Importance of the antithrombin III-heparin complex in the protective reaction to thrombin formation in animal and human blood

The antithrombin III-heparin complex was isolated from albino rat and human blood. The non-enzymatic fibrinolytic activity of the complex during anticoagulation system activation was higher, as compared to analogous activity in normal conditions or in depressed function of anticoagulation system.     

Metabolic and hormonal response to acute cold exposure in newborn pig

Glucose metabolism and changes in plasma insulin, glucagon and catecholamines were studied in unfed newborn pigs during acute cold exposure immediately after birth. When newborn pigs are exposed to a moderate cold external temperature (20 degrees C), they exhibit a transient thermoregulatory response characterized by an increased liver glycogenolysis, an enhanced blood glucose clearance rate (+35%) and a rise in plasma catecholamine concentrations. When the newborn pigs are exposed to a cold external temperature (12 degrees C), they become rapidly (10-12 h after birth) hypothermic and hyperglycaemic. This results from a fall in blood glucose clearance rate (-40%). Muscle glycogenolysis is low in normothermic animals during the 12 h following birth. Muscle glycogenolysis increases after a delay of 6 h in animals exposed to an external temperature of 20 degrees C or 12 degrees C. These data demonstrate that the failure in the thermoregulatory response in the newborn pig exposed to a cold temperature is not the consequence of a lack of mobilization of energy stores, but results from a defect in glucose utilization.     

Kidney function and sulfate uptake and loss in the freshwater bivalve Toxolasma texasensis

Toxolasma texasensis acclimated to an artificial pondwater (PW) maintained a concentration of SO4 in the blood of about 1-2 mmol l(-1) . The anion transport inhibitor DIDS (5,5'-diisothiocyanatostilbene 2, 2'-disulfonic acid) reduced the uptake of 35SO4 from the bathing medium by 54%. The clearance of polyethylene glycol (PEG) injected into the blood of T. texasensis ranged between 0.8 and 1.3 ml g(-1) dry tissue h(-1), and provided an estimate of renal filtration in PW-acclimated animals. The clearance of radioactive 35SO4 simultaneously injected into the same animal was about 16% of the PEG clearance, suggesting that sulfate was being reabsorbed by the kidney. Para-aminohippuric acid was cleared about 4.6 times faster than PEG, indicating that this organic acid was subjected to secretion in addition to filtration. When the normal osmotic gradient was abolished by acclimating T. texasensis to 10% seawater (SW), the PEG clearance decreased to 0.17 ml g(-1) dry tissue h(-1). Sulfate clearance in animals acclimated to PW or 10% SW was the same. However, in mussels acclimated to 10% SW, the calculated amount of SO4 reabsorbed was significantly reduced relative to mussels acclimated to PW. T. texasensis conserved SO4 when acclimated to PW, and reduced reabsorption when acclimated to the sulfate-rich 10% SW. When mussels acclimated to 10% SW were returned to PW, there was a transient increase in sulfate clearance during the first 8 h because filtration exceeded reabsorption.     

Vasopressinergic,Oxytocinergic, and Somatostatinergic Neuronal Activity after Adrenalectomy and Immobilization Stress

Twenty days after bilateral adrenalectomy (ADX) or immediately after the last of three 6-h long immobilization periods, the levels of hypothalamic and neurohypophyseal L-[³⁵S]Cys-labeled arginine vasopressin (AVP), oxytocin (OT), and somatostatin-14 (SRIF) (only stressed animals) were measured simultaneously in male Wistar rats, after third ventricular administration of the labeled precursor, via guide-cannulae. The acetic acid-extracted labeled peptide fractions were purified by two sequential HPLC steps. After a 4 h period of labeling, only L-[³⁵S]Cys-AVP was selectively increased in the hypothalami of ADX-ized rats, compared to the sham-operated animals, possibly reflecting a significant activation of the paraventricular parvocellular (PVC) AVP/corticotropin-releasing factor (CRF) neurons. The increased accumulation of neurohypophyseal L-[³⁵S]Cys-labeled AVP and OT in these animals, without changes in the endogenous levels of these peptides, as measured by UV absorbance, also suggests a moderate activation of the magnocellular (MGC) AVP and OT neurons, as a consequence of adrenal insufficiency. In response to immobilization stress, levels of L-[³⁵S]Cys-OT were selectively increased in the hypothalami and corresponding neurohypophyses, 2 h and 4 h after receiving the label, concomitantly with a statistically significant reduction in the stores of OT in the neural lobes. AVP and SRJF biosynthesis remained unaffected by immobilization; the neurohypophyseal AVP stores likewise remained unchanged. These observations suggest the selective activation of MGC-OT neurons in response to chronic immobilization stress. Selective increases in hypothalamic L-[³⁵S]Cys-AVP in ADX-ized rats, and in hypothalamic L-[³⁵S]Cys-OT in chronically stress-immobilized rats, are presented as a measure of PVC-AVP/CRF and MGC-OT neuronal activation, respectively.     

