Age dependent changes of arterial wall viscoelasticity.

Viscoelastic characteristics (VEC) of old rat aorta (Wistar, 10 months) were obtained by sinusoidal excitation of intraluminal pressure (p) in cylindrical arterial preparations. The pressure excitation frequency (f(exc)) was swept in the range 3-30 Hz up and down at several mean-pressure levels while response volume oscillations were recorded and resonance curves were plotted. Natural frequency (f(0)), dynamic modulus of elasticity (E') and coefficient of viscosity (beta) were estimated from resonance curves and the dependences of VEC on p were drawn. The results showed that f(0) decreased linearly with p whereas our previous data for young rat aorta (Wistar, 4 months) showed independence of f(0) on p. E' increased nonlinearly with p with the values being higher in comparison to young rat aorta. This means stiffening of rat aorta with age in accordance with the known literature data. beta-values increased linearly with p being higher in comparison to young rat aorta, demonstrative of raised intrinsic friction in the wall. VEC values were higher at decreasing f(exc) suggesting that the direction of excitation sweeping also determines the arterial wall biomechanical behaviour. It could be concluded that blood vessels VEC worsen with age, which endangers the arterial wall integrity, especially at higher intraluminal pressure.

P2X3 knock-out mice reveal a major sensory role for urothelially released ATP.

The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X(3) receptor knock-out (P2X(3)-/-) and wild-type control (P2X(3)+/+) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X(3). P2X(3)-positive staining was completely absent in the subepithelial plexus of the P2X(3)-/- mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder-pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X(3)+/+ and P2X(3)-/- mice, although P2X(3)-/- bladder had an increased capacity compared with that of the P2X(3)+/+ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X(3)-/- mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X(3)+/+, but not P2X(3)-/-, were rapidly activated by intravesical injections of P2X agonists (ATP or alpha,beta-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2',3'-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X(3) receptors on a subpopulation of pelvic afferent fibers.

Inhibition of nitrergic relaxations by a selective inhibitor of the soluble guanylate cyclase.

1. The actions of 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a specific inhibitor of the soluble guanylate cyclase (SGC), were investigated in the rabbit anococcygeus muscle. 2. ODQ (1 nM-1 microM) inhibited in a concentration-dependent manner the relaxations induced by electrical field stimulation (EFS; 50 V, 0.3 ms duration, 1 Hz, for 5 s, every 120 s). 3. ODQ (1 microM) also inhibited the relaxations elicited by EFS (50 V, 0.3 ms duration, 1, 2.5, 5, 10 Hz, for 5 s) and sodium nitroprusside (SNP; 1 microM) without affecting those induced by isoprenaline (1 microM), atrial natriuretic peptide (ANP; 100 nM) or an analogue of cyclic GMP (8-pCPT-cyclic GMP; 500 microM). 4. ODQ (1 microM) inhibited the elevations in the concentration of cyclic GMP induced by SNP or EFS, but not by ANP. ODQ did not affect the concentrations of cyclic AMP. 5. Nitrergic relaxation in this tissue appears, therefore, to be mediated via activation of SGC.

Characterization of nitrergic neurotransmission during short- and long-term electrical stimulation of the rabbit anococcygeus muscle.

