Activating effect of substance P on nerve tissue in culture

The effect of substance P on explant development was investigated in organotypic cultures of rat sympathetic ganglia and spinal cord. The pattern of evolution, cellular composition, and dimensions of the growth zone were evaluated on the basis ofin vivo observations. It was found that this peptide exercises a significant growth-promoting effect at a concentrations of 10^–5–10^–12 M for sympathetic ganglia and 10^–5–10^–14 M for spinal cord culture. The growth zone of sympathetic ganglia measured 1.3–1.6 times the control level by the 14th day of culture at all effective concentrations. The area of outgrowth of spinal cord explants increased 2.0–5.2 fold by the sixth day of culture and peak response was recorded at concentrations of 10^–5 and 10^–12 M. This effect resembled response to opioid peptides [1, 3]. The likely physiological significance of regulatory peptides for the processes of nerve tissue development and regeneration is discussed in the light of these findings, together with the part played by the nociceptive/antinociceptive system in processes of histogenesis and repair.Institute of Experimental Cardiology, All-Union Cardiologic Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 610–615, September–October, 1986.     

Effects of opioid peptides and naloxone on tissue from the central and peripheral nervous system in culture

A study was made of the effects of opioid peptides (leu-enkephalin and dalargin AE-1, its synthetic analog) and of naloxone, an opiate receptor blocker, on organotypic cultures of spinal cord and spinal ganglia cells. The cellular composition and size of explant outgrowth was estimated according to in vitam morphological observations. It was found that all the opioid peptides tested at concentrations of 10^–9-10^–10M exercise a clear-cut growth-promoting effect on cultures from the spinal cord as well as those from the peripheral nervous system [4, 5]. Naloxone at a concentration of 10^–5-10^–6 M does not block peptide action, but itself stimulates growth. It was also proved that opioid peptides act as trophic factors for spinal ganglia nerve cells, increasing their survival in culture. Endorphins can thus serve as growth factors for tissues of the peripheral as well as the central nervous system. The likely processes lying at the root of the growth-promoting and trophic effects of endorphins on nerve tissue are discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 227–233, March–April, 1986.     

Electrical activity of the sympathetic ganglia during the depressor reflex

Tonic activity of neurons of the superior cervical sympathetic ganglion was recorded by the "sucrose gap" method and in the 4th and 5th lumbar sympathetic ganglia with the aid of focal nonpolarizing electrodes in acute experiments on anesthetized cats and rabbits. The preganglionic fibers of the ganglia were left intact. Stimulation of the depressor nerve not only sharply inhibited the tonic activity of the ganglia but also led to the appearance of electropositive potentials of 0.7 ± 0.2 mV in the superior cervical ganglion and 20–250 µV in the lumbar ganglia. The amplitude of this potential was unchanged by atropine (1 · 10^–6M). A similar effect occured without stimulation of the depressor nerve, after division of the preganglionic fibers or blocking of their conduction; it is attributed to the cessation of preganglionic tonic impulses which induce not only spikes, but also many EPSPs in neurons of the ganglion. Their frequency in the lumbar ganglia was 4/sec. Summation of these EPSPs leads to constant electronegativity of the ganglion surface relative to the postganglionic fibers, and its disappearance is recorded as a positive potential. Stimulation of the depressor nerve thus does not induce IPSPs in the ganglion; consequently, the inhibition of synaptic activity observed under these circumstances is located in the CNS and not in the ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 6, No. 5, pp. 519–524, September–October, 1974.     

Modulation of excitation transfer in the intracardiac ganglionic system by the opioid peptide dermorphin

The aim of the study was to determine whether opioid peptides could modulate the intracardiac ganglionic excitation. It has been shown that postganglionic impulse activity recorded in the frog intracardiac nerve was inhibited by endogenous amphibian opioid peptide-dermorphin (10(-12)-10(-6) M). The inhibition was blocked by naloxone (10(-5) M). Dermorphin (10(-6) M) had no influence on transitory impulse activity in the intracardiac postganglionic sympathetic fibers. It is suggested that opioid peptides take part in the mediatory process in the intracardiac ganglia.     

