Agata Woldan-Tambor

Cytotoxic Activity of Pyrovalerone Derivatives, an Emerging Group of Psychostimulant Designer Cathinones.

The growing popularity of novel psychoactive substances (NPS) has aroused the concerns of public health specialists. The pyrovalerone derivatives are a branch of synthetic cathinones, a very popular group of psychostimulant NPS. Despite numerous case reports of fatal intoxications, little is known about the cytotoxicity of these substances. Therefore, this study was aimed to evaluate the toxic properties of pyrovalerone, its highly prevalent derivative 3,4-methylenedioxypyrovalerone (3,4-MDPV) with its two major metabolites (catechol-MDPV and methylcatechol-MDPV) and the structural isomer 2,3-MDPV, together with newer members of the group, i.e., α-pyrrolidinovalerothiophenone (α-PVT) and α-pyrrolidinooctanophenone (PV9), using model human cell lines for neurons (SH-SY5Y), hepatocytes (Hep G2), and upper airway epithelium (RPMI 2650). We found that the first generation pyrovalerones (pyrovalerone, 3,4-MDPV, and 2,3-MDPV) produced a modest decrease of mitochondrial activity in the three examined cell lines, but were active in lower concentrations than methamphetamine used as a reference psychostimulant compound. Since catechol-MDPV displayed greater toxic potential than the parent compound, we suggest that the toxicity of 3,4-MDPV could be attributed to activity of this metabolite. Strikingly, the two new generation pyrovalerones, α-PVT and PV9, seem to be the most potent cytotoxic compounds: both induced highly pronounced mitochondrial dysfunction; the latter also demonstrated significant damage to cell membranes. The reported in vitro toxic activity of pyrovalerone cathinones against different cell types reinforces existing concerns regarding the health risks associated with the intake of these drugs.

Activation of orexin/hypocretin type 1 receptors stimulates cAMP synthesis in primary cultures of rat astrocytes

The effects of orexins, which are also named hypocretins, on cAMP formation were examined in primary cultures of rat astrocytes. Orexin A, an agonist of OX₁ and OX₂ receptors, stimulated cAMP production with an EC₅₀ value of 0.68 μM and potentiated the forskolin-induced increase in the nucleotide synthesis. [Ala¹¹-D-Leu¹⁵]orexin B, an agonist of OX₂ receptors, was inactive. The effects of orexin A were antagonized by SB 408124, a selective blocker of OX₁ receptors, but were not affected by TCS OX2 29, a selective antagonist of OX₃ receptors. We hypothesized that the activation of OX₁ receptors stimulated cAMP synthesis in primary rat astrocyte cultures.

Light‐induced suppression of nocturnal serotonin N‐acetyltransferase activity in chick pineal gland and retina: A wavelength comparison

Abstract: Effects of white and monochromatic (blue—434 nm, green—548 nm, and red—614 nm) lights on the nighttime retinal and pineal NAT activity were examined in chicks. The potency of the tested lights to suppress NAT activity was similar for the retina and pineal gland, with a following rank order: white > green > blue > red. The studied tissues of chick were far less sensitive to pulses of monochromatic light than the rat pineal gland. The potency of light to decrease pineal NAT activity of rat was: white > green >> blue > red. In chicks, the suppression of the nocturnal NAT activity produced by a short 5‐min pulse of monochromatic light was completely reversible in the pineal gland, and partially reversible in the retina. Our data suggest the existence of some differences between birds and mammals in terms of sensitivity and mechanisms involved in the light‐induced suppression of melatonin biosynthesis.

Histamine in the chick pineal gland : Origin, metabolism, and effects on the pineal function

Nowak JZ, Zawilska JB, Woldan‐Tambor A, Sçk B, Voisin P, Lintunen M, Panula P. Histamine in the chick pineal gland: Origin, metabolism, and effects on the pineal function. J Pineal Res. 1997; 22:26–32.

