A retrospective analysis of Terlipressin infusion in patients with refractory hypotension after cardiac surgery.

AIM: The aim of this retrospective study was to evaluate the effectiveness of Terlipressin in the treatment of severe hypotension in cardiosurgical patients and to assess the differences between the groups of survivors and nonsurvivors. METHODS: The study population was 27 patients who developed hypotension after cardiac surgery. RESULTS: All surviving patients developed refractory hypotension early after extracorporeal circulation. Of the 9 nonsurvivors, 3 also experienced postcardiotomy hypotension, while the remaining 6 developed severe hypotension during sepsis. Terlipressin given continuously significantly increased the mean arterial pressure and reduced the heart rate in both groups. Norepinephrine requirements decreased significantly among survivors only. The mean pulmonary artery pressure and pulmonary capillary wedge pressure levels remained unchanged or increased insignificantly, while several liver markers in the survivor group significantly increased. CONCLUSION: Terlipressin given continuously is a potent vasopressor in patients with norepinephrine-resistant postcardiotomy hypotension; however, Terlipressin treatment failed in patients who developed refractory hypotension during sepsis. We cannot recommend this therapy in such patients as it proved to be hemodynamicaly ineffective and may even worsen the circulatory situation.

Inconsistent role of nitric oxide on lipolysis in isolated rat adipocytes.

Though two isoforms of nitric oxide synthase, iNOS and eNOS, were reported in adipocytes, the role of NO in adipose tissue is still ambiguous. The aims of the present study were 1) to follow the effect of bacterial lipopolysaccharide (LPS), on 24 h-lipolysis in rat epididymal adipocyte culture in relation to iNOS stimulation; 2) to compare LPS-induced NO effects with exogenously NO, delivered as S-nitroso-N-acetylpenicillamine (SNAP), and 3) to examine the possible role of NO signaling agonist in lipolysis mediated by the beta(3)-adrenoreceptor agonist. Lipolysis was measured by glycerol and free fatty acid (FFA) production. The medium nitrite levels were used for the indirect estimation of NOS expression. Adipocyte mitochondrial function was assessed by the MTT test. LPS produced a concentration-dependent increase of NO with a decrease of viability at the highest dose. However, LPS did not affect lipolysis. SNAP did not exhibit significant changes in glycerol, FFA or MTT. BRL-37344 and db-cAMP significantly increased nitrite, glycerol and FFA levels. There was a positive correlation between glycerol release and nitrite production. Moreover, BRL-37344 significantly reduced mitochondrial functions. The pretreatment with bupranolol, beta(3)-antagonist, restored all parameters affected by BRL-37344. These results support a concept that NO fulfils multifaceted role of stimulating lipolysis under physiological conditions (beta-agonistic effect) and modulating the same processes during inflammatory (LPS) processes.

Urea synthesis and cyclosporin a biotransformation in a laboratory scale hepatocyte bioreactor model.

Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture. The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal. For the perifusion of cells, they must be immobilized to prepare a bioreactor model. The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture. Hepatocytes were isolated, immobilized and perifused with William's E culture medium containing 1mM NH(4)Cl and CsA (20 microM). Hepatocytes in the culture were treated in the same way. CsA disappearance from the perifusion or culture media was determined by a HPLC method. Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5+/-0.05 mg h(-1) vs 0.33+/-0.03 mg h(-1), respectively). ALT leakage was lower in the bioreactor model (60 Ul(-1)) as compared to hepatocyte culture (125 Ul(-1)). The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively). The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture. Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes. In addition, the cellular model described here inherits economic and ethical potentials.

Effect of nitric oxide donors on isoprenaline-induced lipolysis in rat epididymal adipose tissue: studies in isolated adipose tissues and immobilized perfused adipocytes.

