Stereoselectivity at alpha-adrenoreceptor subtypes: observations with the enantiomers of WB 4101 separated through their amides of N-tosyl-(S)-proline.

We present a chromatographic method for the separation and determination of the optical purity of the enantiomers of WB 4101 [(+/-)-1], one of the most potent and selective alpha 1-adrenoreceptor antagonists. (+/-)-1 was converted into the amide of N-tosyl-(S)-proline. The two diastereoisomers were separated on silica gel and analysed by HPLC reversed phase. The analytical method described is both accurate and sensitive and allows the optical purity to be determined at very low concentrations and to obtain WB 4101 enantiomers with a purity of more than 99.95%.     

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.     

Effect of calcitonin gene-related peptide on the neuroeffector mechanism of sympathetic nerve terminals in rat vas deferens

In order to evaluate the mode of action of calcitonin gene-related peptide (CGRP) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of CGRP were tested on the electrical stimulated and the non-stimulated preparations of the isolated rat vas deferens. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers, were dose-dependently inhibited by CGRP in concentrations ranging from 0.1 to 10 nM. The inhibitory response produced by CGRP in high concentrations (greater than 2 nM) usually returned to the control level at 20-30 min and were rarely tachyphylactic. The inhibitory action of CGRP was not modified by pretreatment with 10(-7) M propranolol or 10(-7) M atropine. Contractions produced by exogenous norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in unstimulated preparations were not affected by pretreatment with CGRP in a low concentration (less than 2 nM). On the other hand, the contractions were slightly reduced 1 min after pretreatment with CGRP in high concentrations (greater than 5 nM), which recovered in 15 min after constant flow washout. High concentrations of CGRP also caused a concentration-dependent relaxation on the precontracted preparations produced by high potassium (60 mM K+) solution. These results suggest that CGRP in high concentrations (greater than 5 nM) may have a non-specific inhibitory action on the postsynaptic plasma membrane of the smooth muscle cell and a postulated CGRP receptor exists presynaptically in the rat vas deferens and that CGRP may inhibit the release of NE during adrenergic nerve stimulation.     

A simple and convenient approach for evaluation of the parameters of ligand-receptor interaction. Receptor blocking index and its application

A new approach for determination of the parameters for ligand-receptor interaction, which is based on so-called dilution coordinates, was developed earlier. Equations that allow evaluation of not only the affinity of ligand-receptor interaction but also of the amount of free (or occupied by corresponding ligand) receptors were suggested. The most important advantage of this approach as compared with well-known methods is the ability to determine the binding parameters for ligand-receptor interaction even for the cases in which ligand and receptor are already present in a mixture and separation of counterparts from each other is technically difficult or even impossible. Due to this reason, the proposed approach can be especially useful for studying interactions between highly-labile biological receptors and corresponding ligands as found in vivo. In the present paper I continue to consider how to determine the binding parameters for a given ligand-receptor interaction if the value of receptor blocking index is determined experimentally.     

Evidence for the presence of dopaminergic receptors in vas deferens

Specific dopaminergic recognition sites were identified in membranes prepared from rat vas deferens with the ligand (³H)-haloperidol. Specific binding, defined as the difference of (³H)-haloperidol binding in the presence or absence of an excess of unlabelled haloperidol (100 μM), was saturable and a Scatchard analysis of the data revealed a Kd = 21 nM and a Bmax = 74 fmol/mg prot. (+)-Butaclamol was several times more active in displacing (³H)-haloperidol from binding sites than its pharmacologically inactive enantiomer, (?)-butaclamol, demonstrating stereospecificity of binding. Dopamine displaced 50% of (³H)-haloperidol binding at a concentration of approximately 10 μM, while norepinephrine, epinephrine and serotonin were practically ineffective at this concentration. Our results support the notion that there are dopaminergic receptors in the rat vas deferens. We speculate that some of the known effects of dopamine and dopaminergic drugs on sexual behavior may be mediated peripherally and not solely via the CNS as is usually assumed.     

Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens

In order to evaluate the mode of action of galanin (GAL) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of this peptide were tested on the electrical stimulated and the unstimulated preparations of the isolated rat vas deferens in the presence of 10(-7) M atropine. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers were dose-dependently potentiated by GAL in concentrations ranging from 1 to 50 nM. The facilitatory action induced by GAL in high concentrations (greater than 10 nM) usually returned to the control level at 2-3 min and were tachyphylactic. The potentiating action of GAL was not modified by pretreatment with 10(-7) M propranolol. Contractions produced by exogenous norepinephrine (NE) in the unstimulated preparations were not affected by pretreatment with low concentrations (less than 5 nM) of GAL. On the other hand, the contractions were dose-dependently potentiated 1 min after pretreatment with higher concentrations (greater than 10 nM) of GAL, which recovered 15 min after constant flow washout. Contractions developed by exogenous 5-hydroxytryptamine were not affected, or slightly inhibited, by GAL (1-50 nM). In some preparations without electrical stimulation, high concentrations of GAL caused a slight contraction, which was not blocked by pretreatment with 10(-6) M phentolamine and 10(-6) M tetrodotoxin. These results suggest that GAL receptors exist presynaptically in the rat vas deferens and that stimulation of the receptors by GAL potentiates the release of NE from the nerve terminals during postganglionic sympathetic nerve stimulation. Other mechanisms for GAL action, such as influence on neuronal uptake and catecholamine metabolism, cannot be ruled out.     

