Right ventricular systolic to diastolic duration ratio: A novel predictor of outcome in adult idiopathic pulmonary arterial hypertension.

BACKGROUND: The systolic to diastolic (SD) duration ratio reflects global RV performance in pulmonary arterial hypertension (PAH) yet limited data exists on its application to adult non-congenital PAH. We measured SD ratios on echocardiogram in idiopathic PAH (IPAH) to establish its response to pulmonary vasodilator therapy and prognostic value at diagnosis and follow up. METHODS: Incident patients with IPAH undergoing echocardiogram, haemodynamic and exercise assessments were identified within our centre between 2005 and 2018. SD ratios were adjusted for heart rate at diagnosis and follow up. RESULTS: In 98 patients at diagnosis, the mean SD ratio was 1.03 ±â€¯0.37 decreasing to 0.85 ±â€¯0.25, p < 0.001 at follow-up echocardiogram performed at a median interval of 9.0 months. The SD ratio at diagnosis correlated weakly with RV basal diameter (r = 0.24, p = 0.04) and 6MWD (r = 0.23, p = 0.04). At follow up, the mean SD ratio was lower in those receiving combination vs monotherapy pulmonary vasodilator treatment (71 ±â€¯25 vs 92 ±â€¯22% baseline respectively, p < 0.001). After a median follow-up of 4.8 years, 3 patients were transplanted and 23 patients died. The SD ratio at diagnosis and follow up predicted an increased risk of death/transplantation (HR 2.41 (1.09-5.29), p = 0.03; HR 5.02 (1.27-19.77), p = 0.02 respectively), retaining its predictive value at diagnosis in bivariate models with 6MWD (HR 2.18 (1.06-4.08)), WHO Functional Class (HR 2.33 (1.04-5.21)) and TAPSE (HR 2.36 (1.07-5.19)), all p < 0.05. CONCLUSIONS: The SD ratio carries prognostic value at diagnosis and follow up in IPAH. Its further evaluation alongside current PAH risk stratification parameters should be considered.

(-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection.

Group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) modulate neurotoxicity of excitatory amino acids and beta-amyloid-peptide (betaAP), as well as epileptic convulsions, most likely via presynaptic inhibition of glutamatergic neurotransmission. Due to the lack of subtype-selective ligands for group-III receptors, we previously utilized knock-out mice to identify mGluR4 as the primary receptor mediating neuroprotection of unselective group-III agonists such as L-AP(4) or (+)-PPG, whereas mGluR7 is critical for anticonvulsive effects. In a recent effort to find group-III subtype-selective drugs we identified (+/-)-PHCCC as a positive allosteric modulator for mGluR4. This compound increases agonist potency and markedly enhances maximum efficacy and, at higher concentrations, directly activates mGluR4 with low efficacy. All the activity of (+/-)-PHCCC resides in the (-)-enantiomer, which is inactive at mGluR2, -3, -5a, -6, -7b and -8a, but shows partial antagonist activity at mGluR1b (30% maximum antagonist efficacy). Chimeric receptor studies showed that the binding site of (-)-PHCCC is localized in the transmembrane region.Finally, (-)-PHCCC showed neuroprotection against betaAP- and NMDA-toxicity in mixed cultures of mouse cortical neurons. This neuroprotection was additive to that induced by the highly efficacious mGluR1 antagonist CPCCOEt and was blocked by MSOP, a group-III mGluR antagonist. Our data provide evidence for a novel pharmacological site on mGluR4, which may be used as a target-site for therapeutics.

In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article.

There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype-selective mGlu(5) receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu(5) receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety.

Discovery and characterization of non-competitive antagonists of group I metabotropic glutamate receptors.

