[Allosteric effect of serotonin and mianserin on the kinetics of specific [3H]-ligand binding to adrenergic and muscarinic receptors in the rat cerebral cortex membranes].

The effects of serotonin receptor activation (by serotonin) and inhibition (by mianserin) on the properties of the α1-, α2-adrenoreceptors, and muscarinic cholinergic receptors in subcellular membrane fractions from the rat cerebral cortex were studied. Experimental data on the kinetics of specific antagonists binding to adrenergic and muscarinic receptors were analyzed by graphical and mathematical methods. The results suggest the presence of allosteric (cross-talk) interaction. In the control, α1- and α2-adrenoreceptors were represented by a single pool, and muscarinic receptors, by two pools. Two pools of adrenoreceptors with different affinity were detected against the background of serotonin. It was found that mianserin induces the formation of two pools of only (α2-receptors and muscarinic receptors are represented by two pools differing in the main parameters, such as dissociation constants and adrenoreceptor concentrations, in the control and experimental groups. It was shown that the allosteric effect of serotonin and mianserin is manifested in the inhibition of muscarinic receptors. It was assumed that the adrenergic and cholinergic receptors exist as dimers. The interaction between the adrenergic, cholinergic, and serotonergic systems is likely to be implemented at the cell membrane level.

[Intracellular transmission of the cholinergic signal in the chick amnion].

The role of the system of deposited calcium in the mediation of contractile reactions to carbachol in an isolated amnion of 11-13 day old chicken embryo was studied. It was found that thapsigargin (2 microM, 20 min), an inhibitor of the endoplasmic reticulum Ca2+ -ATPases, decreases the tonic reaction to carbachol by 40 +/- 2%. In the presence of U73122 (5-10 microM, 10 min), a phosphoinositide-specific phospholipase C inhibitor, the rhythmic contractile reaction of the amnion to carbachol is blocked, whereas the tonic reactiondecreases to 47 +/- 9% of the initial one. Ryanodine (10 rM, 5 min) inhibits the spontaneous contractile activity of the amnion and decreases the tonic reaction to carbachol to 36 +/- 3% relative to control. In the presense of ryano- dine, nifedipine (0.05 microM) completely blocks the tonic reaction to carbachol. Thus, calcium mobilized from intracellular stores via inositol trisphosphate and ryanodine receptors is involved in realization of contractile reactions, mediated by M3 receptors, in the chick amnion.

[Similarities and differences in the effect of cocaine on alpha-adrenergic and muscarinic response].

Similarities and differences in the effect of cocaine on alpha-adrenergic and muscarinic receptors were shown in three experimental models. The postsynaptic stimulating effect of cocaine through activation of alpha-adrenergic receptors was found in a chicken embryo without innervation and in an innervated rat vas deferens. Cocaine caused an increase of the amount of activated alpha-adrenergic receptors and the appearance of an additional receptor pool and changed their dimerization level. Cocaine acts as an antagonist on muscarinic receptors of the chicken embryo. The inhibition of muscarinic receptors in the rat brain by cocaine leads to a decrease in the number of receptors and their partial monomerization. Thus, cocaine influences both the alpha-adrenergic response and the muscarinic response by its influence on receptors. Experiments on various objects have shown that cocaine activates the alpha-adrenergic response and inhibits the muscarinic response.

[Effect of rabbit adaptation to cold on the depressor muscarinic cholinergic reaction of the arterial pressure of the hind limb vessels and the small intestine in situ and the systemic arterial pressure].

Modification of the main parameters of cholinergic reactions in rabbits, i.e., of the specific sensitivity to agonists (EC50), maximal reaction values (Pm) of arterial pressure in the hind limb vessels and small intestine in situ, and systemic arterial pressure after adaptation to cold for 1, 5, 10, and 30 days has been investigated. The depressor reaction to acetylcholine (muscarinic cholinergic receptors agonist) was established to correspond to the model p = (PmAn)/(EC(50)(n) + An) with n=1. In the control EC50 was equal to 0.85, 1.01, and 1.21 nmol/kg, while Pm equaled 100, 32.6, and 61.2 mm Hg for the hind limb vessels, small intestine in situ, and systemic arterial pressure, respectively. As a result of rabbits adaptation to cold in situ, qualitatively similar modifications in the EC50 and Pm value in the hind limb vessels and in the small intestine occurred with correlation coefficient r = 0.83 for the EC50 parameters and r = 0.74 for the Pm parameters. No correlations between EC50 and Pm in the systemic arterial pressure were observed.

[The significance of extracellular Ca2+ in contractile responses in chick amnion].

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M3 cholinoceptors in an amniotic membrane strip isolated from 11-14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K+, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca2+ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca(2+)-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.

