Further studies on the ethanol antagonism exhibited by 2(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587)

A lipid soluble alpha 1-adrenoceptor agonist 2-(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587) dose-dependently antagonized the hypnotic, hypothermic and respiratory depressant effects of ethanol in C57B1/6 mice. This effect was present whether St 587 was given before or after ethanol. St 587 did not block the pentobarbitone-induced hypnosis. It also did not influence the elimination of ethanol. Combined treatment with a subhypnotic dose of ethanol and St 587 resulted in marked hyperactivity in mice. This effect was completely abolished by pimozide pretreatment. It was inferred that the dopamine released from brain areas by this dose of ethanol together with the norepinephrine receptor activation offered by St 587 resulted in this hyperactivity. Cirazoline, a more potent alpha 1-adrenoceptor agonist than St 587 was relatively more effective than the latter in blocking the ethanol-induced hypnosis in mice. It seems that alpha 1-adrenoceptor stimulation is a major contributing factor to the ethanol antagonism exerted by St 587. This drug might prove to be useful in the treatment of acute ethanol intoxication and in understanding the mode of action of ethanol.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

Dependence on extracellular potassium of the positive inotropic response to St 587, a selective alpha-1 adrenoceptor agonist, in Zucker rat heart ventricle

The effects of St 587, a selective alpha-1 adrenoceptor agonist, were investigated in non obese and obese Zucker rat heart ventricles. In both groups, the numbers and affinity constants for alpha-1 adrenoceptors were found to be similar. At 4 or 10 mM [K]o, St 587 failed to increase the developed tension whereas at 14 mM [K]o, St 587 significantly increased it in both groups of rats. This effect was reversed by prazosin; St 587 also increased action potential duration at 14 mM [K]o. [K]o is thus important for the occurrence of the inotropic effect of St 587 in 12 week-old Zucker rats, either non obese or obese with reduced beta-adrenoceptor responsiveness. This suggests the participation of phosphoinositide metabolism in the mechanism of St 587 inotropic effect in the rat.     

Protection against alloxan-induced diabetes in mice by stimulation of alpha 2-adrenergic receptors

A single intravenous injection of alloxan in mice induced hyperglycemia in a dose dependent fashion. This diabetogenic action of alloxan was prevented by a single intraperitoneal injection of the alpha 2-adrenergic agonists, i.e. oxymetazoline, clonidine or epinephrine 40 min prior to the injection of alloxan. The alpha 1-adrenergic agonists, i.e. methoxamine and phenylephrine, and a beta-adrenergic agonist, isoproterenol, failed to prevent the diabetogenic action of alloxan. The inhibitory effect of clonidine on alloxan-induced diabetes was antagonized by yohimbine or phentolamine, but not by prazosin. Although alpha 2-adrenergic agonists caused a transient hyperglycemia at the time of alloxan administration (40 min after the administration of alpha 2-adrenergic agonists), the plasma glucose level at the time of alloxan injection did not correlate with the anti-diabetogenic effect of alpha 2-adrenergic agents. These results clearly demonstrate that the alpha 2-adrenergic mechanism which inhibits insulin release from pancreatic B cells prevented the diabetogenic action of alloxan in mice.     

Effect of repeated treatment with mirtazapine on the central alpha1-adrenergic receptors.

Mirtazapine (MIR) is an antidepressant which enhances noradrenergic and serotonergic 5-HT1A neurotransmission via antagomism of central alpha2-adrenergic autoreceptors and heteroreceptors. The drugs does not inhibit noradrenaline and serotonin reuptake but blocks the 5-HT, and 5-HT3 receptors and has high affinity only for central and peripheral histamine H1 receptors. The present study was aimed at determining whether repeated MIR treatment induced adaptive changes in the alpha1-adrenergic receptors, similar to those reported by us early for tricyclic antidepressants, The experiments were carried out on male mice and rats. MIR was administered at a dose of 10 mg/kg once or repeatedly (twice daily for 14 days). The obtained results showed that MIR administrated repeatedly potentiated the methoxamine- induced exploratory hyperactivity in rats and clonidine-induced aggressiveness in mice, those effects being mediated by alpha1-adrenergic receptors. MIR given repeatedly (but not acutely) increased the binding (Bmax ) of [3H]prazosin to alpha1-adrenergic receptors in cerebral cortex, however, the ability of the alpha1-adrenoceptor agonist phenylephrine to compete for the these sites was not significantly changed. The above results indicate that repeated MIR administration increases the responsiveness of alpha1-adrenergic system (behavioural and biochemical changes), as tricyclics do. However, the question whether the increased functional responsiveness found in the present study is important for the clinical antidepressant efficacy, remains open.     

A lipophilic, selective alpha1 -adrenoceptor agonist: 2-(2-chloro-5-trifluoromethylphenylimino)imida-zolidine (St 587)