1. Isolated preparations of rabbit anococcygeus muscle were exposed to electrical field stimulation (EFS; 50V, 0.3 ms duration, 0.08-40 Hz) for periods of 1-60 s (short-term EFS) or 10 min-2 h (long-term EFS). 2. Both short- and long-term EFS caused a contractile response which was enhanced by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG), showing that it is modulated by endogenous NO. 3. In preparations treated with scopolamine and guanethidine and in which a constrictor tone was induced by histamine, both short- and long-term EFS resulted in relaxation of the tissue. 4. Such relaxations were reversed by tetrodotoxin (TTX), omega-conotoxin, inhibitors of NO synthase and the NO scavenger, oxyhaemoglobin, indicating that they are neuronal in origin and nitrergic in nature. 5. The relaxations to long-term EFS persisted for the duration of the stimulation and were associated with sustained release of oxidation products of NO (NOx). The EFS-induced release of NOx was decreased by N-iminoethyl-L-ornithine (L-NIO), an inhibitor of NO synthase, and by TTX. 6. Inhibitors of NO synthase, in addition, increased the basal tone of the tissue and reduced the basal output of NOx. The basal output of NOx was also reduced by TTX. 7. Long-term EFS which induces approximately 50% of the maximum relaxation could be enhanced by addition of L-, but not D-, arginine to the perfusion medium. 8. These data show that there is a continuous basal release of NO from nitrergic nerve terminals which maintains a relaxant tone in the rabbit anococcygeus muscle. 9. In addition, NO is released during short- and long-term EFS which further relaxes the preparation and modulates sympathetic transmission. Activation of the L-argimne: NO pathway for periods up to2 h does not exhaust nitrergic transmission in any appreciable way.

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 neurons 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 microM) it decreased the amplitude of contractions and this was not affected by L-arginine; higher concentrations (50-500 microM) 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 microM). The drug decreased the tone of the preparations. However, it potentiated concentration-dependently the nitrergic relaxations. 5. NBT (1 microM) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)-donor sodium nitroprusside (SNP, 0.01-50 microM). However, the effect of NBT (0.1-10 microM) on the electrically induced relaxation was significantly decreased by L-NOARG (10 and 50 microM). The inhibition was of a non-competitive type. 6. Neither L-NOARG (100 microM) nor NBT (1 microM) 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).

Purinergic innervation of the guinea-pig urinary bladder.

1 A number of criteria for considering adenosine 5'-triphosphate (ATP) as a neurotransmitter in the guinea-pig urinary bladder have been examined. In addition, the effect of tachyphylaxis to ATP on the response to non-adrenergic, non-cholinergic nerve stimulation has been re-examined.2 Quinacrine fluorescence histochemistry revealed a population of nerve fibres, ganglion cells, and nerve bundles in the bladder which were not seen in either the iris or vas deferens, where adrenergic and cholinergic nerves predominate. The distribution and morphology of the quinacrine-positive nerves in the bladder were different from those observed with catecholamine fluorescence and cholinesterase histochemistry, and were unaffected by chemical sympathectomy.3 Release of ATP from the bladder during stimulation of intramural excitatory nerves, in the presence of atropine and guanethidine increased to 3-12 times prestimulation levels. Tetrodotoxin abolished both the contractile response and the increase in ATP release resulting from intramural nerve stimulation. There was no increase in ATP release during contraction resulting from direct muscle stimulation following nerve paralysis with tetrodotoxin.4 Sympathectomy with 6-hydroxydopamine did not affect release of ATP in response to intramural nerve stimulation.5 Release of ATP was dependent on the concentration of calcium ion in the medium.6 Contractions in response to non-adrenergic, non-cholinergic intramural nerve stimulation were closely mimicked by ATP, but not by acetylcholine or histamine.7 Adenosine and dipyridamole reduced the contractions to both ATP and non-cholinergic nerve stimulation.8 2-2'-Pyridylisatogen was not a specific blocker of either ATP or intramural nerve stimulation in the guinea-pig bladder. 2-Substituted imidazolines initiated spontaneous activity making it impossible to assess any blocking action that they may have had.9 Prostaglandins (E(1), E(2) and F(2alpha)) gave weak, slow contractions and an increase in spontaneous activity. Both the response to ATP and non-adrenergic, non-cholinergic nerve stimulation were greatly potentiated in the presence of prostaglandins.10 In the presence of indomethacin the response to non-adrenergic, non-cholinergic nerve stimulation was virtually abolished following desensitization to ATP.

Plasma concentrations and clearance of procalcitonin during continuous veno-venous hemofiltration in septic patients.

