Sodium nitroprusside regulates the relaxation of the longitudinal muscle in the gut.

Nitric oxide (NO) has been shown to mediate nonadrenergic-noncholinergic relaxation in gastrointestinal (GI) smooth muscle cells. As GI smooth muscles relaxations are partly dependent on NO, we decided to investigate the effect of sodium nitroprusside (SNP) on the longitudinal muscle contraction of the isolated guinea pig ileum. Increasing concentrations of SNP (10(-10)M, 10(-9)M, 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) reduced ileum contractions stimulated by electrical stimulation (ES) (8-76%; p < 0.05) and by acetylcholine (Ach) (23-62%; p < 0.05) significantly and in a concentration-dependent manner. Furthermore, treatment with an inhibitor of the soluble guanylate cyclase, methylene blue (10 mM), antagonized significantly the relaxing effect of SNP (0-39%; p < 0.05, p < 0.01, p < 0.001 for ES- and 4-27%; p < 0.05 for Ach-induced contractions). The results show that treatment with 1 microM manganese-containing superoxide dismutase (MnSOD) and 10 microM L-arginine (L-arg) caused a significant decrease in SNP induced relaxations (6-55%; p < 0.05, p < 0.001 and 2-46%; p < 0.05, p < 0.01 for ES- and 15-28%; p < 0.05, p < 0.01, p < 0.001 and 12-32%; p < 0.05, p < 0.01 for Ach-induced contractions, respectively). In conclusion, our data suggest that SNP, which releases NO, is able to depress longitudinal muscle contraction of the isolated guinea pig ileum, suggesting that exogenous application of NO inhibits intestinal contractions of smooth muscle cells and that cGMP mediates the response to NO. In addition, MnSOD and L-arg decreased the relaxing effect of SNP on the isolated ileum of the guinea pig.

The effect of ivermectin on convulsions in rats produced by lidocaine and strychnine.

Ivermectin is one of the most commonly used drugs in pharmacotherapy of parasitic diseases in domestic and wild animals caused by parasitic nematodes and arthropods. However, ivermectin and other avermectins very often produce side-effects in hosts. The most dominant clinical symptom of ivermectin toxicity in domestic and wild animals is CNS depression. In nematodes, the target site of ivermectin's action is glutamate-gated chloride-channel receptor and GABA receptor. The depressive effect of ivermectin in mammals might include more than one mechanism; therefore, the anticonvulsive effect of ivermectin against convulsions caused by lidocaine and strychnine was evaluated. Ivermectin antagonized lidocaine- and strychnine-induced convulsions in rats, although these have different mechanisms. In the present study, the anticonvulsive ED50 ofivermectin for lidocaine-induced convulsions was 2.44 mg/kg (95% CL 1.67 to 3.57 mg/kg), whereas for convulsions induced by strychnine it was higher at 4.25 mg/kg (95% CL 2.32 to 3.78 mg/kg). At the same time, both anticonvulsive doses are significantly lower then the observed LD50 of ivermectin (18.20 mg/kg). Furthermore, flumazenil (0.1 and 0.2 mg/kg), an antagonist of benzodiazepine receptors, antagonizes just one part of these anticonvulsive effects of ivermectin. Our results show the significant anticonvulsive properties of ivermectin and support the findings that ivermectin in the CNS of mammals produces multiple inhibitory effects, probably through participation in the function of GABA-sensitive and GABA-insensitive chloride channels.

Xylazine, an alpha 2-adrenergic agonist, induces apoptosis of rat thymocytes and a thymocyte hybridoma line in vitro.

