Histamine-Independent Modulation of the Neuropeptide Y-Induced Pressor Response by α-Trinositol in the Pithed Rat

Abstract: The modulatory effects of α-trinositol (D-myo-inositol-1.2.6-trisphosphate; PP 56) on the systemic arterial blood pressor responses induced by neuropeptide Y, preganglionic nerve stimulation, phenylephrine and vasopressin were studied in pithed rats. Intravenous administration (within 2 min.) of α-trinositol reduced the neuropeptide Y-induced increase in mean arterial pressure within a defined dose range without altering the heart rate. The influence of α-trinositol on the neuropeptide Y-induced pressor response in the presence of non-selective as well as H₁- and H₂-selective histamine antagonists (diphenhydramine, mepyramine and cimetidine respectively) were investigated. The maximal increase in mean arterial pressure induced by neuropeptide Y as well as the duration of the pressor response was enhanced after non-selective (diphenhydramine) or H₁-selective (mepyramine) histamine blockade. The enhancement of the neuropeptide Y-induced pressor response by the H₁ specific antagonist mepyramine was significantly more pronounced compared to the H₂-selective agent. The exaggerated increase in mean arterial pressure in response to neuropeptide Y after histamine blockade was inhibited by α-trinositol to a similar extent as without such pretreatment. We conclude that neuropeptide Y interacts with histamine in the pithed rat and that this action may partially offset the pressor actions of the peptide. The neuropeptide Y-induced pressor responses may be inhibited by α-trinositol within a defined dose range indicating that this non-peptide agent may act as a functional inhibitor to neuropeptide Y in vascular tissue.     

α-Trinositol: A Functional (Non-receptor) Neuropeptide Y Antagonist in Vasculature

Neuropeptide Y is a sympathetic co-neurotransmitter released with noradrenaline upon sympathetic nerve stimulation. This study describes the ability of a synthetic inositol phosphate, α-trinositol (d-myo-inositol 1,2,6?triphosphate; PP 56) to antagonize vasoconstrictor responses to neuropeptide Y in-vitro as well as in-vivo. In human and guinea-pig isolated arteries α-trinositol potently (10 nM to 1 μM extracellular concentration) suppressed the constriction evoked by neuropeptide Y alone, the potentiation by neuropeptide Y of noradrenaline-evoked constriction, and the neuropeptide Y-induced inhibition of relaxation. Moreover, in the pithed (areflexive) rat, a non-adrenergic portion of the pressor response to preganglionic sympathetic nerve stimulation was sensitive to α-trinositol. As studied in the recently cloned human (vascular-type) Y1 receptor, the action of α-trinositol does not occur through antagonism at the neuropeptide Y recognition site nor does it induce allosteric changes of this receptor. However, we found α-trinositol to inhibit the rise in intracellular Ca²⁺ as well as inositol triphosphate concentrations induced by neuropeptide Y. It is, therefore, proposed that α-trinositol represents a non-receptor, but yet selective antagonist of neuropeptide Y in vasculature, opening up the possibility to investigate involvement of neuropeptide Y in sympathetic blood pressure control and in cardiovascular disorders.     

Differential Effects of Increasing Doses of α-Trinositol on Cerebral Blood Flow Autoregulation

Abstract: The effect of neuropeptide Y inhibition with α-trinositol on the cerebral blood flow autoregulation was studied in Wistar Kyoto rats. α-Trinositol was tested in two doses: one dose (5 mg kg^-1 hr^-1) selectively affecting neuropeptide Y and one higher dose (50 mg kg^-1 hr^-1) affecting both neuropeptide Y and the adrenergic response. The cerebral blood flow was measured with the intracarotid ¹³³xenon injection method in halothane nitrous oxide-anaesthetized animals. Blood pressure was raised by norepinephrine infusion and lowered by controlled haemorrhage in separate groups of rats. In addition we examined the effect of α-trinositol on neuropeptide Y-induced contraction of cerebral vessels in vitro. The in vitro study demonstrated inhibition of neuropeptide Y-induced contraction with a α-trinositol dose selective of neuropeptide Y. The in vivo study demonstrated that cerebral blood flow autoregulation was preserved after both doses of α-trinositol. α-Trinositol in the low neuropeptide Y-selective dose (5 mg kg^-1 hr^-1) did not affect the blood pressure limits of cerebral blood flow autoregulation, but the higher dose (50 mg kg^-1 hr^-1) of α-trinositol shifted the upper blood pressure limit of cerebral blood flow autoregulation towards lower blood pressures, an effect probably due to inhibition of both the adrenergic and neuropeptide Y systems.     

