G-protein-linked serotonin receptors in mouse kidney exhibit identical properties to 5-HT1b receptors in brain

The serotonin 1b (5-HT1b) receptor is thought to mediate both pre- and postsynaptic actions of serotonin. Until recently 5-HT1b sites were thought to be present only in rodent brain. We now report the presence of high-affinity [125I]iodocyanopindolol [( 125I] ICYP) binding sites in the mouse renal medulla with properties identical to those of brain 5-HT1b receptors. In vitro receptor autoradiography demonstrates that [125I]ICYP binding is highly localized to the outer stripe of the renal medulla. Association and dissociation kinetics, saturation analysis and competition displacement analyses indicate that renal medullary [125I]ICYP binding sites exhibit identical properties with brain 5-HT1b receptors. Incubation of renal medullary or brain membranes with guanylimidodiphosphate results in a decreased affinity of 5-HT1b sites for 5-HT and [125I]ICYP; this can be reversed by the addition of a purified mixture of G proteins (Gi/Go). Treatment of brain or kidney membranes with N-ethylmaleimide results in a decrease in 5-HT1b binding which can also be restored by reconstitution with purified G proteins. Adenylyl cyclase from renal medullary homogenates or minces can be stimulated more than 3-fold by forskolin and attenuated by 5-HT. These results indicate that mouse kidney contains high-affinity 5-HT1b receptors with identical properties to those found in brain. These are localized in the outer stripe of the renal medulla and are functionally coupled to adenylyl cyclase inhibitor (Gi) G-proteins.     

Cardiovascular adenosine receptors: Expression,actions and interactions

Intra- and extracellular adenosine levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenge and tissue injury. Extracellular adenosine engages members of the G-protein coupled adenosine receptor (AR) family to mediate generally beneficial acute and adaptive responses within all constituent cells of the heart. In this way the four AR sub-types—A₁, A_2A, A_2B, and A₃Rs—regulate myocardial contraction, heart rate and conduction, adrenergic control, coronary vascular tone, cardiac and vascular growth, inflammatory–vascular cell interactions, and cellular stress-resistance, injury and death. The AR sub-types exert both distinct and overlapping effects, and may interact in mediating these cardiovascular responses. The roles of the ARs in beneficial modulation of cardiac and vascular function, growth and stress-resistance render them attractive therapeutic targets. However, interactions between ARs and with other receptors, and their ubiquitous distribution throughout the body, can pose a challenge to the implementation of site- and target-specific AR based pharmacotherapy. This review outlines cardiovascular control by adenosine and the AR family in health and disease, including interactions between AR sub-types within the heart and vessels.     

Histamine receptors: subclasses and specific ligands

In this review the three main types of histamine receptors are discussed together with their specific ligands. For the classical H1-receptors much emphasis is put on the mechanism by which the receptor is stimulated. For the H1- and H2-receptor the review includes information on the several models available for establishing agonistic or antagonistic activity. In the section on the H3-receptor the ligands are discussed as well as the possible physiological role of this receptor. In the final paragraphs some less well defined activities are presented.     

Calcitonin gene-related peptide: multiple actions, multiple receptors.

Calcitonin gene-related peptide (CGRP) shows diversity both in its effects and its receptors. It is likely to have roles as a neurotransmitter, neuromodulator, local hormone and trophic factor. Its effects include rapid changes in neuronal activity, relaxation of many types of smooth muscle, actions on metabolism and changes in gene expression. Receptor heterogeneity has been revealed from experiments comparing agonist potency ratios and antagonist affinities. The evidence from these approaches is reviewed in this article and a speculative receptor classification scheme is proposed. Some of the likely future directions for CGRP research are discussed.     

Visuo-motor imagery of specific manual actions: A multi-variate pattern analysis fMRI study

An important human capacity is the ability to imagine performing an action, and its consequences, without actually executing it. Here we seek neural representations of specific manual actions that are common across visuo-motor performance and imagery. Participants were scanned with fMRI while they performed and observed themselves performing two different manual actions during some trials, and imagined performing and observing themselves performing the same actions during other trials. We used multi-variate pattern analysis to identify areas where representations of specific actions generalize across imagined and performed actions. The left anterior parietal cortex showed this property. In this region, we also found that activity patterns for imagined actions generalize better to performed actions than vice versa, and we provide simulation results that can explain this asymmetry. The present results are the first demonstration of action-specific representations that are similar irrespective of whether actions are actively performed or covertly imagined. Further, they demonstrate concretely how the apparent cross-modal visuo-motor coding of actions identified in studies of a human "mirror neuron system" could, at least partially, reflect imagery.     