We have investigated the mechanism of inhibition of the new group I mGluR antagonists CPCCOEt and MPEP and determined that both compounds have a non-competitive mode of inhibition. Furthermore using chimeric/mutated receptors constructs we have found that these antagonists act at a novel pharmacological site located in the trans-membrane (TM). Specific non-conserved amino acid residues in the TM domain have been identified which are necessary for the inhibition by CPCCOEt and MPEP of the mGlul and mGlu5 receptors, respectively. Using molecular modeling a model of the TM domain was built for both mGlu1 and mGlu5 receptor subtypes. Docking of CPCCOEt and MPEP into their respective model allowed the modelisation of the novel binding site.

Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain.

The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naïve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.

The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors.

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [(3)H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [(3)H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.

Synthesis, molecular modeling and preliminary biological evaluation of 1-amino-3-phosphono-3-cyclopentene-1-carboxylic acid and 1-amino-3-phosphono-2-cyclopentene-1-carboxylic acid, two novel agonists of metabotropic glutamate receptors of group III.

On the basis of a pharmacophore definition of mGlu4 agonists, the two novel semi-rigid derivatives 12 and 13 were designed and synthesized. The preliminary biological evaluation demonstrated that both compounds interact with hmGlu4a, while ineffective at group II receptor subtypes. In particular, derivative 13 is a full hmGlu4a agonist with an EC50 = 17 microM.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding.

Metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors characterized by a large, extracellular N-terminal domain comprising the glutamate-binding site. In the current study, we examined the pharmacological profile and site of action of the non-amino-acid antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt). CPCCOEt selectively inhibited glutamate-induced increases in intracellular calcium at human mGluR1b (hmGluR1b) with an apparent IC50 of 6.5 microM while having no agonist or antagonist activity at hmGluR2, -4a, -5a, -7b, and -8a up to 100 microM. Schild analysis indicated that CPCCOEt acts in a noncompetitive manner by decreasing the efficacy of glutamate-stimulated phosphoinositide hydrolysis without affecting the EC50 value or Hill coefficient of glutamate. Similarly, CPCCOEt did not displace [3H]glutamate binding to membranes prepared from mGluR1a-expressing cells. To elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR1b and the related subtype, hmGluR5a. Substitution of Thr815 and Ala818, located at the extracellular surface of transmembrane segment VII, with the homologous amino acids of hmGluR5a eliminated CPCCOEt inhibition of hmGluR1b. In contrast, introduction of Thr815 and Ala818 at the homologous positions of hmGluR5a conferred complete inhibition by CPCCOEt (IC50 = 6.6 microM), i.e., a gain of function. These data suggest that CPCCOEt represents a novel class of G protein-coupled receptor antagonists inhibiting receptor signaling without affecting ligand binding. We propose that the interaction of CPCCOEt with Thr815 and Ala818 of mGluR1 disrupts receptor activation by inhibiting an intramolecular interaction between the agonist-bound extracellular domain and the transmembrane domain.

The synaptic potential mediated by metabotropic glutamate receptors is not associated with a substantial elevation of cytosolic free calcium concentration in Purkinje cells.

Brief tetanic stimulation of parallel fibres can evoke a slow excitatory postsynaptic potential (EPSP) in cerebellar Purkinje cells that is mediated by metabotropic glutamate receptors (mGluRs). It is likely that the receptor subtype involved is mGluR1, which couples to the production of diacylglycerol and inositol-1,4,5-trisphosphate (IP3). We therefore examined whether the mGluR-EPSP is associated with an increase in cytosolic free calcium [Ca2+]i using simultaneous Ca2+ imaging and electrophysiological recordings. An mGluR-EPSP could be evoked in all nine Purkinje cells tested. In all but one this potential was not associated with measurable changes in [Ca2+]i whereas single calcium spikes produced large Ca2+ transients. In the one Purkinje cell where [Ca2+]i was elevated, the rise was estimated to be roughly 20-fold smaller than that produced by a single Ca2+ spike.

Signal transmission in the parallel fiber-Purkinje cell system visualized by high-resolution imaging.