The present investigation was directed to study the effect of in vitro or ex vivo NO donors, sodium nitroprusside and molsidomine, using isolated sliced adipose tissue or in the form of immobilized and perfused adipocytes on the basal and isoprenaline-stimulated lipolysis. The results demonstrated that 1) in vitro application of sodium nitroprusside to perfused adipocytes or molsidomine to sliced adipose tissues affects isoprenaline-induced lipolysis in two ways, an increase in lipolysis at low isoprenaline concentrations (which means the sensitization of adipose tissues to adrenergic effect by NO) and decreased adrenergic agonist-stimulated lipolysis at higher concentration of isoprenaline (a decrease in the maximum lipolytic effect of isoprenaline), 2) low concentrations of molsidomine alone induced lipolysis from adipose tissue which attained more than 60% of that by isoprenaline (pD2 value for molsidomine = 11.2, while pD2 for isoprenaline = 8.17) while sodium nitroprusside did not affect the basal lipolysis significantly, 3) in vivo administration of molsidomine for 2 days reduced the maximum lipolytic effect of isoprenaline and (only non-significantly) increased the sensitivity to low doses of isoprenaline. In conclusion the present data demonstrate that NO plays an important role in adrenergic lipolysis in adipose tissues and further investigations are needed to unravel the exact role of NO in lipolysis.

Potential application of immobilized and perfused hepatocytes in environmental toxicology studies.

Conventional cellular models have contributed significantly to the understanding of many aspects of cell physiology and molecular biology. In these models cells are metabolically less active, due to the inefficient oxygenation and waste product buildup. Therefore perfusion methods for the cells are expected to improve cell activities. Cells have to be fixed in or on an appropriate inert carrier or support, which enables cellular perfusion, maintains integrated cellular functions and makes a bioreactor. Since isolated hepatocytes are extensively used in biomedical studies including those dealing with environmental pollutants or toxins and in xenobiotic biotransformation investigations, an efficient hepatocyte perfusion model has to be available for researchers. This research article is focusing on the value of hepatocyte immobilization as a laboratory bioreactor model and is shedding light on its potentiality in research related to public health. We demonstrate the application of this cellular model as a means to study representative phase I and phase II biotransformation reactions using hexobarbital hydroxylation and 7-ethoxycoumarin deethylation and 4-chloro 2-dinitrobenzene glutathione. Both phase I and phase II drug biotransformation in hepatocytes was demonstrated in this study non-destructively to the cells and in an efficient way. In spite of the aforementioned advantages, immobilized hepatocytes yet have relatively limited applications compared to conventional hepatocyte cellular systems. Reasons for this discrepancy are discussed. This cellular system may become popular due to the better performance of immobilized hepatocytes as compared to conventional hepatocyte culture and due to economic and ethical reasons. Naturally its applicability will cover several biomedical areas including basic research in environmental toxicology and other public health issues.

Preparation of functionally active immobilized and perfused mammalian cells: an example of the hepatocyte bioreactor.

In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion. The rat hepatocytes are isolated from the liver. A 1.8% low-gelling agarose solution is prepared in warm Krebs-Henseleit solution. The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing. Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix. This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver. Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals.

Application of the hepatocytes bioreactor to xenobiotic biotransformation.

This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase I and phase II of biotransformation reactions. To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/l) and hexobarbital (0.2 mmol/l) were selected. For phase II reactions, glutathione transferase activity was evaluated by using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/l). Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time. The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension. At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension. The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension.

Adrenergic lipolysis in immobilized and perfused adipocytes.

The method of cellular immobilization and perfusion was applied to adipocytes. The lipolytic effect of isoprenaline, whose action is produced as a result of receptor-drug interaction, was followed. An agarose solution kept at at 37 degrees C was mixed 1:1 with the cell suspension. Thereafter, adipocytes were immobilized in the agarose threads. The lipolytic effect of 0.1 ml of isoprenaline (1 x 10(-4) mol/l), that was rapidly introduced to the cell perfusion inlet in a non-recirculating system, was monitored by assessing glycerol production. The immobilized and perfused adipocytes exhibited significant lipolytic activity. After reaching the maximum effect, 0.1 ml of propranolol (1 x 10(-3) mol/l) that was applied to the bioreactor inlet, abolished the isoprenaline effect. The present data demonstrate the potential applicability of immobilized perfused adipocytes for various kinds of studies.

[The effect of ascorbic acid on adrenergic lipolysis]. / Ucinek kyseliny askorbové na adrenergní lipolýzu.