Pharmacological analysis of the actions of SKF 82526 on cardiovascular dopamine receptors

We have performed experiments with SKF 82526, a selective dopamine (DA1) receptor agonist to determine whether this compound would activate either ganglionic and/or central DA receptors. Bilateral hindlimb perfusion was carried out at controlled flow rates and changes in hindlimb perfusion pressure were recorded to evaluate the action of SKF 82526 on vascular resistance in anesthetized dogs. Intracisternal administration of SKF 82526 (10 and 40 micrograms/kg) did not produce any changes in blood pressure, heart rate or hindlimb vascular resistance. When the same doses were administered i.v., SKF 82526 produced hypotension and a decrease in perfusion pressure in the innervated limb, whereas perfusion pressure in the denervated limb was not altered. Intravenous SKF 82526 did not produce any changes in heart rate. When given into the lower abdominal aorta, SKF 82526 caused a dose-dependent decrease in perfusion pressure only in the innervated hindlimb, no significant changes in perfusion pressure occurred in the denervated limb. The hypotensive and the hindlimb vasodilatory actions of SKF 82526 could be antagonized by RS-sulpiride. It was discovered that SCH 23390, a selective DA1 receptor antagonist was most potent in blocking the hypotensive action of i.v. SKF 82526; however, it did not influence the neurogenic hindlimb vasodilation produced by intra-aortic SKF 82526. On the other hand, R-sulpiride, another selective DA1 receptor antagonist significantly antagonized the hypotensive as well as hindlimb vasodilatory actions of SKF 82526. S-sulpiride, a selective DA2 receptor antagonist, was least effective in blocking hypotension and did not influence the hindlimb vasodilatory action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine-like actions of aminoglycosides at recombinant N-methyl-D-aspartate receptors.

Recent pharmacological studies have led to the hypothesis that aminoglycoside-induced ototoxicity is an excitotoxic process mediated, at least in part, by a polyamine-like modulation of N-methyl-D-aspartate (NMDA) receptors. To explore this hypothesis, we compared the effects of several aminoglycosides (neomycin B, kanamycin A, streptomycin, and dihydrostreptomycin) with spermine on recombinant NMDA receptors of defined composition expressed in Xenopus oocytes. Like spermine, aminoglycosides potentiate agonist-induced responses in the presence of both saturating glycine ("glycine-independent") and subsaturating glycine ("glycine-dependent") concentrations on NR1A/2B receptors. Likewise, aminoglycosides and spermine potentiated the agonist responses under glycine-dependent conditions on NR1A/2A receptors. Despite these similarities, several prominent differences were observed between spermine and aminoglycosides as well as among individual aminoglycosides. For example, neomycin B, streptomycin, and kanamycin A, but neither spermine nor dihydrostreptomycin, potentiated glycine-dependent responses on NR1A/2C receptors. Differences between spermine and aminoglycosides also were observed with respect to the voltage dependence of polyamine-like actions. For example, under glycine-dependent conditions, potentiation at NR1A/2B receptors by spermine was voltage dependent, decreasing as the holding potential was changed from -35 to -85 mV; in contrast, potentiation induced by aminoglycosides at NR1A/2B receptors was voltage independent. No direct relationships emerged between the effect of an aminoglycoside at a specific NMDA receptor subtype and the ototoxicity of these antibiotics.     