We investigated the synaptic transmission in the parallel fiber-Purkinje cell system at high spatio-temporal resolution by using voltage-sensitive dyes and an imaging system. In rat cerebellar slices, cut in the frontal plane or in a plane of the cerebellar surface, local electrical stimulation induced volleys of action potentials in the parallel fibers; subsequent postsynaptic responses from Purkinje cells were observed along the volleys' entire trajectories. Furthermore, the formation of an ordered spatial gradient in parallel fiber conduction velocity across the depth of the molecular layer during postnatal development was observed. In preparations of adult, but not of immature rats, the conduction velocity of parallel fibers in the deep molecular layer was faster than in its more superficial regions. Our observations demonstrate that parallel fibers can mediate Purkinje cell excitation effectively and over considerable distances in a well-organized spatio-temporal manner, thus supporting the classical view of the physiological role assigned to the parallel fibers.

Activation of metabotropic glutamate receptors induces an outward current which is potentiated by methylxanthines in rat cerebellar Purkinje cells.

The responses of slice-cultured Purkinje cells to trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) were examined by intracellular recording techniques and fura-2 microfluorometry. Bath-application of t-ACPD (100 microM, 30 s), a selective agonist of metabotropic glutamate receptors (mGluRs), to Purkinje cells voltage-clamped near their resting potential -65 to -60 mV) consistently induced a transient inward current, followed by a slower outward current (Iout). This outward current was characterized by a linear current-voltage relationship in the range from -130 to -60 mV and accompanied by a significant decrease in membrane conductance. The extrapolated reversal potential of Iout was positive to 0 mV. When t-ACPD was applied for 60 s or more it became apparent that Iout emerged in parallel to the wash-out of t-ACPD. Microfluorometric fura-2 measurements in combination with electrophysiological recordings were used to assess the relation between Iout and intracellular free calcium concentration ([Ca2+]i). In contrast to the inward current that was associated with a transient elevation in [Ca2+]i. Iout was not correlated with an elevated [Ca2+]i. When t-ACPD was applied in the presence of caffeine (5 mM), Iout was reversibly enhanced in amplitude. Caffeine affected neither the t-ACPD-induced calcium signal nor the resting [Ca2+]i. While longer applications of caffeine alone induced outward currents with a current-voltage relationship similar to that of Iout, short applications (30 s) of caffeine had no detectable effect per se but still were effective in enhancing Iout when applied in conjunction with t-ACPD. 3-Isobutyl-1-methylxanthine (IBMX, 0.5 mM), a more selective and potent phosphodiesterase inhibitor than caffeine, exhibited caffeine-like effects at a 10-fold lower concentration. We propose that Iout is generated by a transient inhibition of an inward current that is tonically active at rest and largely voltage-independent in the range tested. Our observations provide evidence for an involvement of cyclic nucleotide second messenger systems in the regulation of this current.

Responses to Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells: Induction of an Inward Current.

The responses to activation of metabotropic glutamate receptors (mGluRs) of Purkinje cells in rat cerebellar slice cultures were investigated using intracellular recordings in single-electrode voltage-clamp mode combined with microfluorometric measurements of cytosolic free calcium using fura-2. Purkinje cells were perfused with saline containing 0.5 microM tetrodotoxin and 10 microM bicuculline and voltage-clamped at -60 mV. Bath-applied trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 50 - 100 microM), a selective agonist of mGluRs, induced a transient inward current that was followed by an outward current. The response induced by t-ACPD was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, up to 40 microM). In contrast, inward currents caused by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1 - 2 microM) were completely abolished, while inward currents caused by quisqualate (0.25 microM) were only partially depressed by CNQX (5 - 40 microM). The inward current induced by t-ACPD was unaffected by external Ba2+ (1 mM), tetraethylammonium (10 mM) and Cs+ (1 mM), and was associated with an increase in apparent input conductance of the cell membrane. The extrapolated reversal potential of inward currents induced by t-ACPD was +18 mV while Cl- currents induced by muscimol reversed at -66 mV. Inward currents induced by t-ACPD, but not those induced by AMPA, were associated with a rise in cytosolic Ca2+ concentration and suppressed by intracellular injection of a calcium chelator. Replacement of external Na+ by choline or Li+ depressed the inward current and resulted in a slower decay of the Ca2+ signal.