We determined the elimination characteristics of procalcitonin (PCT) during continuous veno-venous hemofiltration (CVVHF) and the resulting effect on PCT plasma levels. A prospective study was conducted in patients with sepsis and acute oliguric renal failure, treated with CVVHF using a polysulfone membrane (Baxter Renaflo II PSHF 1200). Patients had sepsis and PCT plasma levels > 4 ng ml(-1) (n = 26). PCT was measured in the pre- and post-filter plasma and the ultrafiltrate at 0, 5, 10, and 15 min and 1, 2, 4, 6, 12, and 24 h after setup of CVVHF. PCT sieving coefficient was 0.24. Elimination of PCT, however, depended on the duration of filtration, because filter adsorption was the main mechanism of PCT clearance during the first hour of hemofiltration, finally increasing to a clearance of PCT into the ultrafiltrate of 2.8-5.5 mL/min after 2 h. PCT plasma levels were not significantly altered during CVVHF (96% of the initial concentration after 24 h, P = 0.72). Similar to what has been observed with cytokines and other proteins of a comparable molecular weight, PCT is removed from the plasma during CVVHF, but plasma PCT levels are unchanged. Thus, PCT can be used as a diagnostic parameter even in patients with acute renal failure undergoing CVVHF.

Differential metabolism of dynorphins in substantia nigra, striatum, and hippocampus.

To map the proteolytic enzymes metabolizing dynorphins in brain structures, size-exclusion chromatography linked to electrospray ionization mass spectrometry was used. Enzymes extracted from rat hippocampus, striatum, and substantia nigra were tested for their capability of converting dynorphin-related peptides. Dynorphin A was the most resistant to proteolytic conversion, whereas Big dynorphin and dynorphin B-29 were slowly converted to dynorphin A and dynorphins A and B, respectively. Dynorphin B and alpha-neoendorphin were the least resistant. Dynorphin B was rapidly converted to Leu-enkephalin in the striatum and hippocampus but to Leu-enkephalin-Arg6 in the substantia nigra. alpha-Neoendorphin was converted to Leu-enkephalin in all tissues investigated.

Biotransformation of nociceptin/orphanin FQ by enzyme activity from morphine-naive and morphine-treated cell cultures.

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated. The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion chromatography. The effect of chronic morphine treatment of the cells (5 days) on NOFQ biotransformation was also studied. It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1-13. The pattern of NOFQ biotransformation was quite similar for all three cell cultures. However, different proportions of the formed peptides were noted. The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+. Dynorphin A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1-12 and the shorter peptides N1-5, N1-6. Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific. Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products.

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex.

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg6 and to a lesser extent to Leu-enkephalin. Five day treatment with 10 microM morphine increased the conversion to Leu-enkephalin-Arg6 by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B > alpha-neoendorphin > > dynorphin A. The Km for the generation of Leu-enkephalin-Arg6 from dynorphin B was 40 microM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1-13) > A(2-13) > A(1-17) > A(2-17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for kappa-opioid receptors) and enkephalin peptides (selective for delta-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.

The use of the slowly degradable analog, alpha, beta-methylene ATP, to produce desensitisation of the P2-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder.

alpha, beta-Methylene ATP has advantages over ATP in producing desensitisation of the P2-purinoceptor since it is degraded more slowly than ATP and does not initiate synthesis of prostaglandins. Following desensitisation of the excitatory P2-purinoceptors in the guinea-pig urinary bladder, the excitatory responses to non-adrenergic, non-cholinergic nerve stimulation were abolished, while those to acetylcholine and histamine were little affected. This result is consistent with the purinergic nerve hypothesis.

Profile of prostaglandins generated in the detrusor muscle of rat urinary bladder: effects of adenosine triphosphate and adenosine.