Reactivity of glutathione with α,β-unsaturated ketone flavouring substances

The relative reactivities of a number of alpha, beta-unsaturated ketones used as flavourings were determined using glutathione as the nucleophile. Monosubstitution at the beta-position of the alpha, beta-unsaturated system impeded nucleophilic addition by approximately 1000 times. Beta-Disubstitution reduced reactivity by more than 100,000 times. Endocyclic alpha, beta-unsaturated ketones were generally less reactive than alicyclic analogues. By way of comparison, the most reactive flavouring investigated, 2-octene-4-one, was consumed by glutathione about 700 times less rapidly than was methylvinyl ketone. Methylvinyl ketone was found to condense with guanylic acid 240,000 times more slowly than with glutathione. It is concluded that alpha, beta-unsaturated ketones used as flavourings generally possess low electrophilicity.     

Mechanical Response of Rat Myocardium to Dibutyryl Cyclic AMP in Relation to Effects of α- and β-Adrenoceptor Stimulators

Abstract: Dibutyryl cyclic AMP, and α- and β-adrenoceptor stimulators are all able to elicit inotropic effects, α- and β-Adrenoceptor stimulation are known to change each myocardial contraction-relaxation cycle differently. In order to elucidate the myocardial function of cyclic AMP the effects of dibutyryl cyclic AMP on the contraction-relaxation cycle of isolated rat heart papillary muscle were examined and compared to the effects of α- and β-adrenoceptor stimulation, respectively. Dibutyryl cyclic AMP (in the presence of propranolol) increased developed tension (T_max) by 18%, rate of tension rise (T′_max) by 46%, rate of tension fall (T′_min) by 62% and onset-rate of relaxation (T″_min) by 136%. These changes in the contraction-relaxation cycle were strikingly similar to those produced by isoprenaline (β-adrenoceptor stimulation). The response to dibutyryl cyclic AMP, however, developed much slowlier than did the response to isoprenaline. The latter effect was associated with cyclic AMP elevation in a way indicating a trigger function for cyclic AMP. The α-adrenoceptor stimulation (by phenylephrine combined with propranolol), however, increased measures both for contraction and for relaxation by about the same degree, and the effects occurred without changes of cyclic AMP contents. Phenylephrine alone (combined α- and β-adrenoceptor stimulation) elicited a substantial cyclic AMP elevation but gave mechanical effects only slightly different from the pure α-adrenergic response. Thus cyclic AMP effects did not seem to be fully expressed in this case. As a whole, the results indicate that the effects of both dibutyryl cyclic AMP and of isoprenaline are mediated by the cyclic AMP-system while α-adrenoceptor stimulation involves other mechanisms.     

Renal Lesions Induced by α-Methyltyrosine Methylester and α-Methyltyrosine

The toxicity of α-methyltyrosine methylester (H 44/68) and α-methyltyrosine (H 9/88) in the rat was investigated in a four week subacute study. Both compounds induced renal lesions with formation of calculi in the urinary tract. The renal lesions were manifested by deposition of tubular crystals associated with regressive and inflammatory changes. The main sites of crystal deposition were the collecting tubules and the papillary ducts. The calculi consisted of α-methyltyrosine. The investigation indicates that α-methyltyrosine methylester is metabolized in the body to the less soluble α-methyltyrosine and that α-methyltyrosine is excreted through the kidneys. Crystals of α-methyltyrosine are formed in the renal tubules and urinary tract probably due to a supersaturation of the urine.     

Separation of δ-, γ- and α-tocopherols by CEC

In this study capillary electrochromatography (CEC) was used for the separation of three tocopherols (TOHs), namely delta-, gamma- and alpha-TOH and the antioxidant compound, butylated hydroxytoluene (BHT). The CEC experiments were carried out using an octadecylsilica (ODS) stationary phase packed, in our laboratory, in a fused-silica capillary (100 microm I.D., 365 microm O.D. x 33 cm of total length and 24.6 or 8.4 cm effective length). The mobile phase was composed by a mixture of methanol (MeOH) and acetonitrile (ACN), at different concentrations and 0.01% (w/v) of ammonium acetate. Retention time (t(R)), retention factor (k), resolution (R(s)) of the three TOHs were strongly influenced by the organic solvent composition of the run buffer and by the effective length of the capillary. Optimum experimental conditions were found even employing the short effective length of the capillary achieving the baseline separation of the studied analytes in a relatively short time (less than 5 min). The optimized method was applied to the qualitative analysis of vitamin E (alpha-TOH) present in a human serum extract.     