Presynaptic delta opioid receptors regulate ethanol actions in central amygdala

Endogenous opioid systems are implicated in the reinforcing effects of ethanol consumption. For example, delta opioid receptor (DOR) knockout (KO) mice show greater ethanol consumption than wild-type (WT) mice (Roberts et al., 2001). To explore the neurobiological correlates underlying these behaviors, we examined effects of acute ethanol application in brain slices from DOR KO mice using whole-cell patch recording techniques. We examined the central nucleus of amygdala (CeA) because the CeA is implicated in alcohol reinforcement (Koob et al., 1998). We found that the acute ethanol effects on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were greater in DOR KO mice than in WT mice. Ethanol increased the frequency of miniature IPSCs (mIPSCs) significantly more in DOR KO mice than in WT mice. In CeA of WT mice, application of ICI 174864 [[allyl]2-Tyr-alpha-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH], a DOR inverse agonist, augmented ethanol actions on mIPSC frequency comparable with ethanol effects seen in DOR KO mice. Superfusion of the selective DOR agonist D-Pen(2),D-Pen(5)-enkephalin decreased the mean frequency of mIPSCs; this effect was reversed by the DOR antagonist naltrindole. These findings suggest that endogenous opioids may reduce ethanol actions on IPSCs of CeA neurons in WT mice through DOR-mediated inhibition of GABA release and that the increased ethanol effect on IPSCs in CeA of DOR KO mice could be, at least in part, due to absence of DOR-mediated inhibition of GABA release. This result supports the hypothesis that endogenous opioid peptides modulate the ethanol-induced augmentation of GABA(A) receptor-dependent circuitry in CeA (Roberto et al., 2003).     

Studies on the cardiovascular actions of central histamine H1 and H2 receptors

Histamine administered i.c.v. to conscious freely moving rats results in dose-related pressor responses and bradycardia. The selective H1 agonist pyridylethylamine (PEA) and the H2 agonist impromidine (IMP) were utilized to characterize the central receptor subtypes involved in the central cardiovascular actions of histamine. Blood pressure and heart rate were monitored directly via indwelling carotid catheters, and drugs were administered i.c.v. through permanently implanted cannulas. Central administration of PEA or IMP produced dose-dependent pressor responses and bradycardia. The H1 antagonist chlorpheniramine blocked the pressor response, but not the bradycardia produced by PEA. In contrast, the H2 antagonist BMY-25405 blocked both the increase in blood pressure and the bradycardia induced by IMP. Crossover experiments were carried out to examine the specificity of the antagonists' actions. Chlorpheniramine failed to block the actions of IMP and BMY-25405 likewise was ineffective in blocking the actions of PEA. These results suggest that both central H1 and H2 receptors mediate the central pressor effects of histamine, and that PEA and IMP are selectively acting at their respective receptors in the brain. The heart rate effects appear to be mediated directly by central H2 actions, as BMY-25405 was capable of blocking the heart rate changes. The finding that IMP is less potent in the central nervous system, compared to its known greater potency in the periphery, suggests that peripheral and central H2 receptors may be pharmacologically distinct.     

Antigen-induced aggregation and modulation of receptors on hapten- specific B lymphocytes

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.     

Involvement of peripheral-type benzodiazepine receptors in the proconvulsant actions of pyrethroid insecticides

It has been demonstrated previously that select Type II pyrethroids are potent proconvulsants in the rat and that the proconvulsant actions of deltamethrin are blocked by administration of PK 11195, an antagonist of the peripheral-type benzodiazepine receptor (PTBR). The present investigation has extended these findings to include various Type I pyrethroids as proconvulsants. Additionally, the proconvulsant activity of cismethrin was reversed by administration of PK 11195. Pyrethroid displacement of specific [3H]Ro5-4864 binding to rat brain membranes was investigated to further define the interaction of pyrethroids with the PTBR. Both Type I and Type II pyrethroids potently inhibited [3H]Ro5-4864 binding with affinities ranging from nanomolar to micromolar. The ED50 values for the proconvulsant effects of both Type I and Type II pyrethroids were significantly correlated with their respective IC50 values as inhibitors of [3H]Ro5-4864 binding. [3H]Ro5-4864 saturation isotherms performed in the presence of fixed concentrations of deltamethrin or cismethrin showed that these pyrethroids increased the observed Kd values for [3H]Ro5-4864 with no change in the maximum number of binding sites. However, Schild plot analysis of the effect of deltamethrin on [3H]Ro5-4864 affinity was nonlinear with the Kd shift approaching a limiting value. Considered together these results suggest an allosteric effect of pyrethroids on [3H]Ro5-4864 binding, and provide additional support for the involvement of the PTBR in the proconvulsant actions of pyrethroids.     

