Screening method for compounds acting at super low concentrations

At present, there are no simple and reliable methods for selection of compounds with physiological activity in super-low concentrations (SLC). The screening system with high predictability is proposed. At the beginning, experiment is carried out on infusorians--spirostoma. Their motion activity, behavior, living ability and shape are registered at concentrations of substances in a range of 10(-3)-10(-13) mol/l. If the effect is detected on spirostoma, then experiments are performed on laboratory animals or their isolated tissues. Specific deviations on injection substance in SLC are detected, depending on compounds class. Nine representatives of different classes of biologically active compounds are investigated in the work: carbofos, deltametrin, 3-quinuclidinyl benzylate, atropine, phenosan, fenazepam, dopamine receptor antagonist SCH-23390, ciprine, and hexenal. It is accepted that if compound action in SLC is observed on spirostoma, then effects in super-low doses are also detected in a whole animal.     

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.     

Effect of serotonin on the activity of the neurons involved in the realization of the avoidance reflex of the snail

Bath application of 10(-5) mol/l of serotonin (5-HT) elicited a 50% increase of summary EPSPs recorded in command neurones for avoidance behaviour. No significant changes of rest potential and input resistance were seen in these cells. 5-HT evoked an increase of spontaneous level of firing in motoneurones involved in the same reflex, as well as an increase in the number of spikes which paralleled increase of EPSPs to the same stimulus in command neurones. In sensory cells, presynaptic to the command neurones, application of 5-HT evoked a significant increase of excitability and of input resistance. Monosynaptic EPSPs recorded in the command neurones showed a 40% increase after serotonin application. It is concluded that the major locus of plastic changes evoked by 5-HT application in the neuronal chain underlying avoidance reflex is the synaptic contact between sensory and command neurones.     

Modulation of the mitotic activity and population of the mast cells in the oral mucosa by substance P

To assess the effect of substances inducing mast cell degranulation (substance P and granuliberin R) on the mitotic indices of the gingiva stratified epithelium, basal cells from rats were studied in vivo. Seventy Lewis male rats were used in the study. The rats received injections of either 0.1 ml 0.9% NaCl (l0 rats), or substance P (10(-4), l0(-6), 10(-8) g/ml) (30 rats), or granuliberin R (10(-4), l0(-6), 10(-8) g/ml) (30 rats) into their mandibular gingiva in the vicinity of the right mental foramen. The mitotic index of keratinocytes was established after the kolchicine arrest (2 hours prior to material collection i.p. injection). The number of cells in metaphase was counted on 1000 consecutive basal layer cells after hematoxilin and eosin section staining. Mast cells were revealed using pinocyanol erythrosinate according to Bensley. Numerical density and morphometric features were analyzed. Substance P and granuliberin R injected into the gingiva affect the mast cells and the basal cell proliferation of the gingival epithelium. The diminished mitotic activity of basal layer cells was accompanied by degranulation and/or migration of mast cells under the basal membrane of the epithelium. After administration of high doses of granuloliberin R, mast cells were found in the deep connective tissue alligned towards the epithelium. A neuromediator from the trigeminal nerve (substance P) and substances from mast cells actively interfere in the proliferation of oral keratinocytes and the activity of connective tissue cells.     

Upregulation of cyclooxygenase-2 and thromboxane A2 production mediate the action of tumor necrosis factor-alpha in isolated rat myenteric ganglia

Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-alpha (100 ng/ml) hyperpolarized the membrane (from -31.0 +/- 2.7 mV under control conditions to -61.2 +/- 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 +/- 0.7 mV under control conditions to 27.5 +/- 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (10(-6) mol/l), a stable thromboxane A2 agonist. The TNF-alpha action was inhibited by 1-benzylimidazole (2 x 10(-4) mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 10(-4) mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-alpha. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-alpha.     

