Effect of thyroliberin on contractility and electrical activity of isolated bovine lymphangions

The role of regulatory peptides is the least explored part in the field of humoral regulation of lymphatics. In this paper we continue a systematic investigation of their effects on the lymphatic vessels of various animals. The effect of thyroliberin was studied on bovine mesenteric lymphatics. The isolated lymphatics contractility and smooth muscle cell electrical activity were investigated. Thyroliberin in ultra low concentrations (1 x 10(-13)-1 x 10(-18) M) exerts a considerable stimulating effect. The mechanism of the theroliberin ultra-low concentrations action and a possibility of the medical usage of the obtained results, are discussed.     

On the "receptor" mechanism of the effect of biologically active substances in ultra low concentrations

On the basis of antioxidant (alpha-tocopherol and phenosan potassium salt) and peptide (thyroliberin) effects on the lipid peroxide oxidation (LPO) and lipid structural parameters of the endoplasmic reticulum membranes in wide concentration range (10(-20)-10(-4) mol/l) in vitro the possibility concerning a proposed role of "super-affine" receptors in the mechanism of biologically active substances (BAS) action in ultra low doses (ULD) is discussed. Because these substances modulate investigated processes in the membranes which have not ordinarily receptors the conclusion about availability of non-receptor component in the mechanism of BAS effect in ULD and a low probability of "super-affine" receptor existence has been done.     

The effectiveness of ultralow doses and concentrations of biologically active compounds]

Numerous data of literature are analysed on the biological activity of ultra-low (10(-12)-10(-19) M) concentrations and corresponding doses of same bioregulators. Our own data are presented on the modulation of lymphatic vessel contractility by peptides (thyroliberin, defensin, and tuftsin) in concentrations ranging from 10(-13) to 10(-16) M. Hypothetic mechanisms of these phenomena are discussed.     

Neuropeptide modulation of lymphatic smooth muscle tone in the canine forelimb

Neurokinin A and B are putative inflammatory mediators. We assessed their ability to alter prenodal lymphatic resistance. Intralymphatic neurokinin A (3.0 x 10(-6), 3.0 x 10(-5) and 3.0 x 10(-4) mol l(-1)) significantly constricted lymphatics at the two highest doses. Preliminary experiments suggested that neurokinin B might dilate lymphatics. To test this, lymphatic pressure was increased by norepinephrine (3.1 x 10(-6) mol l(-1)). Neurokinin B (2.7 x 10(-4) mol l(-1)) was then infused intralymphatically during norepinephrine infusion. Norepinephrine increased perfusion pressure from 5.6 +/- 0.6 mmHg to 12.1 +/- 1.4 mmHg. Subsequent infusion of neurokinin B significantly decreased lymphatic perfusion pressure from 11.9 +/- 1.3 mmHg to 9.9 +/- 1.1 mmHg. These data indicate that neurokinin A and B can alter lymphatic resistance and are consistent with the hypothesis that lymph vessel function may be subject to modulation by neurokinins.     

A new mechanism of action of vasopressin on the neuronal membrane]

It is shown in this work that vasopressin at the concentrations of 1 x 10(-16) to 1 x 10(-6) mol/l decreased statistically significant the amplitude of the electrosensitive sodium and calcium ionic currents of the mollusc's Lymnaea stagnalis neuronal membrane. This peptide increased the amplitude of the fast potassium current at the concentration of 1 x 10(-16) and 1 x 10(-15) mol/l. It decreased the fast potassium current and did not change the slow potassium current at the concentrations more than 1 x 10(-9) mol/l.     

Endothelin-mediated constriction of prenodal lymphatic vessels in the canine forelimb

Endothelin is a 21 amino acid peptide which is produced by the vascular endothelium and is believed to be the mediator of endothelium-dependent vasoconstriction. In the current study we assessed the ability of synthetic human endothelin-1 to affect prenodal lymphatic vessel contractility in the canine forelimb. Intralymphatic infusion of endothelin at 1.09 x 10(-9), 1.09 x 10(-8) and 1.09 x 10(-7) M significantly constricted lymphatic vessels as evidenced by dose-dependent increases in lymphatic perfusion pressure. The increase in lymphatic perfusion pressure seen during intralymphatic infusion of endothelin at 1.09 x 10(-8) M during the intra-arterial infusion of phentolamine was not significantly different from that seen prior to phentolamine, indicating that endothelin-mediated lymphatic constriction is not alpha-receptor mediated. Intra-arterial infusion of endothelin at three infusion rates significantly increased forelimb arterial, systemic and lymphatic perfusion pressures. The constriction seen when endothelin (1.09 x 10(-8) M) was infused intralymphatically in the intact lymphatic system was not significantly different from that observed when only the prenodal lymph vessel was perfused. This indicated that the lymph nodes and efferent lymph vessels do not contribute significantly to the lymphatic constriction produced by endothelin. These data are consistent with the hypothesis that endothelin may modulate lymphatic function under either normal or pathophysiological conditions.     