In our previous experiments, we demonstrated that xylazine, an alpha 2-adrenergic agonist, stimulated proliferation of thymocytes triggered by concanavalin A. In contrast, higher concentrations of xylazine were inhibitory. In this work, we studied the mechanisms involved in immunosuppression of xylazine and found that the compound at concentrations between 100 microM and 500 microM induced apoptosis of rat thymocytes in vitro. In addition, xylazine at concentrations higher than 50 microM also induced apoptosis of a thymocyte hybridoma (BWRT8) and increased apoptosis of the line triggered by T cell receptor (TCR) cross-linking. Apoptosis was confirmed by morphological analysis staining with merocyanine 540 and propidium iodide and in cases of BWRT8 by fragmentation of DNA. The mechanisms of xylazine-induced apoptosis of the BWRT8 hybridoma were further examined. We demonstrated that the process in both nonactivated and activated (TCR cross-linking) BWRT8 cells was not prevented by yohimbine (a selective alpha-adrenergic antagonist) and by antibodies to Fas and Fas-L. In contrast, cell death was completely blocked by a caspase inhibitor, z-Val-Ala-Asp (OMe)-CH2F. Cyclosporine, a calcineurin blocker, partly inhibited the xylazine-induced apoptosis of activated BWRT8 cells.

Immunomodulatory effect of xylazine, an alpha(2) adrenergic agonist, on rat spleen cells in culture.

Xylazine is an adrenergic alpha(2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 microM) and inhibitory (50-500 microM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another alpha(2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha(2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 microM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha(2) adrenoreceptors.

Xylazine, an alpha 2-adrenergic agonist, modulates proliferation of rat thymocytes in vivo and in vitro.

The effect of xylazine, an alpha 2-adrenergic agonist, on proliferation of rat thymocytes in vivo and in vitro was examined. It was found that the agonist administered to rats in vivo at doses of 2.5 mg/kg and 5 mg/kg stimulated thymocyte proliferation to suboptimal (0.625 microgram/ml) concentrations of concanavalin A (Con A). A similar effect was confirmed in vitro when lower concentrations of xylazine (5 microM) were added to cultures of thymic cells from intact animals in the presence of both suboptimal and optimal (2.5 micrograms/ml) Con A concentrations. Higher doses in vivo (25 mg/kg) and in vitro (50 microM, 100 microM and 250 microM) significantly inhibited proliferation of thymocytes to Con A. The phenomenon was followed by a decrease in interleukin-2 (IL-2) production (in vivo and in vitro) and down-regulation of IL-2 receptor alpha (IL-2R alpha) expression (in vitro). The exogenous IL-2 completely restored the inhibitory effect of xylazine in vivo on thymocyte proliferation. However, a minimal influence of the cytokine on the xylazine-inhibited thymocyte proliferation in vitro was observed. Stimulatory effect of xylazine on proliferation of thymocytes was probably mediated through alpha 2-adrenoreceptors since it was blocked by yohimbine, an alpha 2-adrenoreceptor antagonist. It seems that the pathways involved in inhibition of thymocyte proliferation by xylazine are more complex because the xylazine-suppressed thymocyte proliferation was potentiated by yohimbine.

Urinary excretion of phenobarbital and its metabolite p-hydroxyphenobarbital in convulsing and non-convulsing patients.

As part of an investigation of phenobarbital (PB) pharmacokinetics in patients with status epilepticus (SE), urinary excretion of PB and its main metabolite, hydroxyphenobarbital (HPB), was studied in patients who had an episode of SE, as well as in non-convulsing ones. Eleven in-patients were studied:(group 1) five patients (4 M + 1 F; 48 +/- 28 years old; 64 +/- 6 kg body weight; mean +/- SD) with convulsive status epilepticus, and (group 2) six patients (5 M + 1 F; 37 +/- 13 years old; 71 +/- 15 kg body weight) with epilepsy, seizure-free at the moment of PB administration and without established anti-epileptic therapy. All subjects received a single intravenous dose of PB (15 mg/kg) at a rate of 100 mg/min. PB and HPB concentrations were measured by high performance liquid chromatography with UV detection at 220 nm in urine samples collected throughout 24 h. The comparison of pharmacokinetic parameters of urinary excretion of PB and HPB showed a statistically significant difference in the values of recovery of HPB and total barbiturate (higher values in the patients with SE) in 24 h urine. Differences in the excretion of PB between the two groups of patients--higher values in the patients who had had an episode of SE, and in urine flow--slightly elevated volumes in the same group, failed to reach statistical significance, probably due to the small number of participants in the study.