The effect of phorbol ester in a wide range of concentrations on the lipid structure of microsome membranes of tumor cells in vitro

The action of 12-O-tetradecanoyl-13-acetate (TPA) in vitro in a wide range of concentration from 10(-3) mol/l down to ultra-low doses 10(-23) mol/l and dilution 10(-24) mol/l on the microsome membranes isolated from tumor--Ehrlich ascite carcinoma (EAC) has been studied by ESR-method using two spin probes: 5- and 16-doxyl stearates (5- and 16-DS) localized in the different regions of lipid bilayer. From the ESR spectra obtained it was calculated the following parameters: an order of the long axis 5-DS (S) related to order of the fatty acids chains in the lipid bilayer; two rotation correlation times (Tc1 and Tc2) of 16-DC to estimate a microviscosity value and structural-sensitive ones. It was found the stage changes of all these parameters (increase and decrease) as compared with control level (the membranes untreated by TPA) depending on TPA concentration into the range of 10(-3)-10(-24) mol/l; in particular, the most significant shape changes of structural-sensitive parameters have been observed at TPA doses below 10(-16) mol/l. It is concluded that tumor membranes are very sensitive to TPA action in vitro in a wide range of concentration included ultra-low doses.     

Molecular basis of the biological activity of substances and homeopathic remedies at zero concentrations

While getting ultra los dosages in serial dilutions interactions between a substance and impurities of a solvent occur parallel with diminishing of concentrations. The concentration of impurities in high dilutions prevails the calculated concentration of substrate. Experiments show that different concentrations of compounds of impurities in a solvent correspond to different substances in different dilutions, including < 10(-24) mol/l. While dissolving three components--a solvent, impurities and a substance--participate in two processes: changing of concentration and changing of composition. Changed chemical composition of impurities of a solvent reflects specificity of physicochemical properties of a dissolving substance and is the molecular basis for biological activity of solutions with a concentration lower than 10(-24) mol/l.     

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.     

Both endothelium and afferent nerve endings play a role in acetylcholine-induced renal vasodilation

We investigated the nature and signaling pathways of endothelium- and sensory-nerve ending-derived substances involved in acetylcholine-induced vasodilation in rat isolated perfused kidney. Endothelial denudation by Triton X-100 (0.2%, 0.1 ml) or depletion of afferent nerve endings by capsaicin (10(-6) mol/l) attenuated acetylcholine-induced vasodilation. When these two agents were administered together, the response to acetylcholine was completely inhibited. CGRP1 receptor blocker CGRP 8-37 (10(-7) mol/l) and adenosine A(2) receptor antagonist ZM 241 385 (10(-7) mol/l) inhibited acetylcholine-induced dilation. When indomethacin (10(-5) mol/l), a cyclooxygenase inhibitor, l-NOARG (10(-4) mol/l), a nitric oxide (NO) synthase inhibitor, and potassium chloride (30 mmol/l), to test EDHF response, were perfused simultaneously, the inhibition was greater than that was observed with each agent alone. Guanylate cyclase inhibitor ODQ (10(-5) mol/l) or protein kinase A inhibitor KT 5720 (5x10(-7) mol/l) inhibited acetylcholine-induced dilation. Gap junction uncoupler 18alpha-glycyrrhetinic acid (10(-4) mol/l) caused an uncontrollable increase in basal perfusion pressure making it impossible to test against acetylcholine-induced dilation. Our data suggest that NO, prostanoids, EDHF, and CGRP released from vascular endothelium and afferent nerve endings participate in acetylcholine-induced vasodilation and their signal transduction molecules include protein kinase A and guanylate cyclase.     