Involvement of nitric oxide in endothelium-dependent arterial relaxation by leptin

Leptin is a polypeptide, mainly produced in white adipose tissue, and increases sympathetic nerve activity. A few studies investigated leptin's effect on peripheral vessels. We examined the vasorelaxant effects of human leptin on rat arteries. Arterial rings were precontracted with 1 x 10(-6) mol/l of phenylephrine, and leptin was superfused. Leptin relaxed phenylephrine-precontracted arterial rings in a dose-dependent manner. ED50 was calculated to 8.4 microg/ml. Removal of endothelium abolished the effects of leptin. Indomethacin (1 x 10(-5) mol/l) did not affect the vasorelaxation by leptin, whereas 1 x 10(-4) mol/l of N(omega)-nitro-L-arginine methyl ester (L-NAME) completely suppressed it. The inhibition was antagonized by 1 x 10(-4) mol/l of L-arginine. Leptin normally relaxed arterial rings during superfusion of K channel blockers, including 3 x 10(-5) mol/l of glibenclamide, 1 x 10(-6) of mol/l apamin, and 5 x 10(-7) mol/l of charybdotoxin. Low Cl(-) solution (8. 3 mmol/l) inhibited leptin-induced relaxation, but endothelium-independent vasodilatation by nitroprusside was not impaired at low Cl(-) solution. These results suggest that arterial relaxation by leptin is mediated by nitric oxide released from endothelium, and Cl(-) plays an important role in leptin-induced nitric oxide release.     

Comparison of cerebrospinal fluid transport in fetal and adult sheep

We quantified cerebrospinal fluid (CSF) transport (conductance) and CSF outflow resistance in late-gestation fetal and adult sheep using two methods, a constant pressure infusion method and a bolus injection technique into the lateral ventricles. No significant differences in CSF conductance (fetus 0.013 +/- 0.002, adult 0.014 +/- 0.003 ml x min(-1) x cm H(2)O(-1)) or CSF outflow resistance (fetus 83.7 +/- 9.8, adult 84.7 +/- 19.7 cm H(2)O x ml(-1) x min) were observed. To confirm CSF transport to plasma in fetal animals, (125)I- or (131)I-labeled human serum albumin (HSA) was injected into the lateral ventricles. The tracer entered fetal plasma with an average mass transport rate of 1.91 +/- 0.47% injected/h (n = 9). In two fetuses, we monitored the tracer appearance in plasma and cervical and thoracic duct lymph after injection of radioactive HSA into the ventricular CSF. As was the case in adult animals, fetal tracer concentrations increased in all three compartments over time, with the highest concentrations measured in lymph collected from the cervical lymphatics. These results 1) indicate that global CSF transport parameters in the late-gestation fetus and adult sheep are similar and 2) suggest an important role for extracranial lymphatic vessels in CSF transport before birth.     

The effect of ultra small doses of thyroliberin on the structural changes in endoplasmic reticulum membranes in vitro

Peptides are known to have the ability of modulating the activity of important regulatory cellular systems. One of them--thyroliberin, i.e. thyreotropin-releasing hormone (TRH), causes changes in the membrane structure and morphology of rat erythrocytes, as well as activates retractive activity of lymphatic vessels in ultra low concentrations (10(-10) to 10(-16) mol/l). In this study we used an electron spin resonance (ESR) method to explore the effect of TRH in a wide range of concentrations (10(-4) to 10(-18) mol/l) on thermo-induced structural transitions and microviscosity of lipid bilayer of the endoplasmic reticulum membrane of mice (C57 bI) liver cells. Two stable free radicals were used as paramagnetic probes: 2,2,6,6-tetramethil-4-capryolyl-1-oxyl and 16-doxyl-stearic acid, that are localized in superficial and deep layers of the membrane respectively. TRH caused a statistically significant change (p < 0.001) in microviscosity of the membrane surface layer. The largest effect (up to 30% decrease) was observed at TRH concentrations of 10(-10) and 10(-16) mol/l. It was also demonstrated that an addition of 10(-4), 10(-10) and 10(-16) mol/l of TRH decreases effective activation energy and temperature (by several degrees) of the thermo-induced structural transitions. The observed changes in the parameters of the membrane surface layer induced by TRH may be essential for its physiological activity, because of the obtained negative correlation (r = 0.99; p < 0.001) between the membrane microviscosity and frequency of lymphatic vessels' contraction. Complex changes in the structure of deep hydrophobic layer of the membrane caused by TRH were observed in this study as well. Higher concentrations of TRH (10(-4) and 10(-10) mol/l) produced results that were similar to the effect of TRH on the superficial lipid layer of the membrane, whereas the effect of ultra low TRH concentration (10(-16) mol/l) was reversed for microviscosity, number and activation energy of structural transitions in contrast with the case of surface layer. The results of this study suggest presence of a nonspecific factor in the effect of TRH on structural characteristics of the lipid component of biological membranes. It is possible, that the change of structural properties of biological membranes may be a part of the mechanism of TRH action at ultra low concentrations.     

