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In order to find new hypotensive drugs possessing higher activity and better selectivity, a new series of fifteen 5,5-dimethylhydantoin derivatives (1–15) was designed. Three-step syntheses, consisting of N-alkylations using standard procedures as well as microwaves, were carried out. Crystal structures were determined for compounds 7–9. All of the synthesized 5,5-dimethylhydantoins were tested for their affinity to α1-adrenergic receptors (α1-AR) using both in vitro and in silico methods. Most of them displayed higher affinity (Ki < 127.9 nM) to α1-adrenoceptor than urapidil in radioligand binding assay. Docking to two subtypes of adrenergic receptors, α1A and α1B, was conducted. Selected compounds were tested for their activity towards two α1-AR subtypes. All of them showed intrinsic antagonistic activity. Moreover, for two compounds (1 and 5), which possess o-methoxyphenylpiperazine fragments, strong activity (IC50 < 100 nM) was observed. Some representatives (3 and 5), which contain alkyl linker, proved selectivity towards α1A-AR, while two compounds with 2-hydroxypropyl linker (11 and 13) to α1B-AR. Finally, hypotensive activity was examined in rats. The most active compound (5) proved not only a lower effective dose than urapidil but also a stronger effect than prazosin. [ABSTRACT FROM AUTHOR]

P/Q-type Ca2+ flux into nerve terminals via CaV2.1 channels is essential for neurotransmitter release at neuromuscular junctions and nearly all central synapses. Mutations in CACNA1A, the gene encoding CaV2.1, cause a spectrum of pediatric neurological disorders. We have identified a patient harboring an autosomal-dominant de novo frameshift-causing nucleotide duplication in CACNA1A (c.5018dupG). The duplicated guanine precipitated 43 residues of altered amino acid sequence beginning with a glutamine to serine substitution in CaV2.1 at position 1674 ending with a premature stop codon (CaV2.1 p.Gln1674Serfs*43). The patient presented with episodic downbeat vertical nystagmus, hypotonia, ataxia, developmental delay and febrile seizures. In patch-clamp experiments, no Ba2+ current was observed in tsA-201 cells expressing CaV2.1 p.Gln1674Serfs*43 with β4 and α2δ-1 auxiliary subunits. The ablation of divalent flux in response to depolarization was likely attributable to the inability of CaV2.1 p.Gln1674Serfs*43 to form a complete channel pore. Our results suggest that the pathology resulting from this frameshift-inducing nucleotide duplication is a consequence of an effective haploinsufficiency.

Bladder cancer (BC) is a worldwide common disease with a high mortality rate. Recognizing the dynamic changes in plasma that proteases and their inhibitors undergo might be valuable in understanding the carcinogenesis of invasive bladder cancer and in identifying BC patients with poor prognosis. This study aims to determine the activity of the proteolytic enzyme system and their inhibitors in patients with BC. In this paper, the total proteolytic activity, the activity of matrix metalloproteases (MMPs) and serine proteases was analyzed by the method of caseinolytic activity. For detection of activity of some inhibitors of proteolysis, we used the unified method for determining the activity of alpha-1-antitrypsin (α1A) and alpha-2-Macroglobulin (α2M) in human plasma. The level of medium-mass molecules (MMM) was assessed spectrophotometrically by applying the Nikolaichik method.

The P/Q-type CaV2.1 channel regulates neurotransmitter release at neuromuscular junctions (NMJ) and many central synapses. CACNA1A encodes the pore-containing α1A subunit of CaV2.1 channels. In humans, de novo CACNA1A mutations result in a wide spectrum of neurological, neuromuscular, and movement disorders, such as familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2), as well as a more recently discovered class of more severe disorders, which are characterized by ataxia, hypotonia, cerebellar atrophy, and cognitive/developmental delay. Heterologous expression of CaV2.1 channels has allowed for an understanding of the consequences of CACNA1A missense mutations on channel function. In contrast, a mechanistic understanding of how specific CACNA1A mutations lead in vivo to the resultant phenotypes is lacking. In this review, we present the zebrafish as a model to both study in vivo mechanisms of CACNA1A mutations that result in synaptic and behavioral defects and to screen for effective drug therapies to combat these and other CaV2.1 channelopathies.

The P/Q-type CaV2.1 channel regulates neurotransmitter release at neuromuscular junctions (NMJ) and many central synapses. CACNA1A encodes the pore-containing α1A subunit of CaV2.1 channels. In humans, de novo CACNA1A mutations result in a wide spectrum of neurological, neuromuscular, and movement disorders, such as familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2), as well as a more recently discovered class of more severe disorders, which are characterized by ataxia, hypotonia, cerebellar atrophy, and cognitive/developmental delay. Heterologous expression of CaV2.1 channels has allowed for an understanding of the consequences of CACNA1A missense mutations on channel function. In contrast, a mechanistic understanding of how specific CACNA1A mutations lead in vivo to the resultant phenotypes is lacking. In this review, we present the zebrafish as a model to both study in vivo mechanisms of CACNA1A mutations that result in synaptic and behavioral defects and to screen for effective drug therapies to combat these and other CaV2.1 channelopathies. [ABSTRACT FROM AUTHOR]

A series of ring A-modified analogs of nantenine as well as structural variants in ring C were synthesized and evaluated for antagonist activity at 5- HT2A and α1A receptors. Halogenation improves 5-HT2A antagonist potency in molecules containing a C1 methoxyl/C2 methoxyl or C1 methoxyl/C2 hydroxyl moiety. Bromination or iodination (but not chlorination) with the latter moiety also significantly increased α1A antagonist potency. Homologation or contraction of ring C adversely affected antagonist activity at both receptors, implying that a six-membered ring C motif is beneficial for high antagonist potency at both receptors. Molecular docking studies suggest that the improved antagonist activity (by virtue of improved affinity) of C3-halogenated aporphines in this study is attributable to favorable interactions with the C3 halogen and F339 and/or F340. [ABSTRACT FROM AUTHOR]