α-Alkylmercapto- und α-Arylmercapto-alkylisocyanate

α-Alkylmercapto- and α-Arylmercapto-alkylisocyanates α-Alkylmercapto- and α-arylmercapto-alkylisocyanates 2 were prepared from α-halosulfides 1 by reaction with silver cyanate. With ammonia, primary or secondary amines they give the corresponding α-mercaptoalkyl-ureas 4 ; with alcohols, the α-mercaptoalkylurethans 3.     

DES—Nα1—ACETYL—α—MELANOTROPIN:

Application of the 2-methylsulfonylethyloxycarbonyl group for temporary amino protection enables the synthesis from one precursor of des-Nα¹-acetyl-α-MSH, the two mono N-acetylated forms (in positions I and II) and the diacetyl form of this tridecapeptide amide. The free tridecapeptide amide, although structurally unrelated to the normal substrate, was recognized by an enzyme occurring in calf eye-lens tissue. The product of the enzymatic reaction was exclusively α-MSH. Partial sequences derived from the N-terminus were less rapidly acetylated or not at all, depending on their chain length. The enzyme, therefore, appears to direct its activity to free N-terminal α-amino groups of peptides exceeding a certain critical chain length. Acetylation of -amino functions did not occur.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

Pharmacological characterization of A-131701, a novel α1-adrenoceptor antagonist selective for α1A- and α1D-compared to α1B-adrenoceptors

New compounds selective for α_1A-adrenoceptors in the prostate may offer enhanced efficacy for benign prostatic hyperplasia (BPH), with fewer side effects than current treatment. A-131701 (3-[2-((3aR,9bR)-cis-6-methoxy-2,3,3a,4,5,9b,hexahydro-[1H]-benz[e]isoindol-2-yl)ethyl]pyrido[3′,4′:4,5]thieno [3,2-d]pyrimidine-2,4(1H,3H)-dione), from a novel class of benz[e]isolindole pyridothienopyrimidines and pyridothienopyrazines, is selective for α_1a- and α_1d-adrenoceptors in radioligand binding studies (0.22 nM at α_1a-, 0.97 nM at α_1d-) compared to α_1b-sites (2.5 nM) and in isolated tissue bioassays (pA₂ values of 8.9–9.0 for α_1A-receptors in rat vas deferens or canine prostate strips, 9.1 at α_1D-sites (rat aorta)), compared to 7.9 at α_1B-sites (rat spleen). A-131701 also potently blocked radioligand binding to α₁-adrenoceptors in canine and human prostatic membranes, but was considerably weaker at α₂-adrenoceptors. In isoflurane-anesthetized dogs, A-131701 antagonized epinephrine-induced increases in intraurethral pressure (IUP) with a pseudo-pA₂ value of 8.17. In spontaneously hypertensive rats, A-131701 caused transient decreases in mean arterial blood pressure (MABP) and transient tachycardia. The area under the curve (AUC₀→₆₀ min) for the hypotensive response was dose-related, with a log index value for A-131701 of 5.33, suggesting a selectivity of >600-fold comparing IUP to MABP effects. In pentobarbital-anesthetized dogs, A-131701 was more potent in blocking phenylephrine (PHE)-induced increases in IUP (pseudo-pA₂ = 8.0) compared to concurrently measured MABP (pseudo-pA₂ = 7.2), or sixfold selective. Doses greater than 1,000 nmol/kg i.v. of A-131701 were required to lower blood pressure by 10 mm Hg in these dogs (pED₁₀ =. 5.57), indicating a uroselectivity ratio of >250, superior to doxazosin, terazosin, or tamsulosin. Thus, A-131701 is selective for α_1A- and α_1D- vs. α_1B-adrenoceptors in vitro, and prostatic function vs. blood pressure effects in vivo, which may provide therapeutic advantages in the treatment of BPH. Drug Dev. Res. 44:140–162, 1998. © 1998 Wiley-Liss, Inc.     