Action of opioid peptides on nerve tissue growth and regeneration processes in the rat

Using organotypic cultures of the sympathetic ganglia and spinal cord from rats, studies have been made of the effect of opioid peptides on the development of the nervous tissue. It was found that endogenous opioid peptides (leu- and met-enkephalins, beta-endorphin) within the concentrations investigated (10(-9)-10(-10) M), stimulate the growth of neurites, affect the rate of migration and proliferation of the glial and fibroblast-like cells. The effect was observed at the 2nd--5th days of cultivation, depending on the object investigated. Naloxone, a blockator of the opiate receptors, does not abolish the stimulating effect of the opioid peptides. Using clonal line of fibroblast-like cells L6, it was shown that leu-enkephalin decreases the sensitivity to contact inhibition of growth. On the basis of the data obtained, it is suggested that endogenous opioid peptides act as non-specific factors of growth regulation in the development and regeneration of the nervous tissue. Taking into account the role of endorphins in the activity of noci-antinociceptive system possible significance of these compounds in post-injury reparation is discussed.     

Stimulating action of endorphins on the development of sympathetic ganglia in culture

The effect of gamma-and beta-endorphins, leu- and met-enkephalins on the growth and differentiation of nervous tissue was studied in organotypic cultures of the sympathetic ganglia of newborn rats. The growth of explants in living cultures and preparations stained by the Holmes method was analyzed. It was established that endorphins are capable of stimulating the growth of neurites from the explant, of increasing the number of glial and fibroblastoid cells in the zone of the growth. The mean value of the maximal magnitude of the zone of the growth in normalcy was 464 +/- 136 micron, that on addition of leu- and met-enkephalins, gamma- and beta-endorphins 879 +/- 161, 900 +/- 160, 959 +/- 170, and 1056 +/- 137 microns. The growth effect induced by endorphins was demonstrated within a wide dose range--from 10(-7) to 10(-14) M. Naloxone did not inhibit the stimulant action of the peptides. It is suggested that opioid neuropeptides can be used as a source of nonspecific growth factors for nervous tissue.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Effects of the drug balis-2 on the growth of sympathetic ganglia from different strains of rat in culture

The growth-promoting properties of balis-2, produced by microbiological synthesis, were investigated in organotypic cultures of upper cervical ganglia from newborn Wistar, Wag, and August rats. Maximum size of the growth zone was calculated, as well as the density of neurite-glial bundles and numbers of catecholamine-containing fibers within this zone. It was found that ganglia cultures from Wistar and Wag strains grow at 2.0–2.1 times the rate of those from the August strain. Balis-2 exerts an activating action at concentrations of 0.001 and 0.0001%, mainly affecting neurite growth. Level of response was found to vary from one strain to the next: peak response, at 2.3–2.6 times control level was recorded in rats of the August strain, while growth in ganglia culture increased 1.8–2.0-fold against the controls in the remaining two strains under the effects of this substance. In this way it was first shown that balis-2 has neurotrophic properties when used on sympathetic ganglia in culture. In view of the fact that rats belonging to the August strain have a high level of plasma catecholamines and are classified as an emotional species according to their reaction to stress, the subject of how processes of nerve tissue regeneration connect up with the state of the sympathoadrenal system is discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Institute of Physical and Organic Chemistry, Rostov University, Krasnodar. Translated from Neirofiziologiya, Vol. 20, No. 4, July–August, 1988, pp. 539–546.     