Dense-cored membranous structures in smooth muscle cells of the vas deferens of the rat

Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior.     

Blockade of tyramine-induced responses by short-acting 2-halogenoethylamines in the rat vas deferens.

A comparative study was made of the blockade of responses of the rat vas deferens to Norepinephrine (NE) and tyramine (TYR) by short-acting 2-halogenoethylamines (2-HEA). The recovery of NE-induced responses after blockade by 2-HEA was rapid and complete within 120 min. The recovery of responses to TYR was considerably slower and remained incomplete (<50%) at the end of 180 min. Pretreatment of tissues by cocaine (10⁻⁵M/5 min) or TYR (5×10⁻⁴M/5 min) prior to exposure to 2-HEA did not alter the magnitude of initial blockade or recovery of NE-induced responses but increased the magnitude of TYR-induced responses. Pretreatment by Desipramine (DMI) did not protect TYR-induced responses from blockade by 2-HEA. In control tissues, cocaine (10⁻⁵M/10 min) was devoid of any effect on TYR-induced responses while DMI produced concentration-dependent inhibitory effects lasting for 180 min.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional distribution of dopamine, 5-hydroxytryptamine,and noradrenaline in the rat vas deferens

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididimal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2×80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT whilep-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

The presence and longitudinal distribution of the glutathione S-transferase in rat epididymis and vas deferens

The presence of the glutathione S-transferases, enzymes that catalyse the conjugation of glutathione with a variety of compounds, is reported here, for the first time, in the mammalian epididymis–vas deferens. These glutathione S-transferases, approx. 50% of those from rat liver on a per-mg-of-protein basis, are resolved by isoelectric focusing into six peaks, each with a characteristic isoelectric point and substrate specificity. By these same criteria, the first three peaks (pI 8.9, 8.2 and 7.8) can be identified as transferases B, A and C respectively. The fifth peak (pI7.2) may correspond to transferase M; the fourth (pI7.5) and sixth (pI7.0) peaks do not correspond to previously described transferases. The distribution of transferase activity towards any one substrate studied differs in sequential sections of the epididymis and vas deferens; in addition, the longitudinal-distribution pattern differs for each of the three substrates studied. Isoelectric focusing of the cytosol fractions of the different sections further substantiates these observations. The potential significance of these enzymes and of their distribution in terms of epididymal function, maturation of spermatozoa, is discussed.     

Factors involved in the generation of tension during contraction to high potassium in the rat vas deferens

A large number of studies indicate that K⁺-induced contractions of smooth muscle depend on extracellular calcium. If these contractions depend exclusively on extracellular calcium then contractile responses to 140 mM K⁺, which are larger than the response to 35 mM K⁺, should be associated with a larger influx of ⁴⁵Ca. This is not the case in the vas deferens from reserpine pretreated rats. During a 2 min interval, ⁴⁵Ca influx induced by 140 mMK⁺ was identical to that produced by 35 mM K⁺. This suggests that a second mechanism may be involved in responses to high K⁺. Indeed, 140 mM K⁺ caused an approximately 300% increase above control in the formation of inositol trisphosphate (IP₃) in tissues prelabelled with ³H-myoionositol whereas 35 mM K⁺ did not increase IP₃. IP₃ is thought to cause the release of calcium from internal stores which is consistent with our finding of an increase in ⁴⁵Ca efflux into calcium-free medium from tissues prelabelled with ⁴⁵Ca and stimulated with 140 mM K⁺. Stimulation with 35 mM K⁺ did not influence ⁴⁵Ca efflux. We conclude that in the rat vas deferens high K⁺ promotes tension development by smooth muscle by a dual mechanism: influx of extracellular calcium and release of calcium from internal stores via a IP₃ mechanism.     

The detection by X-ray microanalysis of noradrenaline in sympathetic nerve terminals of the rat vas deferens

Summary The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.This investigation was supported by research Fellowship No. 7/176(138)77 from the Council of Scientific & Industrial Research, New Delhi to the first author     

Stereoselective effect of phenprocoumon enantiomers on the binding of benzodiazepines to human serum albumin.

The effect of phenprocoumon enantiomers on the stereoselective binding of 3-substituted 1,4-benzodiazepines to human serum albumin (HSA) was studied by chromatography on HSA-Sepharose column. (S)-Phenprocoumon exerts stereoselective allosteric interaction on the binding of benzodiazepines. The structural requirements of enhanced stereoselectivities are similar to those found previously with (S)-warfarin.     

Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by beta2-adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of alpha2-adrenoceptors and abolished by simultaneous blockade of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor-mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12-myristate 13-acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12-myristate 13-acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C-mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely alpha2-adrenoceptors, adenosine A1 and P2Y receptors.     

基底神经节中多巴胺和腺苷受体二聚化及其药理学意义

近年来,大量研究发现G蛋白偶联受体不仅以单体形式,而且以同源或异源二聚体形式存在。腺苷A1受体和多巴胺D1受体以及腺苷A2a受体和多巴胺D2受体分别共存于基底神经节中纹状体向黑质和脚内核投射的神经元以及纹状体向苍白球投射的神经元内。A1／D1、A2a／D2受体形成受体异聚复合体构成了受体一受体之间相互作用的分子基础。腺苷和多巴胺受体之间在细胞水平以及行为水平上拮抗性的相互作用为其在帕金森病、精神分裂症、舞蹈病和药物依赖等疾病的治疗上提供了新的靶向。     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.