Determination of ascorbic acid in individual rat hepatocyte by capillary electrophoresis with electrochemical detection

A method for the direct determination of ascorbic acid (AA) in individual rat hepatocyte based on capillary electrophoresis (CE) coupled with electrochemical detection (ECD) using a new kind of homemade carbon fiber micro-disk bundle electrode has been described. Individual rat hepatocytes were injected into a fused-silica capillary with an inner diameter of 25 microm, and lysed by 0.1% sodium dodecylsulfate (SDS) as cell lysis solution. The following conditions were suitable for the determination of AA: running buffer, 1.83 x 10(-2) mol/l Na2HPO4-1.70 x 10(-3) mol/l NaH2PO4 (pH 7.8); separation voltage, 20.0 kV; detection potential, 0.80 V (vs. saturated calomel electrode (SCE)). The concentration limit of detection (LOD) of the method was 1.7 x 10(-6) mol/l at a signal-to-noise (S/N) ratio of 3, and the mass LOD was 3.0 fmol. The linear dynamic range was from 5.0 x 10(-6) to 5.0 x 10(-4) mol/l with a correlation coefficient of 0.9962 for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the peak current. This method was successfully applied to AA determination in rat hepatocyte. The recovery was between 91% and 97%, and the amount of AA in single rat hepatocyte ranged from 28 to 63 fmol.     

Determination of midecamycin by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of midecamycin using an end-column amperometric detection with a carbon fiber micro-disk bundle electrode at a constant potential of +1.15 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 1.00x10(-3) mol l(-1) Na(2)HPO(4)-3.49x10(-4) mol l(-1) NaOH (pH 11.4) for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The limit of detection is 5.0x10(-7) mol l(-1) or 0.41 fmol (S/N=3). The linear range of the calibration curve is 1.00x10(-6)-1.00x10(-3) mol l(-1). The relative standard deviation is 1.4% for the migration time and 4.9% for the electrophoretic peak current. The method could be applied to the determination of midecamycin in human urine. In this case, a separation voltage of 14 kV was used.     

Rapid,fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples

Capillary zone electrophoresis was employed for the determination of lactate using end-column amperometric detection at a carbon fiber bundle microdisk electrode. The optimum conditions of separation and detection are 3.6 x 10(-3) mol/l Na(2)HPO(4)-1.4 x 10(-3) mol/l NaH(2)PO (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V versus the saturated calomel electrode for the detection potential. The limit of detection is 7.6 x 10(-7) mol/l or 1.7 fmol (S/N=3) and the linear range is 1.7 x 10(-6)-8.2 x 10(-4) mol/l for the injection voltage of 6 kV and injection time of 5 s. The RSD is 1.8% for the migration time and 3.3% for the electrophoretic peak current. The method was applied to the determination of lactate in human saliva. The recovery of the method is between 95 and 109%.     

Neurokinin B causes concentration-dependent relaxation of isolated human placental resistance vessels

Placental neurokinin B (NKB) was recently identified as the causative agent in preeclampsia, a condition characterized by increased maternal and feto-placental vascular resistance. We hypothesized that NKB should constrict placental resistance vessels. Placentas were obtained from normotensive pregnancies. Immediately after delivery, stem villous arteries (300 microm diameter, 1.2 mm long) were dissected from macroscopically normal tissue in cold HEPES-physiological salt solution (PSS), mounted on a wire myograph system, and bathed in HEPES-PSS at 37 degrees C. After determination of the passive-tension internal circumference characteristics, the arteries were set to 90% of the internal circumference they would have under a normal physiological transmural pressure. Cumulative concentration-response curves were constructed for NKB (1 x 10(-12) to 1 x 10(-5) mol/l). Since there was no constrictive response to NKB, cumulative constrictive concentration-response curves were constructed to the thromboxane A(2) mimetic U46619 (1 x 10(-9) to 1 x 10(-5) mol/l). The vessels were then pre-constricted to 80% of maximal response and exposed to cumulative concentrations of NKB (1 x 10(-12) to 1 x 10(-6) mol/l). NKB caused a concentration-dependent relaxation (Maximal response NKB, 51+/-5%, n=5; time control, 12+/-6%, n=4; P<0.05). Removal of the endothelium did not alter the vasodilatory response to NKB. We conclude that, contrary to our hypothesis, NKB causes an endothelium-independent relaxation of the placental resistance vessels. We propose that NKB plays a role in the maintenance of high placental blood flow in normal pregnancy.     