The mutagenic and carcinogenic effects of sulfur-35

In experiments with metaphase plates from blood lymphocytes, conducted during rats' lifetime, a study was made of the mutagenic effect of 35S. Various tumors were diagnosed in the experimental animals after their death. The competitive analysis of the number of stable cytogenetic changes in lymphocytes of the experimental animals at the remote times and tumor occurrence has revealed a highly positive correlation between these indices. Both effects were severest at the highest absorbed 35S dose of less than 10 cGy.     

Brain-blood permeability: TNF-alpha promotes escape of protein tracer from CSF to blood

The objective of this study was to determine the effect of tumor necrosis factor (TNF)-alpha on the efflux of protein from the central nervous system to blood based on assessing the clearance of radiolabeled albumin from the cerebrospinal fluid (CSF) to blood in rats. (125)I-labeled human serum albumin ((125)I-HSA) was injected into a lateral ventricle, and venous blood was sampled hourly to determine the basal CSF protein clearance into the blood. After this, rats were intraventricularly infused with 10 microliter TNF-alpha and 10 microliter (131)I-HSA (n = 6) or 10 microliter saline and 10 microliter (131)I-HSA (n = 6). Venous blood was sampled hourly for 3 h. (131)I-HSA tracer recovery increased threefold in the venous blood and was significantly higher in the spleen, muscles, and skin in animals treated with TNF-alpha. No significant changes were observed in control animals treated with saline. The data suggest that TNF-alpha promotes the clearance of protein macromolecules from the CSF to the venous blood.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Effects of salinity on solute clearance from the freshwater bivalve, Dreissena polymorpha Pallas

Clearance of polyethylene glycol (PEG), inulin, or dextran that had been injected into the hemolymph of the mussel, Dreissena polymorpha, was measured in animals acclimated to pondwater (PW) or 10% seawater (SW). In addition, we measured the clearance of PEG from mussels acutely transferred into 10% SW and following return to PW after acclimation to 10% SW. Clearance values calculated for PW-acclimated mussels ranged from 2.0 to 3.3 ml (g dry tissue ċ h)^-1 and declined to 0.28 ml (g dry tissue ċ h)^-1 in 10% SW-acclimated animals. Transferring mussels into 10% SW resulted in a reduction in PEG clearance from the blood, coincident with the reduction of osmotic gradient. When 10% SW-acclimated mussels were returned to PW the clearance of PEG increased to rates observed in PW-acclimated animals within 1 h. The PEG clearance remained constant during the re-acclimation to PW even though the osmotic gradient declined from about 100 to 30 mosmol kg^-1. Clearance of the solutes used in this study was likely to be a measurement of renal filtration rate. The clearance values appeared to be maximal when the animals were in PW. The limited capacity to increase clearance in the face of an osmotic challenge may be a critical factor in restricting D. polymorpha to freshwater or lower salinity environments with small ranges in salinity.     

A method for assessing phagocyte clearance rates in man.

By comparing the clearance rates of ICG and 99mTc-sulphur colloid from the blood stream, it has been possible to provide a method for assessing the phagocytic activity of the reticulo-endothelial system in man.     

The phagocytic activity of reticuloendothelial system of newborn precolostral pigs to smooth and roughEscherichia coli

The fate of smooth and rough strains ofEscherichia coli in the blood stream of newborn, germ-free, colostrum deprived piglets was studied using blood clearance test with live and radioisotope-labelled (P³²) bacteria. Smooth strains are eliminated at an extremly slow rate (hours) whereas rough strains are taken up within 30 minutes. Specific immune serum accelerates rapidly the clearance rate of smooth strains after bothin vitro andin vivo opsonisation of bacteria. Clearance of smooth liveEscherichia coli from the blood stream cannot be stimulated by an endotoxin treatment of newborn piglets. The possible role of complement system as a nonspecific opsonin in phagocytosis of rough strains is discussed. The results demonstrate that RES cells of newborn precolostral piglets are functionally competent as to the capacity to remove gramnegative bacteria from the blood stream provided specific and/or nonspecific opsonins are available.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

The liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys

Infection with human or simian immunodeficiency virus (SIV) is characterized by the rapid turnover of both viral particles and productively infected cells. It has recently been reported that the clearance of SIV in vivo is exceedingly fast, with half-lives on the order of minutes. The underlying mechanism or site responsible for this rapid clearance, however, remains unknown. To investigate this issue, we chose to infuse infectious SIVmac239 grown from autologous peripheral blood mononuclear cells that were radioactively labeled by [(35)S]methionine and [(35)S]cysteine. This approach eliminates from the viral membrane alloantigens that may have a significant impact on viral clearance. In addition, this approach also permits identification of the sites of viral clearance by measuring the radioactive intensity, even if degradation of SIV RNA occurs in tissues. We now report that the half-life of infused SIV in blood is extremely close to estimates from a previous study, in which unlabeled SIV grown in a heterologous cell line was used. The allogeneic effect due to the presence of human antigens on the surfaces of virions may, therefore, play a minimal role in the high rate of virion clearance. Moreover, close to 30% of infused radioactivity was found in the liver and measureable amounts were detected in the lungs (5.4%), lymph nodes (3.0%), and spleen (0.4%). The detection of a significant proportion of infused virus in the liver suggests that viral clearance from circulation is mediated by a common, nonspecific mechanism, such as the phagocytic functions of the reticuloendothelial system. The rapid clearance and degradation of exogenously infused virions may pose a major obstacle for gene therapy with viral vectors, unless strategies to overcome the rapid in vivo elimination of these particles are developed.     

Complex Compound of Heparin and Glutamic Acid: Synthesis and Effect on Hemostatic Indices in vitro and in vivo

A complex compound of high-molecular heparin and glutamic acid has been synthesized. This compound has anticoagulant, fibrinolytic, and antithrombotic properties in vitro. Single intravenous or chronic peroral introduction of the complex in normal animals imitates the activation of anticoagulation system. When the anticoagulation system in depressed, the complex restores or even activates the anticoagulant–fibrinolytic background of the serum it. The obtained data are discussed in physiological terms.     

Cardiovascular responses to exercise and muscle metaboreflex activation during the recovery from pacing-induced heart failure.

Rapid recovery of resting hemodynamics from tachycardia- or arrhythmia-induced heart failure (HF) has been demonstrated in both humans and animals. However, little is known about cardiovascular responses to exercise in animals or about reflex control of the cardiovascular system during exercise while recovering from HF. Inasmuch as the reduced cardiac output (CO) during exercise in HF has been shown to lead to underperfusion of active skeletal muscle and tonic activation of the muscle metaboreflex, an improved CO during exercise in subjects recovering from HF may lead to higher skeletal muscle blood flows and to relief of this metabolic stimulus. We investigated cardiovascular responses to graded treadmill exercise and metaboreflex activation [evoked by imposed graded reductions in hindlimb blood flow (HLBF) during mild and moderate exercise] in chronically instrumented dogs during control, mild to moderate HF (induced by rapid ventricular pacing), and recovery from HF. Most hemodynamic responses to graded exercise returned to control within 24 h of disconnecting the pacemaker. After 2 wk of recovery, CO and HLBF at each workload were significantly higher than control. In addition, whereas the increase in CO that normally occurs with metaboreflex activation was markedly attenuated in HF, it completely returned in the recovery experiments. We conclude that cardiovascular responses to graded exercise during the recovery from pacing-induced HF return rapidly to near or above control and that the increased CO and HLBF in recovery likely relieved the metabolic stimulus and tonic metaboreflex activation that may have occurred during moderate exercise in HF.     

Endothelin receptor A antagonism attenuates renal medullary blood flow impairment in endotoxemic pigs

Background

Endothelin-1 is a potent endogenous vasoconstrictor that contributes to renal microcirculatory impairment during endotoxemia and sepsis. Here we investigated if the renal circulatory and metabolic effects of endothelin during endotoxemia are mediated through activation of endothelin-A receptors.

