by relaxation to baseline tension without washing off the drug (fade). The contractile effect of a subsequent addition of 5-HT, 4 min after a priming dose of 5-HT, was markedly reduced. Increasing priming doses of 5-HT caused proportional increases in the 5-HT Emax50, up to a priming dose that completely abolished the contractile effects caused by further additions of 5-HT. The auto-blockade of the 5-HT responses was selective to drugs acting on serotonergic receptors. 5-HT did not antagonize the effects of acetylcholine, dimethylphenylpiperazinium, nicotine, histamine, prostaglandin E2, substance P or angiotensin II. N-methyl-5-HT was the most potent analogue in mimicking the effects of 5-HT to produce auto-blockade and fade of the contractile responses. Other structural analogues of 5-HT that shared the 5-HT blocking effect were 5-methoxytryptamine, 5,6-dihydroxytryptamine, and N,N-dimethyltryptamine although these compounds were considerably less potent than 5-HT as blockers or as agonists. Results suggest that fade and the auto-blockade are part of a common effect and are discussed in relation to a model of drug-induced selective receptor desensitization.

Single-dose tolerance to antinociception, and physical dependence oin β-endorphin in mice

Abstract β-Endorphin (B-EN) injected intracerebroventricularly in mice produced a rapid onset, dose-dependent antinociceptive effect. The median analgesic dose (AD50) 30 min following administration was found to be 270 ng/mouse (3.7 nmoles/kg). B-En produced an acute, single-dose tolerance development was demonstrable with doses twice or more the AD50. Tolerance was maximal at about 12 h following the priming dose and disappeared within 48 h. Tolerance was maximal at about 12 h following the priming dose and disappeared within 48 h. Tolerance was accompanied by some degree of physical dependence as noted by signs of naxolone-precipitated withdrawal similar to those elicitable in the morphine-dependent state. Tolerance development to B-EN was blocked by the simultaneous adminstration of naloxone and also by pretreatment with 0.35 mg/kg actinomycin D or 30mg/kg cycloheximide 30 min before B-EN. It appears that single-dose tolerance to B-En was initiated by processes similar to those involved with tolerance resulting from chronic adminsitration of morphine.

CONTRACTILE EFFECT OF MORPHINE AND RELATED OPIOID ALKALOIDS, β‐ENDORPHIN AND METHIONINE ENKEPHALIN ON THE ISOLATED COLON FROM LONG EVANS RATS

1 Morphine and related synthetic surrogates as well as β‐endorphin and methionine enkephalin caused a contractile response of the longitudinal musculature of the terminal colon of Long Evans rats. 2 The muscular contraction caused by the narcotic analgesics exhibited stereospecificity, with levorphanol being about 50 times more potent than dextrorphan and (−)‐methadone 4 times more potent than (+)‐methadone. In addition, the rank order in potency of a homologous series of N‐alkyl substituted norketobemidones demonstrated that the activity of these compounds in eliciting contractile responses corresponded to that for analgesic efficacy in the rat and also correlated to the ability of these derivatives to inhibit the muscular twitch evoked by electrical stimulation of the guinea‐pig ileum. 3 Naloxone blocked the contractile response of the opiates following competitive kinetics; the naloxone pA2 values for morphine, etorphine, levorphanol and methadone were very close, in spite of the marked differences in potency of these agents. 4 The contractile effect of morphine on the rat colon was abolished by incubation of the tissues with tetrodotoxin 2.0 × 10−7 M or by decreasing the external Ca2+ level 100 fold. Increasing the external Ca2+ concentration caused an apparent non‐competitive antagonism of the response to morphine. 5 Pretreatment of the tissues with hexamethonium 8.3 × 10−5 M caused a modest antagonism of the morphine effect while atropine 5.8 × 10−7M did not significantly modify the morphine contractile effect. In contrast, methysergide 10−5 M caused a 10 fold increase in the morphine EC50. 6 Colons from rats rendered tolerant‐dependent on morphine were markedly less sensitive to the contractile effects of morphine than those from placebo‐treated controls. Tolerance to morphine was also accompanied by an increased sensitivity to the contractile effects of 5‐hydroxytryptamine (5‐HT). 7 A marked increase in the spontaneous muscular activity of segments of the terminal colon of rats chronically treated with morphine was found to occur upon removal of the residual morphine in the tissues by repetitive washings. The spontaneous activity was arrested by applications of morphine, suggesting that physical dependence can be demonstrated in vitro in this particular preparation. 8 It is concluded that the opiate‐induced contractile response is mediated via stereospecific, naloxone‐sensitive, opiate receptors and that the muscular response involves the activation of a 5‐HT neurone in the nerve terminals of the colon.

Reactive Oxygen Species Potentiate the P2X2 Receptor Activity through Intracellular Cys430

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69 residue segment in the C terminal, termed P2X2b, as the donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera but was absent in oocytes expressing P2X4/2b chimera. Site-directed mutagenesis experiments revealed that the Cys430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H2O2) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents but not P2X2bR and P2X2a–C430A and P2X2a–C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H2O2 potentiation. Alkylation of Cys430 residue with methylmethane-thiosulfonate also abolished the mercury and H2O2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys430 residue is an intracellular P2X2aR redox sensor.

Endothelin reduces microvascular blood flow by acting on and venules of the hamster cheek pouch

Superfusion of the cheek pouch with 0.1–10 nM endothelin (E) produced a concentration-related reduction in the clearance of 22Na+ used as an indicator of microvascular plasma flow. The median effective concentration was about 2 nM. The time course of E action was also concentration related. Superfusion with 10 nM E for 10 min caused a greater than 80% reduction in 22Na+ clearance: the rate at which the action of E started was significantly faster than the rate at which its action ended. Recovery did not exceed 70% even though the tissue was superfused with drug-free buffer for 90 min. The E-induced reduction in 22Na+ clearance was associated with vasoconstriction, as determined by intravital microscopy. Arterioles of 4th branching order were more sensitive to E action than arterioles of 1st or 2nd order; however, the constriction lasted considerably longer in the latter vessels. E-induced venular constriction followed a pattern analogous to that of arterioles of the same category, with the exception that the finer venules responded the least. Pretreatment of the cheek pouch with 300 nM nifedipine diminished but did not abolish the 1 nM E-induced reduction in 22Na+ clearance. and the recovery of clearance upon E washout was not accelerated by nifedipine.