The effect of ascorbic acid on basal and adrenergic lipolysis was studied in rat epididymal adipose tissue in vitro. When adipose tissue was incubated with isoprenaline (ISO) for 1.5 h, the concentration-lipolytic effect curves of ISO were practically the same in the presence or absence of ascorbic acid used in concentration 100 and 1000 micrograms/ml. The lipolytic effect of ISO was not substantially altered even in the experiments in which adipose tissue was incubated with ISO for 4.5 h, but ascorbic acid (1000 micrograms/ml) was added only 1.5 h before the end of incubation. On the other hand, the contact of adipose tissue for 4.5 h with high concentration of ascorbic acid (1000 micrograms/ml) induced significant decrease of maximum adipokinetic effect of ISO. Contrary to catecholamine stimulated lipolysis, the basal rate of lipolysis was enhanced by ascorbic acid. Inhibition of maximum lipolytic effect of catecholamines (isoprenaline and noradrenaline-NOR) and oxedrine (isopropylnoroxedrine) was also seen when adrenomimetics were added to incubation medium 1.5 h before the end of 4.5 h incubation of adipose tissue with ascorbic acid. Decreased reactivity of adipose tissue to catecholamines persisted when the tissue, after 3 h incubation with ascorbic, was transferred into fresh medium with ISO and ascorbic acid. Preincubation of ascorbic acid (3h) in the incubation medium without adipose tissue, followed by subsequent addition of the tissue and catecholamines (NOR, ISO) and 1.5 h lasting incubation, did not influence the adipokinetic effect of NOR and ISO. These facts indicate that ascorbic acid decreases the lipolytic effect of catecholamines as a result of its effect on adipose tissue but not on the incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of alpha adrenergic agents on lipolysis in rat adipose tissue; the effect of nonselective alpha adrenomimetics and alpha adrenolytics in an experiment in vitro]. / Vliv alfa adrenotropních látek na lipolýzu z krysí tukové tkáne; pusobení neselektivních alfa adrenomimetik a alfa adrenolytik za pokusných podmínek in vitro.

It is now generally accepted, that in most of the animal species the adrenergic control of lipolysis from adipose tissue is mediated by adrenergic beta 1 and alpha 2 receptors. Therefore, the problem we deal with in this article is concerned with the role of alpha adrenergic receptors in lipolysis in the rat adipose tissue because only in this species the function of alpha adrenergic receptors in lipolysis is still not clear. In in vitro experiments with epididymal adipose tissue of adult rats (where we followed up the release of free fatty acids into the albumin medium) we found: 1) Alpha adrenergic blocking agents phentolamine and phenoxybenzamine had no influence on the lipid mobilizing effect of adrenergic agonists. This effect was absent also when drugs with the strong alpha sympathomimetic effects, e.g. norepinephrine and noroxedrine, were used. 2) On the other hand, alpha adrenergic agonist phenylephrine was able to antagonize in rat adipose tissue the lipid mobilizing effect of beta adrenergic agonist isoproterenol. 3) From our experiments it can be concluded, that phenylephrine acts as a competitive dualist on beta adrenergic receptors, it reduces the effect of full agonist (e.g. isoproterenol) to the level of its own maximum lipolytic effect. No participation of alpha adrenergic receptors in described effects of phenylephrine were detected, because neither the lipolytic, nor the lipolysis blocking effects of phenylephrine were influenced by alpha adrenergic blocking agents. Our studies using alpha adrenergic agonist phenylephrine or nonselective alpha adrenergic blocking agents did not indicate the presence of any alpha adrenergically controlled lipolysis in the rat adipose tissue. However, the role of antilipolytic alpha 2 adrenoceptors in rat adipose tissue cannot be completely excluded, some of these receptors were found on the membranes of rat adipocytes. Our future study deals with this problem.

[Quantitative parameters of drug-receptor interactions and receptor reserves. I. Models I-3-R and I-3-S]. / Uvahy o kvantitativních parametrech farmakon-receptorové interakce a o receptorové rezerve. I: Modely I-3-R a I-3-S.

