Xylazine enhances porcine myometrial contractility in vitro: possible involvement of alpha 2-adrenoceptors and Ca2+ channels

The effects of xylazine on porcine myometrial contractility were studied in vitro using uterine strips to determine the alpha 2-adrenergic influences during the diestrous stage of the estrous cycle. Xylazine (10(-8)-10(-5) M) caused a dose-dependent increase in the amplitude of myometrial contractility. The alpha 2-adrenoceptor antagonists idazoxan and yohimbine (10(-8)-10(-6) M) blocked the effects of xylazine in a dose-dependent manner. Yohimbine was approximately 10 times more potent than idazoxan in this regard. In contrast, an alpha 1-adrenoceptor antagonist prazosin (10(-7) and 10(-6) M) did not block the xylazine-induced increase in myometrial contractility, but a higher dose of prazosin (10(-5) M) did reduce the effects of xylazine. When the porcine uterine strips were pretreated with Ca2(+)-free Tyrod's solution or verapamil, a Ca2+ channel blocker, the effects of xylazine on myometrial contractility were completely abolished, whereas those of carbachol were only moderately reduced. The results suggest that the xylazine-induced myometrial contractility is mediated by alpha 2-adrenoceptors and that this effect is mediated, at least in part, by Ca2+ channels, whereas the effect of carbachol is attributed to an increase in both Ca2+ entry and release of Ca2+ from intracellular pools.     

Existence of two types of postjunctional alpha-adrenoceptors in the isolated canine internal carotid artery

The pharmacological characteristics of postjunctional alpha-adrenoceptors in isolated canine internal carotid arteries were investigated by the use of selective agonists and antagonists for alpha 1- and alpha 2-adrenoceptors. Norepinephrine, phenylephrine, and xylazine caused concentration-dependent contractions in the helical strips. The contraction induced by 10(-4)M xylazine was significantly smaller than that produced by 10(-4)M norepinephrine or 10(-4)M phenylephrine. The contraction induced by 10(-4)M phenylephrine was almost the same value as that induced by 10(-4)M norepinephrine. Phentolamine (10(-8) and 10(-7)M) caused a parallel shift to the right of the concentration-response curve to norepinephrine. The contractile responses to low concentrations of norepinephrine were significantly suppressed by pretreatment with an alpha 2-antagonist such as yohimbine (10(-9) and 10(-8)M) or DG5128 (10(-7) and 10(-6)M). On the other hand, the responses to higher concentrations of norepinephrine were mainly reduced by low concentrations of an alpha 1-antagonist, prazosin (3 x 10(-10) and 3 x 10(-9)M). These results suggest that both alpha 1- and alpha 2-adrenoceptors are located on the plasma membrane of smooth muscle cells in canine internal carotid arteries and that the norepinephrine-induced contractions at low and high concentrations are mainly mediated by activation of alpha 2- and alpha 1-adrenoceptors, respectively.     

Presynaptic alpha-adrenoceptor mediated inhibition of the neurogenic cholinergic contraction in the isolated intestinal bulb of the carp (Cyprinus carpio)

The effects of norepinephrine, epinephrine and clonidine on neurogenic cholinergic contraction were examined in the presence of a beta-adrenoceptor blocking agent, carteolol (5 X 10(-6) M), in the isolated intestinal bulb of the carp. Norepinephrine, epinephrine (10(-9)-10(-6) M) and clonidine (10(-8)-10(-5) M) inhibited the contraction induced by low frequency (2 or 5 Hz) transmural stimulation (TMS) without inhibiting the contraction induced by acetylcholine (ACh, 6 X 10(-8)-4 X 10(-7) M). Methoxamine (10(-4) M) and phenylephrine (10(-4) M) showed no such inhibitory effect on the TMS-induced contraction. The inhibitory effects of catecholamines and clonidine were decreased by phentolamine (5.4 X 10(-6) M) and yohimbine (10(-7)-10(-6) M) but not by prazosin (7 X 10(-7)-10(-6) M). Nicotine (10(-6)-10(-4) M) and serotonin (3 X 10(-8)-3 X 10(-6) M) caused contraction of the intestinal bulb indirectly by releasing endogenous ACh. This contraction was inhibited by norepinephrine, epinephrine and clonidine in a concentration-dependent manner. The present results suggest that catecholamines and clonidine inhibit cholinergic transmission via the activation of a presynaptic alpha-adrenoceptor (presumably of alpha-2 type) located on the cholinergic nerve terminals innervating the smooth muscle of the intestinal bulb of the carp.     