Amperometric detection of perphenazine at a carbon fiber micro-disk bundle electrode by capillary zone electrophoresis

A simple method for determination of perphenazine by capillary zone electrophoresis with amperometric detection is described. The optimum conditions of separation and detection are 1.50 x 10(-3) mol/l Na(2)B(4)O(7)-1.0 x 10(-3) mol/l NaOH (pH 9.9) for the buffer solution, 18 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, and 0.80 V versus saturated calomel electrode for the detection potential, respectively. The limit of detection is 5.0 x 10(-8) mol/l or 44 amol (S/N=3). The linear range of the calibration curve is 1.00 x 10(-7) to 1.00 x 10(-4) mol/l. The relative standard deviation is 1.5% for the migration time and 2.9% for the electrophoretic current at peak maximum. The method is applied to the determination of perphenazine in human urine.     

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.     

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Thyroliberin and its analog lacking hormonal properties stimulate respiratory center activity in the frog Rana temporaria

It has been shown that thyroliberin and its synthetic analogue PR-546 injected into the lymphatic sacs (4.10(-7)-4.10(-9) g/kg) or applied to the medulla oblongata in the bulbar frogs (4.10(-10)-4.10(-12) g/kg) significantly increase the rate of motor respiratory volleys in the hypoglossal nerve. In preparations with the irregular level of the activity of the respiratory center, these drugs enhanced stabilization of the initial level of the rhythm of motor respiratory discharges.     

The sensitivity of experimental tumor systems to ultra low doses of doxorubicin

Biological effect of doxorubycin in standard (10(-3) mol/l) and ultra low doses (10(-5)-10(-20) mol/l) against some "signal" animal tumor systems--Lewis lung carcinoma, 755 adenocarcinoma, B-16 melanoma, Ehrlich carcinoma and L1210 leukemia was studied. The all models were very sensitive to the action of the drug in standard dose. Solid tumors' growth inhibition by 80-95% as well as increasing in life span of mice with L1210 leukemia by 86% in comparison with control and surviving of animals with Ehrlich carcinoma had been revealed. It had been shown that the drug in the area of ultra low doses occurred the following effects: inhibition of Lewis lung carcinoma growth by 80-95% compared to control after administration of the all tested ultra low doses; increasing of the life span of the animals with Ehrlich carcinoma and L1210 leukemia by 86-123% and 6-23%, correspondingly, upon the action of all tested ultra low doses; inhibition of B-16 melanoma growth by 50 and 70% after administration of the drug in doses 10(-20) mol/l and 10(-5) mol/l, correspondingly as well as deceleration of 755 carcinoma growth by 40% compared to control after action of the drug in the dose 10(-20) mol/l; stimulation of the B-16 melanoma growth by 20% relative to control after 10(-10) mol/l dose injection and enhancement of tumors sizes by 20-60% above control levels as a result of treatment of mice with 755 carcinoma by the drug in such ultra low doses as 10(-5) and 10(-15) mol/l. So, it was found that all tested tumor systems revealed certain sensitivity to the some ultra low doses of the drug. At the same time it was shown that doxorubycin in ultra low doses displayed alternative character of its biological effect, directivity of which varied according with the dose level and tumor strain.     

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.     

The immune response to GHRH, relationship to conformation

Antibodies to GHRH1-44, GHRH1-29, and proinsulin were induced in guinea pigs. GHRH1-44 forms 7 S and 10 S complexes with antibodies. It is a divalent antigen. The sequence 30-44 bound 85% of the antibodies to GHRH1-44 with high affinity (3.8 +/- 0.9 x 10(9) l/mol). The fragment 1-29 bound with low affinity (0.6 +/- 0.3 x 10(9) l/mol) 15% of the antibodies (2p less than 0.001). Antibodies to GHRH1-29 had low affinity towards the native hormone (0.4 +/- 0.2 x 10(9) l/mol) and the region 1-29 (0.3 +/- 0.2 x 10(9) l/mol). Antibodies to proinsulin bound linear C-peptide with lower affinity (0.3 +/- 0.2 x 10(9) l/mol) than the C-peptide loop in proinsulin (3.4 +/- 0.9 x 10(9) l/mol). It is concluded that the conformation of the epitopes on the sequence 1-29, recognized during the immune response, i.e. on the cell membrane, is different from the conformation of GHRH1-29 or GHRH1-44 in aqueous solution.