[The effect of cocaine on kinetics of alpha1-adrenergic contractile response in different portions of the rat vas deferens].

The effect of cocaine (10 microM) on the kinetics of contractile response to noradrenaline (NA) was studied in the rat epididymal and prostatic vas deferens. Cocaine caused an acute increase in vas deferens adrenergic sensitivity primarily due to blockage of NA neuronal capture. The presynaptic action prevailed in the epididymal half: the EC50 value decreased 32-fold with a slight increase of the maximum adrenergic response more evident in the prostatic half. In the presence of cocaine, the prostatic contraction to NA was mediated not by a single pool of alpha1-adrenoceptors like in epididymal vas deferens but by two. Its high affinity pool had the same affinity as alpha1-adrenoceptors of the epididymal half, the affinity value of the low one was 36-fold less, and the total maximal response of both pools increased 4.5-fold. The differences in cocaine effect on the rat epididymal and prostatic vas deferens contractions to NA appear to be caused by the different sources of Ca2+ involved in these responses.

[Muscarinic acetylcholine receptors of the chick amnion].

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M1, methoctramine for M2, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M3, and tropicamide for M4 receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC50), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 microM), activator of adenylate cyclase (AC), was unidirectional with beta-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC- or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M3 acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M3 acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M3-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.

[Effect of elevated extracellular K+ on the kinetics of alpha 1-adrenergic contractile response in the rat vas deferens].

Increase in KCl concentration from 5.5 to 15.5 mM in physiological solution produces potentiation of contractions in response to phenylephrine-mediated by alpha 1-adrenoceptors in the rat vas deferens. The effect of partial depolarisation is dosedependent and reversible. Analysis of the kinetics of adrenergic contractile response to phenylephrine in presence of 15.5 mM KCl shows a decrease of EC50 from 6.63 +/- 0.97 to 3.76 +/- 0.47 microM, i. e. the increase in sensitivity of the organ to agonist. Graphic and mathematic analysis also showed that contractile response to phenylephrine as in control was mediated by a single pool of alpha 1-adrenoceptors without changing the maximum response (Pm) and the Hill coefficient (n). It can be assumed that potentiating effect of hyperpotassium solution upon alpha 1-adrenergic contractions is operated by a mechanism which allows greater recruitment of voltage-operated Ca2+ channels by agonist.

[Effect of cold adaptation of alpha- and beta-adrenergic responses of blood pressure in hindlimb and small intestine in situ and systemic blood pressure in rabbits].

Changes in the main parameters of alpha- and beta-adrenergic responses, sensitivity to agonists (EC50) and maximum response (Pm) of hindlimb and small intestinal blood pressure in situ and systemic blood pressure were studied in rabbits adapted to cold for 1-30 days (daily exposures to -10 degrees C for 6 h). The responses to phenylephrine, noradrenaline. adrenaline, clonidine (alpha-agonists), and isopropylnoradrenaline (beta-agonist) corresponded to the equation p = (PmAn)/(EC50n + An) with n = 1 and n = 2, respectively. Cold adaptation induced reciprocal changes in the response of both EC50 and Pm to alpha-agonists and in the response of Pm alone to isopropylnoradrenaline. The significant differences of the parameters from control observed during the first 5 days of adaptation gradually decreased by day 30. After 10 days of adaptation, the efficiency (E = Pm/2EC50) of response to alpha- and beta-agonists of adrenoceptors significantly increased.

[Changes of alpha1-adrenergic and muscarinic cholinergic responses of arterial blood pressure in the rabbit during adaptation to cold].

Changes in major parameters of alpha1-adrenergic and muscarinic cholinergic responses of arterial blood pressure (EC50 and P(m)) were studied in rabbits adapted to cold for 1-30 days (daily cold exposures for 6 hours at -10 degrees C). It was shown that responses to noradrenaline, adrenaline (agonists of alpha1-adrenoceptors) and acetylcholine (agonist of muscarinic cholinoreceptors) could be expressed by equation p = (Pm- A(n))/(EC50(n) + A(n)) with n = 1. Adaptation to cold induced radically different changes in the major parameters of adrenergic and cholinergic responses. In the noradrenergic responses, the parameters EC50 and Pm gradually decreased within 1-30 days of adaptation. In the adrenaline responses, Pm increased from 1 to 30 days of adaptation, EC50 decreased to 10 day and increased on the 30th day. In the muscarinic cholinergic response, the Pm value decreased from 1 and 10 days but returned to control values on the 30th day. IC5O did not differ from the control 1 day, decreased to 10 days and increased on the 30th day.

[Influence of carbachol on the kinetic parameters of the contractile response to noradrenaline in the rat vas deferens].