Substance P and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization and effects on uterine contractility.

Immunoreactivity to the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, SP immunoreactivity (SP-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns, with nerves immunoreactive for CGRP being the more numerous. Immunocytochemical double labeling studies revealed SP coexisted with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., SP-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. SP (10(-4) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP(1-37) and CGRP(8-37) had no effect on basal uterine tension. While CGRP(1-37) (10(-7) M) reduced SP-stimulated (10(-5) M) uterine contraction by 56%, CGRP(8-37) had no effect on SP-stimulated uterine contraction. However, CGRP(8-37) (10(-6) M) significantly reduced the ability of CGRP(1-37) (10(-7) M) to inhibit SP-stimulated uterine contraction. These results demonstrate that SP- and CGRP-I are present in, and coexist in some uterine nerves, presumably afferent nerves. The first 7 amino acids are necessary for the inhibitory effect of CGRP(1-37) on stimulated uterine contraction. In addition, CGRP(8-37) acted as an antagonist to this inhibitory action. SP and CGRP could be coreleased from afferent fibers in an "efferent fashion" and influence uterine contractility. SP having a contractile effect and CGRP having a relaxing effect.     

Time-dependent potentiation of contractile response to norepinephrine in canine isolated cerebral arteries.

The time course of contractile responses to alpha-adrenoceptor agonists was investigated using various arteries isolated from dogs and monkeys. The contractile response to norepinephrine was increased during the time course of the experiment in canine basilar and internal carotid arteries, whereas the response of isolated canine external carotid arteries and monkey internal carotid arteries did not change significantly. Treatment with 10(-7) M propranolol, 5 x 10(-6) M cocaine plus 10(-5) M hydrocortisone, or 5 x 10(-5) M acetylsalicylic acid did not significantly affect the time-dependent potentiation of the norepinephrine-induced contraction in canine internal carotid arteries. The time-dependent enhancement in the response to norepinephrine was also observed in the arterial preparations from which the endothelial cells were removed. The contractile response of canine internal carotid arteries to phenylephrine did not alter significantly throughout the experiments. On the other hand, the responses to clonidine and xylazine were markedly enhanced with time. Significant potentiation of the norepinephrine-induced contraction was observed in canine internal carotid arteries treated with 10(-8) M prazosin, whereas 10(-8) M yohimbine attenuated the time-dependent potentiation. These results suggest that the contractile responses of isolated canine basilar and internal carotid arteries to norepinephrine are potentiated during the course of the experiment, which is likely to be related, in part, to an enhancement in alpha 2-adrenoceptor mediated contraction.     

Effects of norepinephrine on lung liquid production by in vitro lungs from fetal guinea pigs

Lungs from near-term fetal guinea pigs (61 +/- 2 days of gestation) were supported in vitro for 3 h; lung liquid production was monitored by a dye dilution method. Untreated control preparations produced fluid at 1.38 +/- 0.30 mL x kg(-1) body weight x h(-1), with no significant change (ANOVA; regression analysis); those given 1.24 x 10(-9) or 1.24 x 10(-8) M norepinephrine during the middle hour showed no significant change, but those given concentrations between 5.24 x 10(-8) and 1.24 x 10(-5) M all showed significant reductions or fluid reabsorption (based on 42 fetuses). The responses showed a linear relationship with the log concentration (r = 0.97). They appeared to involve alpha-adrenoreceptors, since responses to 10(-7) M norepinephrine were unaffected by 10(-6) M propranolol, but those to 10(-7) and 1.24 x 10(-6) M norepinephrine were abolished by 10(-6) and 1.78 x 10(-5) M phentolamine, respectively (based on 48 fetuses). Activation was through alpha2-adrenoreceptors, since responses to 10(-7) and 10(-5) M norepinephrine were abolished by 10(-4) M yohimbine, but not by 10(-5) M prazosin (based on 60 fetuses). The results show that norepinephrine is able to reduce lung liquid production when at plasma levels present at birth, and that it can produce reabsorption; unlike epinephrine, there was no reduction in responses at high concentrations. This work reintroduces a neglected factor, norepinephrine, into possible controls of lung liquid reabsorption, and opens up the potential for neural controls.     

Mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation

The mechanism of adenosine-induced vasodilation in rat diaphragm microcirculation was investigated using laser Doppler flowmetry. Adenosine (10(-5), 3.2 x 10(-5), and 10(-4) M), the nonselective adenosine agonist 5'-N-ethylcarboxamido-adenosine (NECA) (10(-8)-10(-7) M), the specific A(2A) agonist 2-p-(2-carboxyethyl)phenyl-amino-5'-N-ethyl carboxamidoadenosine (CGS-21680) (10(-8)-10(-7) M), and the adenosine agonist with higher A(1)-receptor affinity, R-N(6)-phenylisopropyladenosine (R-PIA) (10(-7), 3.2 x 10(-7), and 10(-6) M) elicited a similar degree of incremental increase of microcirculatory flow in a dose-dependent manner. The ATP-dependent potassium (K(ATP)) channel blocker glibenclamide (3.2 x 10(-6) M) significantly attenuated the vasodilation effects of these agonists. Adenosine-induced vasodilation could be significantly attenuated by the nonselective adenosine antagonist 8-(p-sulfophenyl)-theophylline (3 x 10(-5) M) or the selective A(2A) antagonist 4-(2-[7-amino-2-(2-furyl)[1,2, 4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol (ZM-241385, 10(-6) M), but not by the selective A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (5 x 10(-8) M). Adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A, 10(-5)M) effectively suppressed the vasodilator response of adenosine and forskolin. These results suggest that adenosine-induced vasodilation in rat diaphragm microcirculation is mediated through the stimulation of A(2A) receptors, which are coupled to adenylate cyclase activation and opening of the K(ATP) channel.     

Galanin and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization, and effects on uterine contractility.

Immunoreactivity to the neuropeptides galanin (GAL) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, GAL immunoreactivity (GAL-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns and cervix, with nerves immunoreactive for CGRP being more numerous. Immunocytochemical double-labeling studies revealed GAL coexists with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., GAL-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. GAL (10(-5) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP had no effect on basal uterine tension, but CGRP (10(-7) M) reduced GAL-stimulated (10(-7) M) uterine contraction by 92.5%. These results demonstrate that GAL- and CGRP-I are present in, and coexist in, some uterine nerves, presumably afferent nerves. GAL and CGRP could be released from afferent fibers in an "efferent fashion" and influence uterine contractility, GAL having a contractile effect and CGRP having a relaxing effect.     

Reversal of ketamine/xylazine anesthesia in the rabbit with yohimbine

Ketamine and xylazine used in combination have been shown to be effective, easily administered, cost efficient agents for surgical anesthesia in the rabbit. The effect of xylazine on the central nervous system has been shown to be mediated through alpha-2 adrenergic receptors. Yohimbine, an alpha-2 adrenergic antagonist has been shown to reverse xylazine induced depression and partially antagonize ketamine in other species. We evaluated the antagonistic effect of yohimbine on ketamine/xylazine anesthesia in the rabbit. Six New Zealand White rabbits were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg) to establish baseline parameters including respiratory rate, heart rate, and palpebral, pedal and postural reflex activity. Fourteen days later each rabbit was subjected to the same anesthetic regimen followed 30 minutes later by the intravenous administration of yohimbine (0.2 mg/kg). The duration of anesthesia estimated by the time elapsed between the loss and return of the palpebral reflex was reduced in the yohimbine treated trial (means = 29.7 +/- 1.9 minutes) compared to the control trial (means = 67.0 +/- 13.5 minutes). The palpebral reflex returned within 5 minutes following yohimbine treatment. Our results indicated that yohimbine is an effective antagonist of ketamine/xylazine anesthesia in the rabbit. Yohimbine decreases anesthetic duration after intravenous administration and also may aid in the control of undesirable anesthetic effects and overdosage.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Inhibitory effect of ginger (Zingiber officinale) on rat ileal motility in vitro