Physostigmine and modulators of nitric oxide system on the mean arterial pressure of the spontaneously hypertensive rat.

1. A slow intravenous infusion of L-arginine (3 mg kg-1) lasting one hr produced significant hypotension in urethane-anaesthetized spontaneously hypertensive rats (SHRs). 2. A slow intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME) (3 mg kg-1 h-1) did not produce any significant change in the mean arterial pressure during infusion. After stopping infusion of L-NAME, a slowly developing increase of the mean arterial pressure was observed during the following 40 min. 3. The pressor response to physostigmine (20, 40 and 80 micrograms kg-1, IV), injected during a slow intravenous infusion of either L-arginine or L-NAME, was not changed. 4. L-arginine and L-NAME depressed the pressor responses to physostigmine, if physostigmine was injected after the end of a 1-hr infusion. 5. Acute pretreatment with increasing doses of physostigmine markedly affected the blood pressure response to L-arginine (i.e., L-arginine-caused hypotension was more pronounced), but only slightly that to L-NAME. 6. In conclusion, L-arginine, as a donor of NO, produced hypotension by itself and also decreased, but not significantly, the central cholinergically-mediated hypertension (CCMH) produced by physostigmine. It is quite possible that the peripheral NO released by L-arginine antagonized the increased adrenergic activity in the CCMH. This does happen in normotensive rats, but to a lesser degree than in SHRs, as shown in the current experiments. 7. Also, our results show that inhibition of endogenous NO biosynthesis using L-NAME does not necessarily lead to pressor response in vivo, at least in SHRs. It is concluded that L-arginine-nitric oxide pathways operate in SHRs, as well as in normotensive Wistar rats, but their role in modulating cholinergically-mediated regulation of the mean arterial pressure is less pronounced in SHRs than in normotensive animals.

The effect of nickel chloride on the isolated hemidiaphragm of the rat.

1. NiCl2 (cumulative concentrations of 0.56-1.91 mmol 1(-1)) produced concentration-dependent depression of tension developed (Td) and the maximum rate of rise of tension (dT/dt max) of isometric contraction of the isolated rat hemidiaphragm, during direct subtetanic (DST) electrical stimulation, only. EC50 values for NiCl2-induced depression of Td and Dt/dt max were 0.88 +/- 0.06 and 0.83 +/- 0.13 mmol 1(-1), respectively. NiCl2 did not significantly change either parameter of the isometric contraction during direct single-pulse (DSP) electrical stimulation. 2. Maximal depression of Td and dT/dt max, produced by a single concentration of NiCl2 (1 mmol 1(-1)) during DST electrical stimulation was obtained 20 min after addition of the drug in the bathing medium. 3. In the normal Tyrode solution, addition of CaCl2 (final concentration of 5.86 mmol 1(-1)) almost completely antagonized the depressant effect of NiCl2 (1 mmol 1(-1)) on Td and dT/dt max during DST electrical stimulation. In the calcium-free solution, the depression both of Td and dT/dt max produced by NiCl2 (1 mmol 1(-1)) was significantly more pronounced in comparison with the effect of NiCl2 in the normal Tyrode solution. 4. L-calcium channel activator, Bay K 8644 (25 mumol 1(-1)), significantly potentiated both Td and dT/dt max during DST electrical stimulation, but NiCl2 (1 mmol 1(-1)) decreased both parameters of the isometric contraction even in the presence of this concentration of Bay K 8644. On the other hand Bay K 8644 (25 mumol 1(-1)) did not antagonize NiCl2-induced depression of Td and dT/dt max. 5. Verapamil (2.5 mumol 1(-1); 45 min of incubation) and lidocaine (0.10 mmol 1(-1); 30 min of incubation) significantly potentiated the depression of Td and dT/dt max, produced by NiCl2 (1 mmol 1(-1), during DST electrical stimulation. The addition of CaCl2 (final concentration of 7.20 mmol 1(-1)) in the bathing medium only partially antagonized the depressant synergistic action of both verapamil or lidocaine and NiCl2 on Td and dT/dt max. 6. Forskolin (cumulative concentrations of 2.60-44.20 mumol 1(-1)) fully antagonized NiCl2-induced depression of both Td and dT/dt max; propranolol (1 mumol 1(-1)) did not abolish this antagonizing action of forskolin. Also, NiCl2 (cumulative concentrations of 0.56 -1.54 mmol 1(-1)) did not change potentiating effect of forskolin (23.4 mumol 1(-1)).(ABSTRACT TRUNCATED AT 400 WORDS)