On a simple model of a reaction-chain from the pharmacon-receptor complex (RA) formation the formation of a ternal complex with substrate (S) and its activation to a product the authors investigate the meaning of the term "affinity" and "internal activity" and the behaviour of the model with substrate and velocity limits at various sites. If K1 = dissociation constant of the complex RA, K2 = dissociation constant of the complex RAS, then at S1 much greater than R1 (i.e. without receptor reserve) the obtained value ED50 described usually as KA = K1K2: (K2 + [St]) and if K1 is constant it is variable in relation to the substrate concentration. In this way continuous differences of apparent affinity of the same substance to the same receptor under different biological conditions are possible. At R1 much greater than St (i.e. with receptor reserve) apparent KA = K1K2: (K2 + [Rt]) and thus K1 can be assessed by blocking receptors with an irreversible antagonist. The authors describe the calculation and very simple graphical method for assessment of basic parameters using an irreversible antagonist. After linear plotting of ED50 (= KA) on the chi-axis and maximal effects on the gamma-axis and after extrapolation of the connecting line of the two points (results without antagonist and results with antagonist) the intersecting point with the chi-axis gives value K1. This makes it possible, among others, to assess also the "intrinsic efficacy" of the substance, the ratio of receptors eliminated by the antagonist, prediction of the behaviour of typical curves during gradual elimination of receptors by a blocker and to assess possibly also the half-life of binding of the irreversible blocker with the receptor.

[Closer characterization of alpha-receptor subpopulations in the smooth muscle of the splenic capsule in rats]. / Pokus o blizsí charakteristiku alpha-receptorových supopulací v hladkém svalu krysí capsula lienalis.

Two different subpopulations of adrenergic alpha-receptors -- "high-affinity" (pD2 less than 8) and "low-affinity" (pD2 less than 6) ones -- are involved in alpha-adrenergic contractions of the rat spleen muscle evoked by noradrenaline in vitro. Only the "high-affinity" subpopulation is agonistically influenced by the alpha 1-mimetic phenylephrine (pD2 cca 6, pKA cca 4.8), antagonistically influenced by prazosin (pA2 cca 9); it has a substantial receptor reserve for noradrenaline and a somewhat lower reserve for phenylephrine, it can be desensitized by these mimetics and it requires the presence of exogenous Ca2+ in the medium for evoking the contraction. -- Only the "low-affinity" subpopulation is agonistically influenced by the alpha 2-mimetic UK-14304. Both subpopulations are activated by noradrenaline and competitively blocked by yohimbine. The effect of neither subpopulation is influenced by nifedipine. According to the new classification (11) the "high-affinity" receptors can be described as alpha 1A, the "low-affinity" ones as alpha 2A. Contrarily to current data, the peculiar feature of this second subpopulation is a low affinity for yohimbine (pA2 less than 7) and a certain extent of blockability by phenoxybenzamine.

Changes induced in adrenergic lipolysis by the administration of diethylstilboestrol, oestradiol and clomiphene.

The authors studied changes in adrenergic lipolysis in the epididymal adipose tissue of rats to which diethylstilboestrol and oestradiol combined with the anti-oestrogen clomiphene were administered. The maximum lipid-mobilizing effect of isoprenaline was increased not only by subcutaneously administered diethylstilboestrol, but also by the highest dose of the antioestrogen clomiphene used (p.o., 200 micrograms.kg-1 b.w.). Under the given experimental conditions, with 4 1/2 h incubation of adipose tissue, clomiphene was also effective when added in vitro. Its own oestrogenic effect probably stimulated the lipid-mobilizing action of isoprenaline. On combining the administration of increasing doses of clomiphene (p.o., 1-5 days) with a constant dose of oestradiol (200 micrograms.kg-1, s.c. on the 8th day, i.e. 24 h before the actual experiment), changes in isoprenaline lipolysis depended on the dose of clomiphene. In low doses clomiphene inhibited the stimulating effect of subsequently administered oestradiol on isoprenaline-induced lipolysis, but in large doses (100 and 200 micrograms.kg-1 daily) it potentiated, together with oestradiol, the lipid-mobilizing effect of isoprenaline. The results show that the non-steroid oestrogen diethylstilboestrol and the antioestrogen clomiphene may be included among the hormones capable of altering the response of adipose tissue to sympathomimetics (isoprenaline). We attribute the fact that clomiphene acted either as an antagonist or as an agonist of oestradiol to its combined oestrogenic and anti-oestrogenic effects.