Methods and Findings

A randomized experimental study was performed with anesthetized and mechanically ventilated pigs subjected to Escherichia coli endotoxin infusion for five hours. After two hours the animals were treated with the selective endothelin receptor type A antagonist TBC 3711 (2 mg⋅kg⁻¹, n = 8) or served as endotoxin-treated controls (n = 8). Renal artery blood flow, diuresis and creatinine clearance decreased in response to endotoxemia. Perfusion in the cortex, as measured by laser doppler flowmetry, was reduced in both groups, but TBC 3711 attenuated the decrease in the medulla (p = 0.002). Compared to control, TBC 3711 reduced renal oxygen extraction as well as cortical and medullary lactate/pyruvate ratios (p<0.05) measured by microdialysis. Furthermore, TBC 3711 attenuated the decline in renal cortical interstitial glucose levels (p = 0.02) and increased medullary pyruvate levels (p = 0.03). Decreased creatinine clearance and oliguria were present in both groups without any significant difference.

Conclusions

These results suggest that endothelin released during endotoxemia acts via endothelin A receptors to impair renal medullary blood flow causing ischemia. Reduced renal oxygen extraction and cortical levels of lactate by TBC 3711, without effects on cortical blood flow, further suggest additional metabolic effects of endothelin type A receptor activation in this model of endotoxin induced acute kidney injury.     

The incorporation of 35S-heparin into mast cells in the rat and its release into the circulatory bed

It was shown that the intensity of the clearance of 35S-heparin in the blood is extremely high in the first 30 minutes after the introduction of the label. Further on the intensity of the clearance was decreased and by the 60th minute only 11 per cent of the radioactivity was left and by the 240th minute--3.7%. It was established that during intravenous introduction of 35S-heparin the obese cells are capable of accumulating it from the blood flow.     

Clearance and tissue distribution of functionalized polymeric liposomes from the blood stream of rats

Polymeric liposomes containing a synthetic porphinato-iron-imidazole complex (hemoglobin or red blood cell model) were labeled by introducing 1,2-di[1-14C]palmitoyl-sn-glycero-3-phosphocholine into their polymerized bilayers. After intravenous injection into rats, their clearance from a blood stream was measured. The apparent half-life time (50% disappearance time) was about 14 +/- 2 h. Their tissue distribution was determined with time by whole autoradiographic measurement.     

Steady-state clearance rates of warfarin and its enantiomers in therapeutically dosed patients

Previous studies to identify the pharmacokinetics of R- and S-warfarin have not used steady-state area under the curve (AUC) data during therapeutic doses of racemic warfarin. Instead they have used high single doses of either racemic warfarin or a single enantiomer in volunteers or have taken a single blood sample from anticoagulated patients and assumed full compliance and a steady-state status. In this study, a series of steady-state racemic warfarin, R-warfarin, and S-warfarin serum concentrations, during a 24 h dosage interval, was measured in 10 compliant patients (5 females and 5 males) taking racemic warfarin. The anticoagulation status of all 10 patients according to the International Normalised Ratio (INR) was stable. Their mean (SD) age and weight were 67.0 (9.9) yr and 63.9 (15.4) kg. The mean (SD) clearances derived from steady-state AUC values, following therapeutic dosing, for racemic warfarin, R-warfarin, and S-warfarin were 2.40 (0.82), 2.30 (0.65), and 2.80 (1.17) ml/h/kg, respectively. The mean (SD) ratio of S-:R-warfarin clearance was 1.24 (0.40). Comparison of the clearance measured from the AUC, of these patients, to one point determinations assuming steady state for the samples drawn at either 6, 15, or 20 h after dosage (during the dosing interval) showed some statistical differences. Most single point determinations of warfarin clearance assume that a sample 12 h postdose is equivalent to that of the steady-state concentration, but in this study the steady-state concentration of only 6 patients occurred between 6 and 15 h postdose. This could explain why these studies demonstrate differences in the clearance of R- and S-warfarin compared to the values we have derived from steady-state AUC data using patients with proven compliance and therapeutic doses. Chirality 9:13–16, 1997. © 1997 Wiley-Liss, Inc.     

Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.     

Pharmacokinetics of phosphocreatine in the blood serum of man, dogs and rabbits

It is shown that a single intravenous injection of phosphocreatine to man or to experimental animals is followed by its rapid clearance from blood serum. This clearance is biphasic in nature with a half-life of 3-5 min at the first rapid stage and of 20-33 min at the next slower stage. To maintain the constant phosphocreatine concentration in blood, it is necessary to infuse it at a rate comparable to that of clearance. In particular, in blood serum of man, the phosphocreatine concentration can be kept at the level of 0.2 mM if it is injected at a rate of 60 mg/h per kg bw.