Metaphit, an acylating ligand for phencyclidine receptors: characterization of in vivo actions in the rat

Metaphit, which acylates phencyclidine (PCP) receptors in vitro, was shown to acylate PCP receptors and antagonize the behavioral and electrophysiological effects of PCP in vivo. Metaphit (2 mumol/rat) administered i.c.v. produced PCP-like stereotyped behavior and ataxia in 10 to 20% of rats. At a lower dose, Metaphit (1 mumol/rat) antagonized the ability of PCP to induce stereotyped behavior and ataxia for 3 and 4 days, respectively. The Metaphit-induced antagonism of PCP induction of stereotyped behavior and ataxia was dose-dependent and specific as Metaphit did not antagonize induction of stereotyped behavior by amphetamine. Further evidence for a specific PCP receptor mechanism was the finding that PCP pretreatment blocked the effects of subsequent Metaphit administration. Metaphit also antagonized PCP-induction of stereotyped behavior, but not ataxia, after i.v. administration. Doses of Metaphit that produced long-term antagonism of the behavioral effects of PCP also produced a significant decrease in the maximum binding, but not Kd, of the binding of the PCP analog, [3H]-1-(2-thienyl)cyclohexyl]piperidine, in Metaphit-pretreated rats. The binding of [3H]etorphine and [3H]spiroperidol was not altered significantly by pretreating rats with Metaphit. (-)-Cyclazocine and (+)-SKF 10,047 induced stereotyped behavior and ataxia that was not antagonized by Metaphit-pretreatment. In electrophysiological experiments, Metaphit, like PCP, initially depressed the firing of caudate neurons as does PCP, but then irreversibly inhibited PCP-induced depression of caudate neurons. These results suggest that metaphit antagonized the effects of PCP by selectively acylating PCP receptors and that (-)-cyclazocine- and (+)-SKF 10,047-induced behavioral effects are not mediated primarily by PCP receptors.     

Motor effect of dopamine on human sigmoid colon. Evidence for specific receptors

The effect of dopamine on human sigmoid motility has been studied in 26 subjects. To record mechanical activity of the sigmoid colon, two small, air-filled balloons mounted on a probe introduced through a sigmoidoscope were used. The recordings were made at a distance of 25 and 15 cm from the anal edge. Dopamine was infused for 10 min after a 30-min control infusion of physiologic solution. Mean amplitude, mean duration, mean frequency, percentage of motor activity, and motility index of the pressure waves were determined. The motor response to dopamine was characterized by an increased baseline pressure with phasic waves superimposed. Dopamine produced a significant response at the dose of 5 microgram/kg/min. Alpha and beta antagonizing agents failed to oppose the effect of dopamine, while anticholinergic drugs enhanced its motor action. These studies suggest that dopamine may stimulate the motor function of human large bowel through specific receptors.     

Sites of excitatory and inhibitory actions of alcohols on neuronal alpha2beta4 nicotinic acetylcholine receptors

To define potential alcohol binding sites in the neuronal nicotinic acetylcholine receptor (nAChR) we used cysteine mutagenesis and sulfhydryl-specific labeling. The basis of this strategy is that covalent addition of an alkylthiol group to a cysteine in an alcohol binding site will mimic the action of an irreversibly bound alcohol. Each amino acid in the extracellular region of the second transmembrane segment of the nAChR subunit alpha2 was mutated to cysteine. The resulting alpha2 subunits were coexpressed with wild-type beta4in Xenopus laevis oocytes, and the responses were studied using two-electrode voltage clamp. Of the 11 mutants tested, 2 fulfilled criteria for participation in an alcohol binding site: alpha2(L262C)beta4 and alpha2(L263C)beta4. Covalent binding of propanethiol to these cysteines did not change acetylcholine (ACh) affinity, but modified ACh maximal response in both cases: it increased for alpha2(L263C)beta4 and decreased for alpha2(L262C)beta4. The same modifications on ACh responses were obtained with ethanol on alpha2(L263C)beta4 and octanol on alpha2(L262C)beta4. This suggested that alcohol binding at L263 enhances receptor function, whereas binding at L262 inhibits function. We studied different n-alcohols (ethanol, butanol, pentanol, and octanol), as well as isoflurane and urethane, on these two mutants. Covalent binding of propanethiol to the cysteines revealed changes in the alcohol modulation consistent with an excitatory site (L263) or an inhibitory site (L262) being no longer accessible to alcohol. Thus, n-alcohols appear to act on both sites and their ability to enhance (short-chain), inhibit (long-chain), or produce no effect (intermediate-chain) depends upon their relative action at these two sites.     