[Adverse drug reactions in children]. / Nezeljena dejstva lekova kod dece.

Small children, older than one year of age, can bear as many drugs as mature people can. They can bear some drugs even better than older people, but show unusual sensitivity towards certain drugs. The occurrense of dose-dependent adverse drug effects can be influenced by pharmacokinetics (limited biotransformation-inactivation and elimination of drugs). Only a few drugs can cause adverse effects occurring almost exclusively in children. Even if rare, pediatric adverse drug reactions may be life-treatening. It should be pointed out that many drugs are restricted for children, and many drugs, in spite of the fact that are in clinical practice for several decades, contain no approval for use in children.

The effects of physostigmine, L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on the mean arterial pressure of the rat.

The effects of physostigmine, L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) were investigated in urethane-anaesthetized rats. The drugs were chosen because physostigmine has been known to produce an increase in peripheral adrenergic activity, whereas L-arginine and L-NAME have been known to modulate nitric oxide (NO) production. Slow infusion of L-arginine produced significant hypotension, but only in animals pretreated by physostigmine. L-NAME applied in the same way produced a slow developing increase in blood pressure, but not in animals pretreated by physostigmine. The pressor responses to physostigmine were potentiated if the drug was injected during infusion of L-NAME, and depressed if the drug was injected after stopping L-NAME infusion (in rats not pretreated with physostigmine). It is concluded that L-arginine-NO pathways act in vivo to oppose peripheral vasoconstrictor influences coupled with central cholinergically mediated activation of the adrenergic system, as produced by physostigmine. In this way, NO is part of a general mechanism for blood pressure regulation.

Some new evidence on antifatigue action of aminophylline on the isolated hemidiaphragm of the rat.

1. Aminophylline (cumulative concentrations of 0.036-3.60 mmol/l) produced a concentration-dependent increase in both tension developed (Td) and the maximum rate of rise of tension (dT/dt max) of the isolated hemidiaphragm of the rat both during direct single-pulse and subtetanic stimulation. 2. The repeated series of additions of aminophylline into the bathing medium (the second and the third series) produced even further, more pronounced potentiation of both Td and dT/dt max during subtetanic stimulation only, the potentiation being the strongest after the third series of additions of the drug ("antifatigue effect"). The antifatigue effect of aminophylline was much more pronounced than the antifatigue effect of the equimolar concentrations of caffeine. 3. The presence of intact beta 1-adrenergic receptors seems to be essential for the antifatigue action of aminophylline under our experimental conditions. 4. The antifatigue effect of aminophylline was not affected by reserpine or 6-OHDA pretreatment of rats. 5. In a Ca(2+)-free medium the stimulatory effect of aminophylline on Td and dT/dt max was abolished or depressed (single-pulse and subtetanic stimulation, respectively). After returning the muscle into the medium containing Ca2+, the effect of aminophylline was significantly potentiated during both types of the stimulation. 6. The antifatigue action of aminophylline was preserved even in the presence of nicardipine or its solvent in the bathing medium. 7. In the presence of heparin (which produced a significant depression of both Td and dT/dt max by itself during direct subtetanic stimulation) the stimulatory effects of aminophylline on Td and dT/dt max (the second and third series of additions) were significantly potentiated in comparison with the effects of the first series of additions of aminophylline (with no heparin in the bathing medium). 8. The dose-response curves for the effects of aminophylline in the presence of Ni2+ on Td and dT/dt max during direct single-pulse stimulation were significantly shifted to the right. Ni2+ by itself produced significant and dose-related depression of both Td and dT/dt max during single-pulse and subtetanic stimulation, the subtetanic stimulation being much more sensitive. The antifatigue effect of aminophylline during subtetanic stimulation was preserved in the presence of Ni2+. 9. Our results indicate the important role of the extracellular calcium and the involvement of intact beta 1-adrenergic receptors in the antifatigue action of aminophylline. Also, the potentiating effect of heparin on the antifatigue action of aminophylline is presumably due to the influx of extracellular calcium through L-type Ca2+ channels during subtetanic stimulation. Our results indicate the possibility of the presence of T-type calcium channels (which can be blocked by Ni2+) in the isolated hemidiaphragm of the rat, but they do not seem to be involved in the antifatigue action of aminophylline.