Local structural differences between α and β-elicitins shown by circular dichroism and ultraviolet difference spectroscopy

In order to investigate differences in the conformation of elicitins exhibiting different levels of activity (toxicity to tobacco plants). the environment of the tyrosyl residues in four elicitins has been compared by different spectroscopic methods (difference absorption and circular dichroism). We compared two α-elicitins (capsicein and parasiticein) and two β-elicitins (β-cryptogein and β-cinnamomin), that are 50–100 times more toxic than the α-ones. Thermal difference UV spectroscopy and titration experiments clearly showed the exposure of Tyr-85 by comparison of parasiticein lacking Tyr-85 and the accessibility of its hydroxyl group to the solvent. The adjacent Ty-87 was also suggested to be located at the surface. In β-cryptogein, β-cinnamoniin and capsicein the pK was measured at between 10.5 and 10.8, while in parasiticein it is higher (11.5) owing to a difference in the local environment. Thermal difference UV spectroscopy showed one more exposed tyrosine in β-elicitins than in α-ones. This difference was attributed to Tyr-12, considering the more hydrophilic characteristic of the sequence around residue 13 in β-elicitins and the role of this region in the toxicity. However, no difference in titration behaviour was noted among elicitins concerning Tyr-12. The other two tyrosines also presented an abnormal pK of titration (> 12). In all elicitins Tyr-47 was probably exposed, while Tyr-33 was probably buried and not titrated, except in β-cinnamomin at very alkaline pH.     

17β-Nitro-5α-androstan-3α-ol and its 3β-methyl derivative: Neurosteroid analogs with potent anticonvulsant and anxiolytic activities

Many 17-substituted androstan-3α-ol analogs act as positive allosteric modulators of GABA_A receptors and exert anticonvulsant and anxiolytic-like activity actions in animal models. The endogenous neurosteroid allopregnanolone (17β-acetyl; 1) is among the most potent of these. Here we demonstrate that 3α-hydroxy-17β-nitro-5α-androstane (2b) and its 3β-methyl analog (3α-hydroxy-3β-methyl-17β-nitro-5α-androstane; 2c) modulate GABA_A receptors as assessed by [³⁵S]t-butylbicyclo-phosphorothionate and [³H]flunitrazepam binding with potencies equivalent to or greater than 1. These compounds also had potencies equivalent to or greater than 1 in the pentylenetetrazol and 6 Hz seizure models in the mouse. Furthermore, 2b exhibited anxiolytic-like activity in the elevated zero maze. The 3β-hydroxy, 3α-desmethyl analog (2a) was devoid of activity on GABA_A receptors in vitro but had moderate activity in the seizure models, possibly as a result of epimerization in vivo at the 3-position. This conclusion was supported by the lack of in vivo activity of the 3β-hydroxy, 3α-methyl analog (2d), which is not expected to undergo epimerization. We conclude that nitro can serve as a bioisostere for acetyl at the 17β-position of 5α-androstan-3α-ol, such that the nitro analog fully retains the bioactivity of the endogenous neurosteroid at GABA_A receptors.     

Adenylate Cyclase Modulation by Ammonium Ion: GTP-like Effect on Muscarinic and α2-Adrenergic Receptors

The stimulatory influence of ammonium sulphate on adenylate cyclase activity has been investigated. By competition binding experiments on the β-adrenergic stimulatory receptor in rat myocardial membranes, no influence could be detected of ammonium sulphate neither in receptor coupling to the stimulatory guanine nucleotide binding protein nor in the GTP-induced uncoupling. In order to detect an impaired inhibition instead of an increased stimulation of adenylate cyclase activity by ammonium sulphate the investigation was extended to inhibitory receptors. The same type of effect by ammonium sulphate was detected on both the muscarinic cholinergic receptor in rat myocardial membranes as well as on the α₂-adrenergic receptor in human platelets. The influence of ammonium sulphate noted in competition binding studies and off-kinetics experiments was GTP-like, i.e. causing a decrease in agonist-receptor affinity leaving all the inhibitory receptors in the low affinity state. In conclusion, this paper indicates that the observed stimulatory effect of ammonium sulphate is exerted by the ammonium ion on the inhibitory guanine nucleotide binding protein, impairing the negative control of adenylate cyclase activity.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Reaktionen α,β-ungesättigter γ-Oxosulfone mit Nucleophilen

Reactions of α,β-Unsaturated γ-Oxosulfones with Nucleophiles Bromination of the γ-oxosulfones 1 and subsequent dehydrohalogenation leads to the α,β-unsaturated γ-oxosulfones 3 . These products react with primary and secondary amines to yield the enaminoketones 7 . γ-Oxobissulfones 10 are formed from 3 by addition of the sulfinic acids 9 . Thioles 11 react with 3 to give the γ-oxomercaptosulfones 12 or the thioacetals 14 .     

Reactions with Arylhydrazones of α-Cyanoketones: Synthesis of New α-Arylhydrazononitriles

New α-arylhydrazononitriles were prepared by reaction of the hydrazide 1 and the amidrazone 2 with nucleophilic reagents. β-Bifunctional reagents were condensed with 1 and 2 to yield new heterocyclic hydrazonitriles.