Pharmacological actions of WEB 2086, a new specific antagonist of platelet activating factor

WEB 2086, a thieno-triazolodiazepine, is a potent and specific antagonist of platelet activating factor (PAF) in vitro and in vivo. This compound inhibits PAF-induced human platelet and neutrophil aggregation in vitro (IC50 = 0.17 and 0.36 microM, respectively) but has little or no effect on the action of other platelet aggregating agents. In comparison with kadsurenone, ketotifen or thiazinamium chloride, WEB 2086 was 26 to 200 times more potent in the PAF-induced platelet aggregation. In anesthetized guinea pigs, pretreatment with 0.1 to 2.0 mg/kg p.o. or 0.01 to 0.5 mg/kg i.v. of WEB 2086 inhibits dose-dependently the accumulation and aggregation of 111Indium labeled platelets, bronchoconstriction, systemic hypotension and also the lethal effect due to an i.v. PAF infusion [30 ng/(kg X min)] or intratracheal instillation of PAF (300 micrograms/kg). Under the same experimental conditions in guinea pigs, WEB 2086 given by inhalation achieved a similar anti-PAF activity. In anesthetized rats, the hypotension induced by an i.v. PAF infusion was also reversed (ED50 = 0.052 mg/kg i.v.). The increase in cutaneous vascular permeability due to intradermal PAF (25 ng/site) was inhibited dose-dependently by WEB 2086 (0.025-2 micrograms/site) in rats. Because of its structural relationship to triazolodiazepines, WEB 2086 was examined for anticonvulsant and sedative action. Up to doses of 300 and 800 mg/kg p.o., respectively, no effects were found. In conclusion, WEB 2086 is a potent and specific PAF antagonist with triazolodiazepine structure but without sedative activity.     

General anesthetics potentiate gamma-aminobutyric acid actions on gamma-aminobutyric acidA receptors expressed by Xenopus oocytes: lack of involvement of intracellular calcium.

Potentiation of the gamma-aminobutyric acid (GABAA) receptor-gated Cl- channel response has been suggested to be a primary action of some anesthetic agents. We asked whether the GABAA receptor is a target site common for general anesthetics that are chemically and structurally diverse. This hypothesis was tested in Xenopus oocytes expressing mouse cortical mRNA, and GABA-activated Cl- currents were measured using two-electrode voltage clamping. General anesthetics, including inhalational (halothane, diethylether, enflurane and isoflurane), i.v. (3 alpha-hydroxy-5 alpha-dihydroprogesterone, ketamine and propofol) and alcohol (pentanol) anesthetics, enhanced GABA-induced currents by 56 to 1089% at concentrations that were clinically relevant. The results suggest that potentiation of the GABAA receptor/channel response may be a common action for anesthetic agents. Moreover, anesthetic effects were dependent on GABA concentrations; the enhancement was marked with low GABA concentrations and was exponentially decreased as the GABA concentration increased. Also, anesthetic effects were dependent on anesthetic concentrations. The apparent EC50 of halothane was found to be similar to the anesthetic ED50. We also investigated the role of intracellular Ca++ in mediating anesthetic enhancement of the GABA current. We found that intracellular injection of the Ca++ chelator, EGTA, did not change the enhancement by anesthetics. In addition, these anesthetics alone did not produce significant currents, suggesting that the Ca(++)-dependent Cl- current was not activated by these anesthetics per se. Thus, we found that diverse anesthetics potentiate GABA-induced Cl- currents, but this action is not mediated by a release of intracellular Ca++.