Graphic and mathematical analysis of kinetics of the rat vas deferens contractile response to noradrenaline showed that alpha1-adrenoceptors mediating the contraction were in different functional states. In some organs, these receptors were homogeneous with the Hill coefficient n = 1 (the linear mode of the Scatchard plot), in the others--not homogeneous, with n > 1 (not linear mode of the Scatchard plot). Carbachol increased the contractile response to noradrenaline. As in the control, two types of response were revealed: 1. The Hill coefficient n < 1 (biphasic mode of the Scatchard plot) with two pools (high and low affinity) of alpha1-adrenoceptors, and 2. The Hill coefficient n = 1 (the linear mode of the Scatchard plot). These results suggest that the influence of carbachol is caused by the local interaction of M-cholinergic and alpha1-adrenergic systems.

[Carbachol effect on kinetics of the alpha1-adrenergic contractile response of the rat vas deferens]. / Vliianie karbakhola na kinetiku al'fa1-adrenergicheskoi sokratitel'noi reaktsii semiavynosiashchego protoka krysy.

Analysis of the control kinetics of the rat vas deferens contractile response to phenylephrine showed that alpha 1-adrenoceptors mediating the contraction could be in different functional states. In some organs these receptors represented a single pool with Hill coefficient n = 1 (the linear mode of the Scatchard plot), in others--not a single pool, n > 1 (not linear mode of the Scatchard plot). Activation of muscarinic cholinergic receptors by carbachol exerted a stimulating effect on the alpha 1-adrenergic contractile response especially to low adrenomimetic concentrations and the maximum response was increased. The action of cholinomimetic was accompanied by a decrease of Hill coefficient. When the control represented a single pool of alpha 1-adrenoceptors in the presence of carbachol Scatchard plot became biphasic with Hill coefficient n < 1, in addition to the low affinity pool the high affinity appeared. In case of not homogeneous control pool, in the presence of carbachol a single pool was revealed and n was close to 1. These findings suggest that the stimulatory effect of carbachol is caused by its modulator action on the alpha 1-adrenoceptors states and activating influence on the intracellular effector's system.

[Effect of adaptation to cold on the alpha1- and beta-adrenergic reactions of arterial vessels of the small intestine in rabbits]. / Vliianie adptatsii k kholodu na al'fa1- i beta-adrenergicheskie reaktsii arterial'nykh sosudov tonkoi kishki krolika.

Changes in major paraments of alpha 1- and beta-adrenergic responses (EC50 and Pm) were studied in the intestine arterial blood vessels of rabbits adapted to cold for 1-30 days (daily cold exposures for 6 hours at -10 degrees C). It was shown that responses to phenylephrine, noradrenaline, adrenaline (alpha 1-agonists), isopropylnoradrenaline (beta-agonist) corresponded to the equation p = (Pm.An)/(EC50n + An) with n = 1 and n = 2, respectively. Adaptation to cold induced radically different changes in the major parameters of alpha- and beta-adrenergic responses. In the alpha-adrenergic responses, the parameters EC50 and Pm changed reciprocally. In the beta-adrenergic response, only Pm value changed while EC50 did not differ from the control over the entire period of adaptation to cold. The pronounced differences from the control gradually decreased within 1-30 days of adaptation.

Analysis of ligand-receptor interactions from the molecular level to the whole-body level.

A system for the quantitative analysis of ligand-receptor interactions is presented, based on models of different levels of complexity. For two pools of receptors, binding of a radioactive ligand is described by b = [(Bml x A(nl))/(K(nl)dl + A(nl))] + [(Bm2 x A(n2))/(Kn2(d2) + A(n2))], (1) where b is the number of bound receptors at a ligand concentration [A], Bml and Bm2 are the receptor concentrations. Kdl and Kd2 are dissociation constants for the ligand-receptor complex, and n1 and n2 are Hill coefficients. The magnitude of the physiological response for a system consisting of two discrete pools of receptors with different affinities is given by p = [(Pm x A(nl))/(EC50(nl) + A(nl))] + [(Pm2 x A(n2)/(EC50(n2)2 + A(n2))], (2) where p is the magnitude of the response to an agonist (or antagonist) at concentration [A], Pml and Pm2 are the maximal magnitudes of the responses for the individual pools of receptors, EC50(1) and EC50(2) are the agonist concentrations giving responses of magnitudes Pm1/2 and Pm2/2, and n1 and n2 are Hill coefficients. The parameters of these equations show: the number of pools of receptors with different affinities for the ligand (Kd or EC50), the number of active receptors (Bmax) or the magnitudes of the maximal response (Pmax), and the numbers of ligand molecules binding with the receptor (n, the Hill coefficient). E is the efficiency (E = Bmax/2Kd, or E = Pmax/2EC50) and gives the overall characteristics of the activity of the effector system. This method of analysis can be applied to any biological reactions whose results can be presented quantitatively.