Trans-ACPD-induced Ca2+ signals in cerebellar Purkinje cells.

A combination of intracellular recording and fluorometric measurements of cytosolic calcium [( Ca2+]i) was used to locate changes in [Ca2+]i induced by the specific metabotropic glutamate receptor (mGluR) agonist trans-D,L-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), in Purkinje cells of rat cerebellar slices. Under voltage-clamp conditions, application of t-ACPD (100 microM) induced an inward current accompanied by a large increase in [Ca2+]i located primarily in the soma but also, to a lesser degree, in restricted parts of the dendrites. In contrast, elevations of [Ca2+]i associated with calcium spikes were confined to the dendrites and inward currents of a similar amplitude induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an agonist of ionotropic glutamate receptors, did not raise [Ca2+]i.

Calculation of calcium dynamics from single wavelength fura-2 fluorescence recordings.

We describe techniques for measurements of cytosolic calcium dynamics in single current- or voltage-clamped nerve cells. The calculations of calcium dynamics are based on continuous recordings of fura-2 fluorescence intensity at one excitation wavelength after an initial reference measurement at two excitation wavelengths. We show that such single wavelength recordings are not only sufficient for the calculation of Ca2+ concentrations, but also lead to a superior signal-to-noise ratio at a high temporal resolution. Moreover, this strategy diminishes requirements for the experimental setup, such as a device necessary to switch quickly between excitation filters. We have applied this approach on measurements of cytosolic free Ca2+ in single-electrode voltage-clamped CA3 pyramidal cells in hippocampal slice cultures.

Climbing Fibre Responses in Olivo-cerebellar Slice Cultures. II. Dynamics of Cytosolic Calcium in Purkinje Cells.

The generation of climbing fibre responses in cerebellar Purkinje cells has been analysed in co-cultured slices of rat cerebellum and inferior olive. Complex spikes were evoked in Purkinje cells by climbing fibre activation or by intrasomatic injection of depolarizing current pulses. Microfluorometric measurements of cytosolic free calcium ([Ca2+]i) by means of intracellularly injected fura-2 combined with intracellular recordings revealed that both types of complex spikes were accompanied by a transient rise in [Ca2+]i, which was most prominent at dendritic locations. Synaptically induced Ca2+ transients were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 microM), an antagonist of the ionotropic action mediated by non-N-methyl-d-aspartate excitatory amino acid receptors. Ca2+ transients evoked by injections of depolarizing current pulses were not affected by CNQX. These observations indicate that Ca2+ transients induced by climbing fibre activity are generated by voltage-gated Ca2+ channels, which are activated by a CNQX-sensitive synaptic depolarization.

Muscarinic and beta-adrenergic depression of the slow Ca2(+)-activated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of depolarization-induced cytosolic Ca2+ transients.

Combined intracellular and microfluorometric recording techniques were used to evaluate whether the inhibition by cholinergic or adrenergic transmitters of the Ca2(+)-activated potassium current (IAHP) in hippocampal CA3 pyramidal cells was mediated by an alteration of depolarization-induced change in cytosolic free Ca2+ concentration [(Ca2+]i). Low concentrations of isoproterenol (1-10 microM) and muscarine (0.25-1 microM) reversibly abolished IAHP without affecting concomitant Ca2+ transients or the steady-state [Ca2+]i. Only after application of higher concentrations of muscarine, [Ca2+]i increased; in the presence of potassium channel blockers, muscarine depressed Ca2+ currents and concomitant Ca2+ transients. These observations provide direct evidence that the inhibition of IAHP by isoproterenol and muscarine are not mediated by an alteration of Ca2+ dynamics.