Genetic differences in the resistance of rats to isoprenaline-induced heart lesions.

Two strains of rats were obtained by selective breeding: the IR strain, resistant to isoprenaline-induced myocardial lesions and the IS strain, sensitive to this damage. The IR rats grew more slowly, the weight of their adipose tissue was higher and the weight of m. soleus was less than that of the IS rats. The IR rats had a higher content of triglycerides in the serum and a lower isoprenaline-stimulated lipolytic activity of adipose tissue in vitro. The basal NEFA level in the serum and its rise after the administration of isoprenaline in vivo did not differ between the strains. The IR rats had a higher content of glycogen in the heart and in the muscle. After the administration of isoprenaline the glycogen content decreased more slowly in IR rats. The findings indicate a considerable importance of the glycogen stores in the heart for the resistance of myocardium to damage.

Effect of oestradiol on catecholamine-stimulated lipolysis.

The authors studied the effect of a single in vivo dose of oestradiol (OE) on adrenergic lipolysis in the epididymal adipose tissue of adult and juvenile male rats, and the effect of OE on plasma free fatty acids (FFA), cholesterol and beta-lipoprotein levels at various intervals after its administration. It was found that OE injected 24 h beforehand in vivo (s.c.), in doses of 100 and 200 micrograms X kg-1 body weight, significantly potentiated the lipid-mobilizing action of the catecholamines noradrenaline (NOR) and isoprenaline (ISO) in adult rats (the action of ISO was potentiated more intensively); in addition, the adipose tissue became more sensitive to the action of NOR, but not of ISO. Raising the dose of OE to 400 micrograms X kg-1 did not enhance the potentiation of the lipolytic action of the catecholamines any further; on the contrary, the lipid mobilizing effect of the catecholamines was potentiated less than after half this dose. Following the s.c. injection of an oily OE solution, the lipolytic effect was potentiated after more than 7 h; the potentiation was strongest after 12 h, but only as far as the maximum attainable degree of lipolysis was concerned. Potentiation of adrenergic lipolysis was found only in adult male rats. In male rats weighing 130-150 g the lipolytic effect of catecholamines (in mumol/g adipose tissue) was significantly greater than in adult animals and the pre-administration of OE did not potentiate adrenergic lipolysis any further. Determination of plasma FFA, cholesterol and beta-lipoprotein levels 1, 2, 4 and 6 hours after the s.c. injection of OE showed only nonsignificant changes (an increase in FFA and a decrease in cholesterol). The authors consider it important to distinguish between the effect of OE on catecholamine-stimulated lipolysis in depot adipose tissue and its effect on lipid metabolism. In their opinion, the dose-dependent effect of OE on muscular and metabolic adrenergic reactions could be one of the factors co-reversible for certain side reactions to steroid contraceptives.

Effect of different albumin media on the lipid-mobilizing action of sympathicotropic substances in vitro.

The authors studied the effect of adrenotropic substances on lipolysis in rat epididymal adipose tissue in albumin medium in vitro. On using albumins of different origin (human, bovine), the pD2 values for catecholamines differed by more than one order, in correlation to the type of albumin used. The isopropylnorsynephrine pD2 values did not differ. The addition of ascorbic acid (100 microng/ml) raised the catecholamine pD2 values and completely equalized the pD2 values found in both media. The pD2 values for the synephrine derivative did not alter. The propranolol pA2 values were not negatively affected by the addition of ascorbic acid. Ascorbic acid also produced a mild increase in the maximum lipid-mobilizing values obtained with any of the given substances in either medium. It was concluded in the discussion that catecholamines are oxidized at different rates in different albumin media and that this oxidation can be inhibited by adding ascorbic acid. Ascorbic acid likewise mildly stimulates the maximum lipid-mobilizing effect. The authors recommend the addition of ascorbic acid to albumin medium as a regular component for the study of adrenergic lipid mobilization.