The generation and release of PGE2, PGF2 alpha, PGD2, TXB2 and 6-keto-PGF1 alpha in the rat detrusor muscle were studied by means of radioimmunoassays. The effect of ATP (0.1 mmol/1) and adenosine (0.1 mmol/1) on the content and profile of PGs in the incubation medium was investigated. It was found that PGE2 and 6-keto-PGF1 alpha accounted for more than 80% of the total PG activity. ATP increased the amounts of PGs in the incubation medium (percentage change of the control values, N = 6: PGE2 54.53 +/- 12.69, PGF2 alpha 31.01 +/- 8.82, PGD2 44.52 +/- 12.36, TXB2 17.29 +/- 10.45, 6-keto-PGF1 alpha 36.62 +/- 5.0) but did not change their profile. Adenosine had no effect on either content or profile of the PGs. The results suggest that ATP but ot adenosine may activate PG biosynthesis via P2-purinoceptor-mediated mechanisms.

Direct evidence for ATP release from non-adrenergic, non-cholinergic ("purinergic") nerves in the guinea-pig taenia coli and bladder.

Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis.

A mu-receptor opioid agonist induces AP-1 and NF-kappa B transcription factor activity in primary cultures of rat cortical neurons.

Activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B) represent mammalian transcription factors which bind to distinct enhancer motifs. The specific mu-receptor opioid agonist, Tyr, D-Ala2, Gly, N-Me-Phe4, Gly-ol5 (DAMGO), was found to increase AP-1 and NF-kappa B activity in primary cultures of neurons from rat cerebral cortex. Acute (2 h, 4 h) and long-term (72 h) treatment with DAMGO time-dependently increased the DNA-binding activity of both AP-1 and NF-kappa B and the stimulation could be abolished or inhibited by concurrent incubation with naloxone. However, acute naloxone-precipitated withdrawal did not significantly change AP-1 or NF-kappa B activity. These results indicate a mu-opioid receptor-related co-induction of AP-1 and NF-kappa B transcription factors in cultured cortical neurons.

Endogenous adenosine as a modulator of hippocampal acetylcholine release.

Modulation of acetylcholine release via adenosine receptors was studied in rabbit hippocampal slices, which were preincubated with 3H-choline and then continuously superfused. Electrical field stimulation of the slices elicited a release of acetylcholine, which was inhibited in a concentration-dependent manner by various adenosine receptor agonists. The effects of the agonists were antagonized by the methylxanthines. From the order of potency: cyclohexyladenosine greater than (-)phenylisopropyladenosine [-)PIA) greater than 5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloradenosine greater than (+)phenylisopropyladenosine greater than adenosine, the inhibitory adenosine receptor may be classified as A1-(R1-)receptor. In experiments on rabbit caudate nucleus slices, adenosine receptor agonists only slightly decreased the evoked acetylcholine release. The presence of an inhibitory tone of endogenous adenosine on hippocampal acetylcholine release is supported by the following findings: 1) the methylxanthines theophylline, 8-phenyltheophylline and 3-isobutylmethylxanthine (IBMX) increased the evoked acetylcholine release in concentrations below those required for phosphodiesterase inhibition. 2) Adenosine uptake inhibitors, in contrast, decreased the evoked transmitter release. 3) Deamination of endogenous adenosine by addition of adenosine deaminase to the medium enhanced the acetylcholine release. In conclusion, acetylcholine release in the hippocampus is depressed at the level of the cholinergic nerve terminals by endogenous adenosine via A1-(R1-)receptors.

Nandrolone modulates the non-opioid and opioid analgesia and tolerance/dependence: role of sexual dimorphism.

The aim of this investigation was to study the effect of the doping steroid nandrolone on metamizol and morphine-induced analgesia and tolerance/dependence in rats. Nandrolone per se did not change the basal nociceptive thresholds in both sexes. It diminished the analgesic effect of metamizol in females, revealed by tail flick test, and males, revealed by paw pressure and hot plate tests. In general, the action of nandrolone was to decrease the morphine-induced analgesia in female and male rats. This was strongly manifested by paw pressure and tail flick tests in male, and tail flick tests in female animals. Nandrolone slowed the development of opioid tolerance/dependence. It aggravated the withdrawal syndrome in the females and invigorated aggression in the males. The data provide evidence that anabolic steroid nandrolone might decrease the analgesic action of metamizol or morphine. The doping steroid could modulate opioid tolerance/dependence and the aggressive behavior in a gender dependent manner. The action of nandrolone is most likely due to profound long-term effects on the central nervous system and might be a gateway to addiction of other drugs of abuse.