Arylamine and arylalkylamine N-acetyltransferases in retina, pineal gland, brain and liver of chicks: A comparative study

Regulation of arylamine N-acetyltransferase (A-NAT) and arylalkylamine N-acetyltransferase (AA-NAT) was examined in retina, pineal gland, brain and liver of chicks. Enzyme activities were determined using as substrates p-phenetidine and procainamide for A-NAT, tryptamine and phenethylamine for AA-NAT. The activity of A-NAT in all tissues studied does not appear to be regulated by a light-dark cycle. On the other hand, AA-NAT showed distinct light-dark dependent changes (with high values at night) in the retina and pineal gland, but not in brain and liver. The nocturnal increase of retinal and pineal AA-NAT activity was prevented by cycloheximide; the drug did not affect A-NAT activity in these tissues. Treatment of light-adapted chicks with aminophylline significantly increased AA-NAT activity of the retina and pineal gland, without altering the enzyme activity in brain and liver. In these animals, the activity of A-NAT (procainamide) did not change in any tissue studied, whereas the enzyme activity measured using p-phenetidine as a substrate did decrease but only in the retina. A similar pattern of changes in retinal A-NAT and AA-NAT activities was observed after intraocular injection of d,b-cAMP. The rate of inactivation at 4 degrees C was significantly slower for AA-NAT than A-NAT. NATs from brain and liver displayed the highest and lowest, respectively, liability in the cold. The results indicate that the chick retina contains both A-NAT and AA-NAT. The two enzymes have distinct characteristics and the regulation of their activities is different. The retinal A-NAT is similar to A-NAT present in other tested tissues; however, AA-NAT can be induced at night only in the retina and pineal gland. It is suggested that there are two forms of retinal A-NAT, and that, under specified conditions, the activity of one form (A-NAT; p-phenetidine) may be regulated in an opposite manner to AA-NAT activity.

Histamine-stimulated cyclic AMP formation in the chick pineal gland : Role of protein kinase C

The role of protein kinase C (PKC) in histamine (HA)-stimulated cyclic AMP formation in intact chick pineal glands was investigated. In the pineal gland of chick HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA potently increased cyclic AMP accumulation in a concentration-dependent manner. Treatment of intact glands with PKC inhibitors, i.e. chelerythrine and stautosporine, reduced the stimulatory effect of the HA-ergic compounds on cyclic AMP formation. HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA significantly increased inositol-1,4,5-trisphosphate (IP3) levels in intact chick pineal glands, indicating their activities on phospholipase C and 1,2-diacylglycerol formation. The stimulatory effect of HA on IP3 levels was antagonized by aminopotentidine, a potent blocker of H2-like HA receptors in avian pineal gland. Preincubation of chick pineal glands with a PKC activator, 4 beta-phorbol 12, 13-dibutyrate (4 beta-PDB), enhanced the accumulation of cyclic AMP elicited by HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA. On the other hand, 4 beta-phorbol, inactive on the PKC, was ineffective. Our results point to the possibility that PKC is involved in the regulation by HA of cyclic AMP synthesis in the pineal gland of chick. Furthermore, the cyclic AMP response to pineal HA receptor stimulation can be positively modulated by a concomitant activation of the PKC pathway.

Histamine H2‐like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [3H]tiotidine binding

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA‐ergic drugs on the cAMP‐generating system, and (2) radioreceptor binding of [3H]tiotidine, a selective H2 antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1–1000 μm) potently and concentration dependently stimulated cAMP production in [3H]adenine pre‐labelled slices of chick cerebral cortex, displaying an EC50 value (concentration that produces 50% of maximum response) of 2.65 μm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA ≥  4‐methylHA (H2) ≥ Nα,Nα‐dimethylHA (H3 ≫ H2 = H1) ≫  2‐methylHA (H1) ≫ 2‐thiazolylethylamine (H1) ≥ Rα‐methylHA (H3) ≫ amthamine, dimaprit (H2), immepip (H3, H4). The HA‐evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H2 receptor blockers (aminopotentidine ≥ tiotidine > ranitidine ≫ zolantidine), and not significantly affected by mepyramine and thioperamide, selective H1 and H3/H4 receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non‐competitive mode of action. The binding of [3H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (Kd) = 4.42 nm] and high capacity [maximum number of binding sites (Bmax) = 362 fmol/mg protein]. The relative rank order of HA‐ergic drugs to inhibit [3H]tiotidine binding to chick cerebrum was: antagonists – tiotidine ≫ aminopotentidine = ranitidine ≥ zolantadine ≫ thioperamide ≈ triprolidine; agonists – HA ≥ 4‐methylHA ≫ 2‐methylHA ≥Rα‐methylHA ≈ dimaprit. In conclusion, chick cerebral cortex contains H2‐like HA receptors that are linked to the cAMP‐generating system and are labelled with [3H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian‐specific H2‐like HA receptors or a novel subtype of HA receptors.