Interactions between prostaglandin E2 andd-ala2-met-enkephalinamide on adenylate cyclase activity in the guinea-pig superior cervical ganglion

Crude membrane fractions, obtained from superior cervical ganglia of normal and sympathectomized guinea-pigs, have been used to investigate the role of prostaglandin E₂ andd-ala²-met-enkephalinamide in the modulation of ganglionic adenylate cyclase as well as their functional interrelationship. In ganglia from normal animals the enzyme activity was stimulated and inhibited, respectively, by the prostaglandin (10^–4M) and by the opiate pentapeptide (10^–4M), while little or no effects were observed in denervated preparations. When the two substances were tested in combination, a supra-additive stimulation of adenylate cyclase activity was obtained both in normal and denervated ganglia. In the latter preparation the opiate increased prostaglandin E₂ specific binding, suggesting that the mechanism of supra-additivity could involve interactions at receptors level. Furthermore, the supra-additive stimulation of adenylate cyclase activity by the combination of the two drugs was obtained in a narrow range of concentrations since at low prostaglandin E₂ doses (10^–7–10^–6M) or at very high doses of the opiate (10^–3M), only the inhibitory effect ofd-ala²-met-enkephalinamide was evidenced.     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

TNRNFLRFamide and SDRNFLRFamide modulate muscles of the stomatogastric system of the crab Cancer borealis

The effects of the extended FLRFamide-like peptides, TNRNFLRFamide and SDRNFLRFamide, were studied on the stomach musculature of the crab Cancer borealis. Peptide-induced modulation of nerve-evoked contractions was used to screen muscles. All but 2 of the 17 muscles tested were modulated by the peptides. In several muscles of the pyloric region, peptides induced long-lasting myogenic activity. In other muscles, the peptides increased the amplitude of nerve-evoked contractions, excitatory junctional potentials, and excitatory junctional currents, but produced no apparent change in the input resistance of the muscle fibers. The threshold concentration was 10^–10 M for TNRNFLRFamide and between 10^–9 M to 10^–8 M for SDRNFLRFamide. The absence of direct peptidecontaining innervation to these muscles and the wide-spread sensitivity of these muscles to the peptides suggest that TNRNFLRFamide and SDRNFLRFamide may be released from neurosecretory structures to modulate stomatogastric musculature hormonally. We speculate that hormonally released peptide will be crucial for maintaining appreciable muscle contraction in response to low-frequency and low-intensity motor discharge.Abbreviations cpv muscles cardiopyloric valve muscles - CG commissural ganglion - DG neuron dorsal gastric neuron - dgn dorsal gastric nerve - dvn dorsal ventricular nerve - EJC excitatory junctional current - EJP excitatory junctional potential - FaRPs FMRF-amide related peptides - gm muscles gastric mill muscles - lvn lateral ventricular nerve - mvn medial ventricular nerve - p muscles pyloric muscles - STG stomatogastric ganglion     

Synaptic effects of nicotinic and muscarinic agonists in sympathetic ganglia of the frog

Superfusion of the isolated sympathetic ganglia of the frog with nicotinic agonists (suberyldicholine, tetramethylamonium, and dimethylphenylpiperazinium), as well as acetylcholine in the presence of atropine led to a brief depolarization of the neurons and blockade of synaptic transmission. The muscarinic agonists methylfurmethide (MFM) and methyldilvasen, cis^–, L(+), as well as acetylcholine elicited a stable depolarization which is not accompanied by disturbance in transmission. Oxotremorine at a concentration of 1·10^–5 M did not lead to the depolarization of the post-synaptic membrane, but at a concentration of 1·10^–6 M decreased the quantal EPSP content twofold, which indicates that the presynaptic receptors belong to the M₂ subtype. Inhibition of acetylcholinesterase significantly intensified the postsynaptic effect of MFM: a shift of the concentration-effect curve took place toward the side of lower MFM concentrations. It was shown that the post-synaptic muscarinic receptors of the ganglionic neurons possess varied sensitivity to the enantiomers of methyldilvasen and, consequently, are stereospecific. The identified functional properties of the cholinoreceptors of the ganglionic neurons explain the set of changes in synaptic transmission under conditions of the prolonged presence of a mediator in the synaptic cleft.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 227–234, March–April, 1988.     