Abstract: A set of aporphine analogs related to nantenine was evaluated for antagonist activity at 5-HT2A and α1A adrenergic receptors. With regards to 5-HT2A receptor antagonism, a C2 allyl group is detrimental to activity. The chiral center of nantenine is not important for 5-HT2A antagonist activity, however the N6 nitrogen atom is a critical feature for 5-HT2A antagonism. Compound 12b was the most potent 5-HT2A aporphine antagonist identified in this study and has similar potency to previously identified aporphine antagonists 2 and 3. The ring A and N6 modifications examined were detrimental to α1A antagonism. A slight eutomeric preference for the R enantiomer of nantenine was observed in relation to α1A antagonism. [Copyright &y& Elsevier]

Abstract: A series of C1, C2, C3 and N6 analogs of nantenine (2) was synthesized and evaluated in 5-HT2A and α1A receptor functional assays. Alkyl substitution of the C1 and N6 methyl groups of nantenine provided selective 5-HT2A and α1A antagonists, respectively. The C2 alkyloxy analogs studied were generally selective for α1A versus 5-HT2A. The C3 bromo analog 15 is one of the most potent aporphinoid 5-HT2A antagonists known presently. [Copyright &y& Elsevier]

Abstract: GABA is a major neurotransmitter in the hypothalamus. In particular, neurons in the paraventricular nucleus (PVN) of the hypothalamus receive dense GABAergic inputs from peri-PVN regions. The noradrenergic system has been reported as a modulator of GABAergic transmission to the PVN. Previous electrophysiological and morphological studies support the presence of adrenoceptors on GABAergic neurons innervating the PVN. In this study, we identified three adrenoceptors on GABAergic neurons in the peri-PVN region, focusing on the anterior hypothalamic area (AHA) and rostral zona incerta (ZIr). GABAergic neurons were identified using enhanced green fluorescent protein (eGFP), followed by single cell RT-PCR analysis of the GABA synthetic enzymes, glutamic acid decarboxylase (GAD)65 and/or GAD67. Single cell RT-PCR data revealed the expression of α1A-, α1B- and α2A-adrenoceptor mRNA on GABAergic neurons in AHA and ZIr. Additionally, immunohistochemical studies showed that the immunoreactivities of α1A-, α1B- and α2A-adrenoceptor were colocalized with eGFP-expressing neurons in AHA and ZIr. The present findings suggest the contribution of adrenoceptors to the modulation of GABAergic neurons in AHA and ZIr. [Copyright &y& Elsevier]

Abstract: Several series of dihydrostilbenamide, imidazo[2,1-a]isoindole, pyrimido[2,1-a]isoindole and phthalazinone derivatives were obtained and their vasorelaxant activity was measured on isolated rat aorta rings pre-contracted with phenylephrine (10−5 M). Some phthalazinones attained, practically, the total relaxation of the organ at micromolar concentrations. For the most potent compound 9h (EC50 =0.43μM) the affinities for α1A, α1B and α1D adrenergic sub-receptors were determined. [Copyright &y& Elsevier]

P-type (CaV+2.1) Ca2+ channels are a central conduit of neuronal Ca2+ entry, so their Ca2+ feedback regulation promises widespread neurobiological impact. Heterologous expression of recombinant CaV2.1 channels demonstrates that the Ca2+ sensor calmodulin can trigger Ca2+-dependent facilitation (CDF) of channel opening. This facilitation occurs when local Ca2+ influx through individual channels selectively activates the C-terminal lobe of calmodulin. In neurons, however, such calmodulin-mediated processes have yet to be detected, and CDF of native P-type current has thus far appeared different, arguably triggered by other Ca2+ sensing molecules. Here, in cerebellar Purkinje somata abundant with prototypic P-type channels, we find that the C-terminal lobe of calmodulin does produce CDF, and such facilitation augments Ca2+ entry during stimulation by repetitive action-potential and complex-spike waveforms. Beyond recapitulating key features of recombinant channels, these neurons exhibit an additional modulatory dimension: developmental upregulation of CDF during postnatal week 2. This phenomenon reflects increasing somatic expression of CaV2.1 splice variants that manifest CDF and progressive dendritic targeting of variants lacking CDF. Calmodulin-triggered facilitation is thus fundamental to native CaV+2.1 and rapidly enhanced during early development. [ABSTRACT FROM AUTHOR]

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd[sup 2+] (200 µm), as well as ω-conotoxin MVIIC (5 µm), blocks ∼85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain α[sub 1A] and/or α[sub 1B] subunits. Inhibition of nicotine-evoked dopamine release by conotoxins α-MII and ω-GVIA is additive and indicates that presynaptic α3β2 nAChRs are functionally coupled to α[sub 1A], but not α[sub 1B], calcium channel subtypes. Conversely, insensitivity to α-AuIB and sensitivity to ω-MVIIC indicate that non-α3β2/α3β4-containing nAChRs are functionally coupled to α[sub 1B]-containing calcium channels. In contrast, Cd[sup 2+] blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd[sup 2+]-insensitive component of release is blocked by α-AuIB and therefore appears to be triggered by Ca[sup 2+] flowing directly through the channels of presynaptic α3β4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels. [ABSTRACT FROM AUTHOR]