Ginger (Zingiber officinale rhizome) is a widespread herbal medicine mainly used for the treatment of gastrointestinal diseases, including dyspepsia, nausea and diarrhoea. In the present study we evaluated the effect of this herbal remedy on the contractions induced by electrical stimulation (EFS) or acetylcholine in the isolated rat ileum. Ginger (0.01-1000 microg/ml) inhibited both EFS- and acetylcholine-evoked contractions, being more potent in inhibiting the contractions induced by EFS. The depressant effect of ginger on EFS-induced contractions was reduced by the vanilloid receptor antagonist capsazepine (10(-5) M), but unaffected by the alpha(2)-adrenergic antagonist yohimbine (10(-7) M), the CB(1) receptor antagonist SR141716A (10(-6) M), the opioid antagonist naloxone (10(-6) M) or by the NO synthase inhibitor L-NAME (3 x 10(-4) M). Zingerone (up to 3 x 10(-4) M), one of the active ingredients of ginger, did not possess inhibitory effects. It is concluded that ginger possesses both prejunctional and postjunctional inhibitory effects on ileal contractility; the prejunctional inhibitory effect of ginger on enteric excitatory transmission could involve a capsazepine-sensible site (possibly vanilloid receptors).     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

Inhibitory effect of the plant flavonoid galangin on rat vas deferens in vitro

Flavonoids are phenolic compounds that are widely distributed in higher plants and therefore are ingested by humans and animals with their regular foods, but also have various pharmacological properties. In the present study we have investigated the effect of galangin, a member of the flavonol class, on the contractile response elicited by electrical field stimulation (EFS) in the rat isolated vas deferens. Galangin (10(-8)-3 x 10(-4) M) produced a concentration- dependent inhibition of the EFS-evoked contractile response, with only a minimal inhibitory effect on phenylephrine-induced contractions. The inhibitory effect of galangin was unaffected by atropine (10(-6) M) plus hexamethonium (10(-4) M), a combination of the NK(1) receptor antagonist SR 140333 (10(-7) M), the NK(2) receptor antagonist SR 48968 (10(-6) M) and the NK(3) receptor antagonist SR 142801 (10(-7) M), L-NAME (3 x 10(-4) M), naloxone (10(-6) M) or yohimbine (10(-7) M). However, the vanilloid receptor antagonist capsazepine (10(-5) M) significantly reduced the inhibitory effect of galangin. It is concluded that the galangin inhibits excitatory transmission of the rat vas deferens with a mechanism involving, at least in part, vanilloid receptors.     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.     

Effectiveness of antagonists for tiletamine-zolazepam/xylazine immobilization in female white-tailed deer

A combination of tiletamine-zolazepam/xylazine (TZ/X) is effective in the chemical immobilization of white-tailed deer (Odocoileus virginianus); however, the lengthy duration of immobilization may limit its usefulness. From October to November 2002, 21 captive female deer were assigned randomly to an alpha(2) antagonist treatment to reverse xylazine-induced sedation (seven does per group). All deer were given 220 mg of TZ (4.5+/-0.4 mg/kg) and 110 mg of X (2.2+/-0.2 mg/kg) intramuscularly (IM). Antagonist treatments were either 200 mg of tolazoline (4.0+/-0.4 mg/kg), 11 mg of atipamezole (0.23+/-0.02 mg/kg), or 15 mg of yohimbine (0.30+/-0.02 mg/kg) injected, half intravenously and half subcutaneously, 45 min after the IM TZ/X injection. In addition, 10 other deer (five per group) were immobilized as before and then given tolazoline (200 mg) after 45 min, with either a carrier (dimethyl sulfoxide [DMSO]) or carrier (DMSO) plus flumazenil (5 mg) to reverse the zolazepam portion of TZ. Mean times from antagonist injection until a deer raised its head were different for alpha(2) antagonist treatments (P=0.02). Times were longer for yohimbine (62.3+/-42.7 min) than for either atipamezole (24.3+/-17.1 min) or tolazoline (21.3+/-14.3 min). Mean times from antagonist injection until standing were not different (P=0.15) among yohimbine (112.0+/-56.4 min), atipamezole (89.7+/-62.8 min), or tolazoline (52.6+/-37.2 min). A sedation score based on behavioral criteria was assigned to each deer every 30 min for 5 hr. On the basis of sedation scores, tolazoline resulted in a faster and more complete reversal of immobilization. Flumazenil treatment did not affect recovery.     