Prostacyclin as neuromodulator in the sympathetically stimulated rabbit heart

Prostaglandin E₂ (PGE₂) has previously been shown to inhibit sympathetic neurotransmission in different organs and species. Based on this inhibitory effect and on its reversal by cyclo-oxygenase inhibitors, PGE₂ has been claimed to be a physiological modulator of in vivo release of norepinephrine (NE) from sympathetic nerves. It is now recognized that prostacyclin (PGI₂) is the main cyclo-oxygenase product in the heart. We therefore addressed the question whether PGI₂, within the same preparation, is formed in increased amounts during sympathetic nerve stimulation and has neuromodulatory activity.The effluent from isolated rabbit hearts subjected to sympathetic nerve stimulation or to infusion of NE or adenosine (ADO) was collected, and its content of PGE₂ and 6-keto-PGF_1α (dehydration product of PGI₂) was analyzed using gas chromatography/mass spectrometry, operated in the negative ion/chemical ionization mode. Other hearts were infused with PGI₂ and nerve stimulation induced outflow of endogenous NE into the effluent was analyzed using HPLC with electrochemical detection. Nerve stimulation at 5 or 10 Hz (before but not after adrenergic receptor blockade), as well as infusion of NE (10⁻⁶–10⁻⁵M) or ADO (10⁻⁴M) increased the cardiac outflow of 6-keto-PGF_1α. Basal and nerve stimulation induced efflux of 6-keto-PGF_1α was approximately 5 times higher than the corresponding efflux of PGE₂. PGI₂ dose-dependently inhibited the outflow of NE from sympathetically stimulated hearts, the inhibition at 10⁻⁶M being approximately 40%.On the basis of these observations we propose that PGI₂ is a more likely candidate than PGE₂ as a potential modulator of neurotransmission in cardiac tissue in vivo.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

Sympathetic nervous control of the vasculature in the tail of the Atlantic cod,Gadus morhua

Summary The effects of some drugs and electrical stimulation of the sympathetic chain on the vasculature of the isolated perfused tail of the cod has been studied. Adrenaline, phenylephrine and isoprenaline all constricted the tail vasculature, isoprenaline also producing dilation in some preparations at lower concentrations (10^–9–10^–6M). Electrical stimulation of the sympathetic chain produces vasoconstriction, which can be abolished by phentolamine in concentrations expected to be selective for -adrenoceptor blockade in the cod. A small dilatory response to nerve stimulation was seen in some preparations after phentolamine. Carbachol produced weak and irregular effects, and a contribution by cholinergic fibres therefore seems unlikely. It is concluded that the excitatory nervous control of the cod tail vasculature is solely by adrenergic sympathetic fibres, which act via an -adrenoceptor mechanism.     

Location of efferent sympathetic neurons and afferent neurons in spinal ganglia innervating the heart

Location and numbers of neurons associated with sympathetic innervation of the heart within the right stellate and accessory cervical ganglia, the spinal cord, and spinal ganglia were investigated using horseradish peroxidase retrograde axonal transport techniques in cats. The enzyme was applied to central sections of the anastomosis of the stellate ganglion with the vagus nerve, the inferior cardiac nerve, and the vagosympathetic trunk caudal to the anastomosis. Labeled neurons within the stellate ganglion were located close to the point of departure of the nerves and more thinly distributed in the accessory cervical ganglion. A group of labeled cells was found in the anastomosis itself. Preganglionic neurons associated with sympathetic innervation of the heat were detected at segmental levels T₁–T₅ in the spinal cord. Labeled neurons were diffusely located in the spinal ganglia, concentrated mainly at levels T₂–T₄.Medical Institute, Ministry of Public Health of the RSFSR, Yaroslavl'. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 106–111, January–February, 1989.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.