[Introduction of adrenergic beta-blockers into medical therapy]. / Uvodjenje adrenergickih beta-blokatora u terapiju.

Introduction of adrenergic beta-blockers into medical therapy is an exceptionally good example of how a clear scientific background and a perfect knowledge about physiological and pathophysiological functions may help to plan and accomplish a pharmacological investigation in order to introduce new drugs. Several scientific contributions have helped development of adrenergic beta-blockers: R. Ahlquist with his discovery of adrenergic beta-receptors, U.von Euler with the discovery of the adrenergic neurotransmitter noradrenaline, J. Axelrod with the discovery of catecholamine metabolism, E. Sutherland with the discovery of the molecular nechanism of beta-receptor functioning, and last, but not least, Sir James Black with the discovery and successfull introduction of first adrenergic beta-blockers into medical therapy. Except R. Ahlquist, all others were awarded the Nobel prizes in Physiology or Medicine. Beta-blockers are among the most significant contributions to modern pharmacotherapy. If nothing spectacular happens during the next several yearx of this century, then the adrenergic beta-blockers will remain among the top ten pharmacological discoveries in the 20th century. This Pharmacotherapeutic Symposium on Beta-Blockers shows the achievements in clinical application of adrenergic beta-blockers in various Medical specialities during the last 10 years.

The effect of physostigmine on the haemorrhagic hypovolemia in anaesthetized rabbits.

1. The effects of physostigmine (70 micrograms kg-1, intravenously) on mean arterial blood pressure, blood volume and survival were studied in anaesthetized rabbits subjected to haemorrhagic hypovolemia. 2. It was found that physostigmine increased the mean arterial blood pressure, increased the residual blood volume, decreased the haematocrit values and increased the survival of the animals. 3. The increase of blood pressure might be due to a general adrenergic activation produced by physostigmine, whereas the increase in plasma volume might be due to changes in pre- to postcapillary resistance ratio. 4. The beneficial effect of physostigmine might also be due to antagonism of humoral factors known to aggravate the hypovolemia (e.g. endogenous opioids).

Beta-adrenergic receptors and catecholamines in the rat heart during tourniquet trauma.

Tourniquet trauma produced a decrease in the noradrenaline content in the heart of the rats through the period of tourniquet application (up to 4 hr). In the same period, the content of adrenaline was significantly increased. This relationship between noradrenaline and adrenaline remained the same in the posttraumatic period. Parallel to the observed changes in the catecholamine content of the heart, a significant decrease in the number of the beta-adrenergic receptors (Bmax) and an increase in their affinity (a decrease in KD) was also found in the hearts of rats exposed to tourniquet trauma. These changes remained throughout the posttraumatic period, with one exception: no change 30 min after trauma has been observed. Reapplication of tourniquet was associated with a restoration of the beta-adrenergic receptors and complete survival of the animals. The decrease in the beta-receptors density after trauma might be due to down-regulation produced by increased concentration of adrenaline, a beta-receptor agonist. Meanwhile, some other factors, particularly ischaemia, might also contribute to the observed changes in the beta-adrenergic binding sites.

