Mila Vlaskovska

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on 45Ca2+ uptake and glutamate release. 45Ca2+ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK‐801. K+ depolarization‐induced 45Ca2+ uptake was also reduced by MK‐801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the 45Ca2+ uptake induced by depolarization. Opioid agonists, selective for μ‐, κ‐, and δ‐receptors, inhibited the 45Ca2+ uptake induced by K+ depolarization. The combination of low concentrations of MK‐801 and opioid agonists resulted in additive inhibition of K+‐ induced 45Ca2+ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca2+ uptake under acute, and perhaps also chronic, opiate treatment.

Noradrenergic-nitrergic interactions in the rat anococcygeus muscle: evidence for postjunctional modulation by nitric oxide.

1 The distribution of NADPH‐diaphorase positive and catecholamine‐containing nerve structures, and functional noradrenergic‐nitrergic interactions, were studied in the rat anococcygeus muscle. 2 The morphological findings demonstrated NADPH‐diaphorase positive neurones mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus‐like structures. 3 The nitric oxide synthase inhibitor NG‐nitro‐l‐arginine (l‐NOARG) inhibited concentration‐dependently the nitrergic relaxation, an effect reversed by l‐arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1–10 μm) it decreased the amplitude of contractions and this was not affected by l‐arginine; higher concentrations (50–500 μm) potentiated the contractions, an effect that was prevented by l‐arginine. 4 The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 ± 0.041 μm). The drug decreased the tone of the preparations. However, it potentiated concentration‐dependently the nitrergic relaxations. 5 NBT (1 μm) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)‐donor sodium nitroprusside (SNP, 0.01–50 μm). However, the effect of NBT (0.1–10 μm) on the electrically induced relaxation was significantly decreased by l‐NOARG (10 and 50 μm). The inhibition was of a non‐competitive type. 6 Neither l‐NOARG (100 μm) nor NBT (1 μm) had any effect on the spontaneous or electrically‐induced release of 3H‐radioactivity from the tissues preincubated in [3H]‐noradrenaline. 7 It is concluded that l‐arginine‐NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).

Prejunctional α1-adrenoceptors modify release of [3H]noradrenaline in the guinea-pig vas deferens

1. 1. Several α1- and α2-adrenoceptor agonists and antagonists were examined for effects on spontaneous and stimulus-evoked release of [3H]noradrenaline from sympathetic nerves in guinea-pig vas deferens. 2. 2. Prazosin (0.1 and 1 μM), phentolamine (30 μM) and yohimbine (10 μM) each enhanced the stimulus-evoked release of [3H]noradrenaline. 3. 3. Prazosin and phentolamine increased the spontaneous outflow of [3H]noradrenaline, whereas yohimbine was without effect. 4. 4. Methoxamine (10 μM) and clonidine (0.1 μM) inhibited the stimulus-evoked release of [3H]noradrenaline, whereas only methoxamine (1 μM) decreased the spontaneous outflow of [3H]noradrenaline. 5. 5. The identity of prejunctional α-adrenoceptors in the guinea-pig vas deferens is discussed.

Viscoelastic Characteristics of In Vitro Vital and Devitalized Rat Aorta and Human Arterial Prostheses

The arterial wall viscoelasticity plays an essential role in the vascular responsiveness to vasoactive drugs or pathologies. The aim of this investigation was to derive and compare resonance curve (RC), natural frequency (f0), dynamic modulus of elasticity (E′), and coefficient of viscosity (β) of (i) vital and devitalized preparations of rat thoracic and abdominal aorta, (ii) human arterial prostheses, and to study the histomorphology of vital and devitalized rat aorta. The method of low frequency forced oscillations was employed. RC of vital preparations showed a hardening type of elasticity whereas in devitalized preparations it was of softening type. E′ increased nonlinearly, f0 decreased and β increased linearly with equivalent intraluminal pressure (peqi). Distensibility of abdominal aorta was lower than thoracic aorta. Distensibility decreased with increasing peqi. E′, f0, and β increased significantly after devitalization. It was suggested that postmortem viscoelastic characteristics should not be used directly to specify the vital arteries viscoelasticity. RC of human prostheses showed a softening type of elasticity. Arterial prostheses have low circumferential distensibility with E′-values higher than reported in the literature for human arteries. The method of forced oscillations could be employed for studying the arterial wall biomechanics and viscoelasticity of arterial prostheses.

A structure-activity study of nociceptin-(1-13)-peptide amide. Synthesis of analogues substituted in positions 0, 1, 3, 4 and 10.

A series of analogues of nociceptin, Noc(1-13)NH(2) (an agonist at the ORL1 receptor) was synthesized with following modifications: (1) N-terminal extension with Arg(0); (2) replacement of Gly(3) by basic or polar amino acids-Arg, Asn, Lys(For) or deletion; (3) exchange of Phe(1) or Phe(4) by Phe(NO(2)); (4) substitution of Ser(10) with D-Ser, Pro, D-Pro. The analogs were synthesized by solid-phase methodology using Fmoc-amino acid pentafluorophenyl esters. The affinity for the ORL1 and for the kappa, micro and delta-opioid receptors was investigated by radioligand binding assay and bioactivity by a mouse vas deferens (MVD) assay. The addition of the amino acid residue Arg to the N-terminal enhances the opioid receptor affinity of Noc(1-13)NH(2) while retaining ORL1 receptor affinity at a moderate level. The replacement of Gly in position 3 by the basic or polar amino acids-Arg, Asn, Lys(For) or its deletion led to inactive analogues. The replacement of Ser in position 10 by its D-isomer, Pro and D-Pro resulted in a series of analogues with the following order of activity: Ser(10)>D-Ser(10)>Pro(10)>D-Pro(10). In [D-Ser(10)]Noc(1-13)NH(2), introduction of an additional Phe(NO(2))(4) led to a >60-fold increase of ORL1 affinity, completely attenuating the loss of affinity brought about by Ser(10). In other analogues, introduction of Phe(NO(2))(4) did not change the magnitude of ORL1 binding significantly. Generally, while modifications in position 3 frequently led to a loss of most or all bioactivity, modifications in position 0 (Arg(0)) or 4 (Phe(NO(2))(4)) and 10 (D-Ser(10), Pro(10)) are tolerated.