The effect of vanadate on the electrogenic Na+/K+ pump, intracellular Na+ concentration and electrophysiological characteristics of mouse skeletal muscle fibre

The present study reports a discrepancy between the effects of vanadate on the membrane Na+-K+-ATPase and the Na+/K+ pump of the skeletal muscle. Vanadate in concentration 4 X 10(-6) mol/l which is necessary to block the enzyme Na+-K+-ATPase activity of membrane fractions failed to inhibit the electrogenic Na+/K+ pump of intact muscle cells. The effect of vanadate on the electrophysiological parameters of the muscle fibre membrane required much higher vanadate levels, but again, Na+/K+ pump was still active. Vanadate in concentrations 4 X 10(-4) and 4 X 10(-5) mol/l depolarized the membrane potential and decreased the membrane resistance [apparently in consequence of enhanced passive membrane permeability for Na+ ions]. Action potentials and the electrical excitability of the muscle fibre membrane were reduced by these vanadate concentrations.     

The effect of the small cardioactive peptide (b) on synaptic transmission efficiency and on the excitability of command neurons in the defensive behavior of the edible snail]

Small cardioactive peptide (b) (SCPb) application in concentration 5 X 10(-8) mol/l into the saline surrounding the snail CNS leads to an increase of amplitude of summate excitatory postsynaptic potential in the command neurones (CN) elicited by the intestinal nerve stimulation. Besides, SCPb causes an increase of excitability of the CN. Described effects can change the threshold of the neuronal net underlying avoidance behaviour. The possibility of integrative effects of peptides in low concentration on the behaviour is discussed.     

Regulation of discrete sub-populations of transmitter-identified neurones after inhibition of electrical activity in cultures of mouse spinal cord

Summary The effects of blockade of electrical activity by tetrodotoxin in cultures of mouse spinal cord and dorsal root ganglion on immunohistochemically-identified neuronal sub-populations have been investigated. Some spinal cord neuronal types, such as those storing methionine-enkephalin, substance P or calcitonin gene-related peptide were almost totally depleted after inhibition of electrical activity for 4 days. By contrast, putative substance P- and calcitonin gene-related peptide-immunoreactive dorsal root ganglion neurones were not significantly affected by such treatment. Several other neuronal types were reduced by about 30–40% after exposure to tetrodotoxin. The decrement in methionine-enkephalin-, substance P- and calcitonin generelated peptide-immunoreactive neurones caused by tetrodotoxin was reversible, and, in the case of methionine-enkephalin, could not be elicited after day 30 in culture. Radioimmunoassay of levels of methionine-enkephalin in cultures confirmed the immunohistochemical data. It is concluded, therefore, that exposure to tetrodotoxin selectively reduces peptide immunoreactivity in specific neuronal sub-populations, but that the selectivity is not based on a single known neuronal characteristic such as transmitter phenotype, or a particular structural protein. The action of tetrodotoxin on those cells most severely attenuated is an alteration in transmitter expression rather than a lethal effect. The diminution with time of the ability of tetrodotoxin to attenuate methionine-enkephalin levels may reflect a reduction in the activity-dependent regulation of peptide expression relative to other competing trophic influences.     

Experimental studies of the liberation of plasminogen activator by DDAVP

Infusion of 0,4 micrograms DDAVP/kg for 30 min increased the plasminogen activator activity in blood of experimental animals (dog, rat, rabbit). The plasminogen activator releasing effect was also demonstrated in isolated perfused vascular preparations (pig ear, rat lung) at low doses (10(-9) - 10(6) mol/l). According to these results DDAVP is believed to attack directly the endothelial cell.     

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).     

L-carnitine as a basis of cholinomimetic substances]