Dynamics of Parecoxib or Meloxicam effect on acute postoperative pain after orthopedic surgical interventions: comparative study in hospitalized women and men.

The dynamics of Parecoxib or Meloxicam analgesic effect on acute postoperative pain was studied in 48 patients (22 female and 26 male) sustaining arthroprosthetic (Parecoxib analgesia) or arthroscopic (Meloxicam analgesia) orthopedic surgery. The results show higher postoperative pain and slower dynamics of Parecoxib and Meloxicam analgesia in women than in men, which necessitates supplementation of the applied analgesic medication in the female patients.

In vitro studies of the activity of newly sinthesized nociceptin / orphanin FQ receptor ligand analogues.

Based on template hexapeptides Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH(2) and Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) analogues and corresponding deacylated homologues were synthesized substituting ornithine, diaminobutanoic (Dab) and diaminopropanoic (Dap) acids for lysine at position 6. The aim was to investigate the effect of the newly synthesized compounds on the neurogenic contractions of isolated rat vas deferens. Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH(2) and its analogues manifested a strong inhibitory effect on the neurogenic contractions without effect on the muscle tone, which is characteristic effect of NOP receptor agonists. In contrast, Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) and its analogs manifested a strong inhibitory effect on the muscle tone and negligible effect on the neurogenic contractions which is characteristic effect of NOP receptor antagonists. The most active were the peptides in which Dab or Dap is the substitute. The study reveals that substitution of Lys with shorter amino acids could increase agonist or antagonist activity of the peptide.

Studies on the excitatory neurotransmission in the guinea-pig urinary bladder.

The amplitude of atropine-resistant twich contractions of muscle strips of the guinea-pig urinary bladder was not changed after 90 min continuous electrical stimulation in the presence of 100 microM hemicholinium-3, as compared with the amplitude of the corresponding contractions in control experiments without hemicholinium-3. Addition of 100 microM choline to the medium did not change the amplitude of contractions either. Methionine-enkephalin (1 microM) known as a potent inhibitor of acetylcholine release in autonomic nerves did not change the amplitude of the twich responses. Apamin (1 microM) which inhibited the non-adrenergic, non-cholinergic responses in taenia coli did not change the amplitude of the atropine-resistant contractile responses of the guinea-pig urinary bladder. It is concluded that acetylcholine is not the principle excitatory neurotransmitter in the guinea-pig urinary bladder, although some cholinergic contribution to the twich response exists. The nonadrenergic, non-cholinergic atropine-resistant contractile response of the guinea-pig urinary bladder is apamin-resistant.

Effect of synthetic oxytocin on the motor and bioelectrical activity of the stomach and small intestines (in vivo).

A study is made of the effect of synthetic oxytocin on the mechanograms and electromyograms of the stomach and small intestines of 11 wakeful and 7 chloralose-anaesthesized dogs. Oxytocin was administered intravenously in doses of 0.5 to 2.0 IU/kg for the anaesthesized dogs. The mechanograms were recorded by means of the balloon-kymographic method, the electromyograms-by silver macroelectrodes implanted in the muscle wall. In all animals oxytocin decreased the tone and abolished the peristaltic contractions and the spike-potentials in the stomach and in the intestines for 8 to 15 min. This effect of oxytocin is not eliminated by adreno-, cholino- and gangliolytics. Besides these changes, oxytocin administered in wakeful dogs reduces two or three times the frequency of the basic electric rhythm (BER) in the stomach from 10 s to 3 min and increases the propagation of BER in the small intestine from 10 s to 10 min. The effect of oxytocin on gastric and intestinal BER is eliminated by cholinolytics and gangliolytics and it is not manifested in the small pouch of Heindenhein. A direct myogenic mechanism of oxytocin effect on the activity of the gastro-intestinal smooth muscle is discussed, as well as the effect mediated by vagal nerves and cholinergic receptors.