An improved gas chromatographic determination of valproic acid and valpromide in plasma.

A newly, improved analytical method for determination of valproic acid (VPA) and valpromide (VPD) in human plasma was developed. The method is based on gas chromatographic determination with flame ionization detection, after chloroform extraction of the drugs from plasma. Caprylic acid was used as an external standard. With the extraction procedure chosen, high recoveries for both VPA and VPD were achieved (98-102%), with correlation coefficient of 0.9998 for VPA and 0.9996 for VPD. Sensitivity of the method was also high (2 ng or 2 mg/L), while the linearity was obtained over the range of 5-150 mg/L, with high correlation for both drugs (0.9997 and 0.9995 for VPA and VPD, respectively). Reproducibility of the method was documented with low values of coefficients of variation, both inter-assay and day-to-day values (1.4-4.4 and 1.7-3.7 for VPA and 2.1-5.0 and 2.6-5.1 for VPD, respectively). The method has been used to follow VPA plasma levels in adult epileptic patients on sodium valproate therapy.

Modulating effect of adenosine on the hypertensive response to physostigmine.

The purpose of this work was to study the neuromodulatory role of adenosine on the hypertensive response to physostigmine in the rat. It was found that increasing doses of adenosine, injected intravenously, produced a biphasic blood pressure response, consisting of an initial and short-lasting hypotension, followed by a prolonged hypertension. The hypertensive phase of this response was significantly depressed or even abolished by phentolamine, whereas the hypotensive phase was depressed but not abolished by atropine. If injected intravenously, but immediately before physostigmine, adenosine produced a potentiation of the blood pressure response to physostigmine. This was even more evident if adenosine was injected directly into the carotid artery immediately before the intravenous injection of physostigmine. Neither aminophylline nor 8-phenyltheophylline affected this potentiating action of adenosine. This finding might indicate that this potentiating action does not involve the already known adenosine receptors, or that this action is realized through some other type of receptors and mechanisms not sensitive to xanthine derivatives. It is concluded that adenosine might have a general modulating presynaptic effect on various transmitting processes, including those which take place in the central nervous system during the hypertensive response to physostigmine in the rat.

The life-saving effect of physostigmine in haemorrhagic shock.

The intravenous injection of physostigmine (70 micrograms kg-1) produces a life-saving effect in acute haemorrhagic shock in non-anaesthetized rabbits. This effect is most probably due to a transfer of tissue fluids into circulation. The crucial beneficial effect of physostigmine might be a decrease of the capillary hydrostatic pressure due to changes in pre- to postcapillary resistance ratio. Both lines of defence comprise a normalization of blood pressure and normalization of blood volume, thus saving the life of the animal.

When is the use of alternative biological fluid in pharmacokinetics possible; an example of theophylline.

Special interest in alternative approach to clinical pharmacokinetic research nowadays, especially in pediatric practice, is concerned on biological fluids obtainable by non- invasive methodology. Saliva and urine are the most interesting fluids in this approach. The possibility of using saliva and urine was investigated and evaluated in theophylline (Th) therapy, both in asthmatic children and adults. The investigation on drug presence in saliva (asthmatic children and adults) and in urine (asthmatic children) during Th therapy, confirmed good correlation between saliva and plasma, and between urine and plasma, and provided reliable basis for the conclusion that both saliva and urine can be used in pharmacokinetic research of The elimination in asthmatic children. Only saliva was investigated in adult patients and it was found that it can be used as alternative biological fluid in pharmacokinetic research, but with less reliability than in children.