Acetylcarnitine, though having the same configuration as acetylcholine and Acetyl-beta-methylcholine, is devoid of cholinomimetic properties as long as the carboxylic group is free. Contrary findings are explainable by the lack of uniformity of the test substance, caused by substitution of the carboxylic group and intramolecular cleavage of water or acetic acid from carnitine or acetylcarnitine and by admixtures of active substances, and are attributable to the formation of metabolites in vivo. Already the recrystallization of salts of L-acetylcarnitine and L-carnitine in alcohols causes the formation of active carboxylic esters. The latter can be separated and identified by t.l.c. from the starting substances. At the isolated frog heart (Rana esculenta), neither L-carnitine nor L-acetylcarnitine have muscarine-like effects; higher concentrations of them (0.03-0.15 M) exert positively inotropic effects that increase with concentration and are qualitatively and quantitatively equal for L-carnitine and lower O-acyl-L-carnitines. As betaine, L-carnitine affects the heart rate only at 42 +/- 12 mg/ml, crotonic acid betaine at 22 +/- 7 mg/ml, gamma-butyrobetaine at 15 +/- 8 mg/ml. As a result of carboxyl substitution of betaines, the cholinomimetic properties increase to the level of the stimulation system choline/acetylcholine. The LD50 of L-acetylcarnitine for mice injected s.c. with 8.4 (7.3-9.7) mg/g body weight is within the range of LD50 of L-carnitine. Both substances, even when administered in high doses, give no such symptoms as cholinomimetic substances. Carnitine carboxyl ester, acetylcarnitine carboxyl ester, and other carnitine derivatives, on a molar basis, are 2-10(1) to 2-10(3)-fold more toxic than carnitine and acetylcarnitine. The modes of action of carnitines and their metabolites upon the heart rate are discussed.     

Substance P effects on spinal nociceptive neurones.

In decerebrated spinal cats, the effects of iontophoretically applied acetylcholine (ACh) and substance P were examined on the responses of dorsal horn neurones to noxious stimulation and touch of the skin. Both agents, in amounts that did not have a significant direct effect on the neuronal firing rate, prolonged the response of the cells to noxious stimulation but did not alter that to touch stimulation. The peptide and ACh potentiated the late, but not the early, responses of dorsal horn neurones to sural A_δ and C afferent stimulation. Substance P-induced potentiation of the above responses was observed even when the agent did not produce a significant depolarization of nociceptive cells. In greater amounts, the peptide depolarized the neurones, an effect that was not associated with a detectable change in the membrane resistance. These results indicate that substance P facilitates nociceptive pathways by potentiating the subliminal fringe and, in greater amounts, by depolarizing the cells. The failure by the peptide to potentiate touch-induced excitation of the nociceptive neurones appears not to be due to the selectivity of the drug effect but due to the absence of subliminal fringe.     

Effects of psychotropic drugs of different classes injected in super small doses

Effects of benzodiazepine tranquillizers (phenazepam, flunitrazepam), antidepressants (amitriptiline, imipramine) and nootropic piracetam injected in supersmall dozes were studied in outbred albino rats. It was found that in supersmall doses (10(-12)-10(-14) mol/kg) all these substances exert characteristic for each of these classes specific effects revealed by means of adequate pharmacological techniques. Benzodiazepine tranquillizers increased the number of punished water lickings in the conflict situation test. Antidepressants increased the number of wheel rotations in the test of the forced swimming in a tank with freely rotating wheels and enhanced a correlation between the number of wheel turns during the first and the second five minutes of the experiment. Nootrop piracetam increased the rate of acquisition of the active avoidance reflex in a shuttle box. The effects of all investigated drugs injected in supersmall doses were not accompanied by side effects, characteristic for them at administration in usual dosages. The conclusion is made, that the action of the drugs injected in supersmall doses is an universal property of psychotropic drugs. When administered in supersmall doses the pharmacological substances still exert their specific activity, but are devoid of side effects.     

The role of cGMP in extinguishing the reactions of identified neurons in the edible snail to acetylcholine

Possible role of cGMP is studied in control of extinction of snail neurones RPa4, RPa3 and LPa3 reactions to acetylcholine (ACh), applied rhythmically to neurone soma by means of microiontophoresis. It is shown that guanylate cyclase activators which raise the cGMP level in the cell--Na nitroprusside and Na azide (5,10(-4)-10(-3) mol/l)--intensify at extracellular application the extinction of inward transmembrane current and membrane depolarization in response to ACh. Suggestion is made about participation of cGMP-dependent phosphorylation of membrane proteins in control of the development rate, depth and duration of neurone cholinoreceptors short-term plasticity.     

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.