Effects of specific alpha-adrenoceptive agents on extraneuronal uptake (uptake2) of isoproterenol in perfused rat heart

Effects of specific alpha-adrenoceptive agents (alpha 1-agonist, alpha 1-antagonist, alpha 2-agonist and alpha 2-antagonist) on the extraneuronal accumulation of 3H-isoproterenol in the perfused rat heart were examined. The extraneuronal accumulation of 3H-isoproterenol in the hearts perfused with 3H-isoproterenol (10(-6)M) under COMT inhibition by tropolone (10(-4)M) was about 6 times higher than that of intact COMT. The increase in the accumulation by COMT inhibition was regarded as 100% and the effects of specific alpha-adrenoceptive agents on the accumulation was evaluated. alpha 1-agonists, methoxamine and phenylephrine, did not affect the accumulation. alpha 1-antagonists, prazosin, bunazosin and YM-12617, significantly decreased the accumulation of 3H-isoproterenol and these IC50 values were 2 x 10(-6)M, 3.5 x 10(-6)M and 2.3 x 10(-5)M, respectively. alpha 2-agonists, clonidine and guanabenz, significantly reduced the accumulation and these IC50 values were 3.4 x 10(-5)M and 2.9 x 10(-7)M, respectively. The alpha 2-antagonist, yohimbine, did not affect the accumulation. The present experiments clearly demonstrated that the tested alpha 1-antagonists and alpha 2-agonists inhibited uptake2 in rat heart but the tested alpha 1-agonists and an alpha 2-antagonist did not inhibit it.     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Alpha1 and alpha2 adrenoceptor mediated melanosome aggregatory responses in vitro in Oreochromis mossambica (Peters) melanophores

Effects of specific and non-specific adrenoceptor agonists and antagonists were examined on the isolated scale melanophores of O. mossambica in physiological Ringer solution. The responses were recorded as melanophore size index. It was observed that adrenaline, nor-adrenaline, phenylpropanolamine, clonidine and phenylepherine induced melanosome aggregation in a dose-dependent manner. Denervation of the fish melanophores increased the sensitivity of the melanophores to adrenaline but not to nor-adrenaline. Phentolamine (3.55 x 10(-5) M), prazosin (2.38 x 10(-5) M) and yohimbine (2.821 x 10(-5) M) significantly inhibited the aggregatory responses of the fish melanophores to adrenaline, nor-adrenaline, clonidine and phenylepherine. The blocking effect of yohimbine was significantly higher than that of prazosin. It is concluded that the effect of adrenaline is directly mediated through the receptors and alpha2 adrenoceptors are predominantly involved in the aggregatory responses of this fish melanophores, while alpha1 adrenoceptors presence has been indicated.     

Binding analysis of 1alpha- and 17alpha-dihydrotestosterone derivatives to homodimeric sex hormone-binding globulin

Binding studies of the interaction of immobilized 1alpha- and 17alpha-aminoalkyl derivatives of 5alpha-dihydrotestosterone (DHT) with purified N-deglycosylated homodimeric human sex hormone-binding globulin (SHBG) were performed using a surface plasmon resonance biosensor. These 1alpha- and 17alpha-derivatives with spacers of appropriate lengths between the amine function and the steroid ring skeleton enabled privileged, sterically undisturbed, interactions of either the 17- or 3-characteristic functional groups of DHT with SHBG. The association constants (K(a)1) for the binding of these immobilized DHT derivatives to the first binding site of SHBG, determined by SPR measurements, were 0.16 x 10(7) M(-1) for 17alpha-aminopropyl-17beta-hydroxy-5alpha-androstan-3-one (1), 1.64 x 10(7) M(-1) for 17alpha-aminocaproyl-17beta-hydroxy-5alpha-androstan-3-one (2), and 1.2 x 10(8) M(-1) for 1alpha-aminohexyl-17beta-hydroxy-5alpha-androstan-3-one (3). These values were compared with global K(a) data for the corresponding nonimmobilized DHT derivatives from equilibrium measurements using competitions with a tritiated testosterone tracer: the K(a) values were 1.25 x 10(7) M(-1) for 1, 1.50 x 10(7) M(-1) for 2, and 140 x 10(7) M(-1) for 3, confirming a remarkably high binding affinity of this latter compound for SHBG. A global fitting analysis of the biosensor data revealed that the interaction of the three immobilized steroids with SHBG was best described by a kinetic model assuming two structurally independent binding sites. This hypothesis of a bivalent binding model was also directly suggested by a dual fluorescent signal observed by the flow cytometry analysis of SHBG immobilized as a hybrid complex binding simultaneously two 1alpha-aminohexyl DHT ligands, one formed by 3, covalently coupled to phycoerythrin-labeled latex microspheres, and the other by the same DHT derivative, coupled to a fluorescein derivative (4).