A new compound (St 587) is described, which is a selective α₁ -adrenoceptor stimulating agent with lipophilic properties. This combination of characteristics is novel, since all α₁ -adrenoceptor agonists developed so far are hydrophilic. The α-adrenergic effects of 2-(2-chloro-5-trifluoromethylphenylimino) imidazolidine (St 587), a derivative of clonidine, were examined in several animal models. St 587 (1–10,000 μg/kg, i.v.) induced vasoconstriction in pithed, normotensive rats. This peripheral pressor activity was strongly antagonized by prazosin (0.1 mg/kg), but not affected by yohimbine (1 mg/kg). In intact, pentobarbitone-anaesthetized normotensive rats, St 587 (1–3,000 μg/kg, i.v.) evoked transient pressor responses, but a secondary fall in blood pressure and cardiac frequency was not observed. In pitched rats, St 587 (1–1,000 μg/kg) failed to modify the increase in heart rate produced by electrical stimulation of the cardioaccelerator sympathetic nerve fibres. St 587 (300 and 1,000 μg/kg) did not display central hypotensive activity, when injected into the left vertebral artery of anaesthetized cats. In addition, no hypotensive effect was observed when St 587 was administered i.v. to anaesthetized normotensive rats and cats. In mice, St 587 (10–10,000 μg/kg, i.p.) lacked sedative properties, since it did not prolong the hexobarbitone (75 mg/kg, i.p.)-induced loss of the righting reflex. The overall lipophilicity (log P′) of St 587 in the octanol/buffer (pH=7.4) reference system at 37°C amounted to 1.54. The experimental data suggest that St 587 is a lipophillic compound with selective α₁ - agonistic activity. The inability of St 587 to cause hypotension and sedation provides further evidence for the view that α₁ -adrenoceptors in the brain are not involved in the central hypotensive action and the sedation, caused by clonidine and related drugs. These effects are solely mediated by homogenous populations of α₂ -adrenoceptors.     

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.     

Adrenergic response of splanchnic arteries from cirrhotic patients: role of nitric oxide, prostanoids, and reactive oxygen species

Peripheral and splanchnic vasodilatation in cirrhotic patients has been related to hyporesponsiveness to vasoconstrictors, but studies to examine the vascular adrenergic response provide contradictory results. Hepatic arteries from cirrhotic patients undergoing liver transplantation and mesenteric arteries from liver donors were obtained. Segments 3 mm long from these arteries were mounted in organ baths for testing isometric adrenergic response. The concentration-dependent contraction to noradrenaline (10(-8) to 10(-4) M) was similar in hepatic and mesenteric arteries, and prazosin (alpha 1-adrenergic antagonist, 10(-6) M), but not yohimbine (alpha 2-adrenergic antagonist, 10(-6) M), produced a rightward parallel displacement of this contraction in both types of arteries. Phenylephrine (alpha 1-adrenergic agonist, 10(-8) to 10(-4) M) and clonidine (alpha 2-adrenergic agonist, 10(-8) to 10(-4) M) also produced concentration-dependent contractions that were comparable in hepatic and mesenteric arteries. The inhibitor of cyclooxygenase meclofenamate (10(-5) M), but not the inhibitor of nitric oxide synthesis N(w)-nitro-l-arginine methyl ester (l-NAME, 10(-4) M), potentiated the response to noradrenaline in hepatic arteries; neither inhibitor affected the response to noradrenaline in mesenteric arteries. Diphenyleneiodonium (DPI; 5 x 10(-6) M), but neither catalase (1000 U/ml) nor tiron (10(-4) M), decreased the maximal contraction for noradrenaline similarly in hepatic and mesenteric arteries. Therefore, it is suggested that, in splanchnic arteries from cirrhotic patients, the adrenergic response and the relative contribution of alpha 1- and alpha 2-adrenoceptors in this response is preserved, and prostanoids, but not nitric oxide, may blunt that response. Products dependent on NAD(P)H oxidase might contribute to the adrenergic response in splanchnic arteries from control and cirrhotic patients.     

Presynaptic alpha 2 -adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the alpha 2 -adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective alpha 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the alpha 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in conscious dogs evoked a dose-related rise in plasma GH at doses of 2-8 /micrograms/Kg, but not at 16 and 32 /micrograms/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic alpha 2-receptors, mianserin abolished the GH rise induced by CLON (4 /micrograms/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 /micrograms/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of alpha 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2-32/micrograms/kg iv]; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic alpha 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.     

Effects of androgen on alpha- and beta-adrenergic receptors in membranes from the rat seminal vesicle.

The effects of castration and androgen-replacement on adrenergic receptors in membranes from the rat seminal vesicle were studied. Membranes from seminal vesicles showed saturable and high-affinity binding sites for the beta-adrenergic receptor antagonist, [3H]dihydroalprenolol ([3H]DHA), and the alpha 1-adrenergic receptor antagonist, [3H]prazosin. Castration markedly reduced beta-adrenergic receptors with decreasing the effect of GTP modulating the receptor-ligand affinity, suggesting defects in both the receptor per se and the guanine-nucleotides-regulating mechanism after castration. In contrast, castration increased alpha 1-adrenergic receptors and androgen-replacement reversed this change. The effects of GTP decreasing the alpha 1-receptor binding affinity to the radioligand were observed to a similar extent in the castrated and control membranes. These results demonstrate an inverse regulation by androgen on beta- and alpha 1-adrenergic receptors in membranes of the rat seminal vesicle.     

Regulation of DNA polymerase alpha activity by the alpha 1-adrenergic receptors in proliferatively activated rat liver cells.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.     

Effect of adrenoblockers on the analgesic effect of GABA-positive preparations

The experiments on rats have shown that selective alpha 1 and alpha 2 adrenoceptor blockers (prazosin and yohimbine) and an inhibitor of dopamine-beta-hydrolase FD-008 failed to change the antinociceptive effect of baclofen, a direct GABAB receptor agonist. The antinociceptive effect of THIP and depakin, acting predominantly on GABAA receptors, was significantly reduced by prazosin, FD-008 and yohimbine in vocalization test. In tail-flick test the analgetic effect of THIP and depakin was not altered by prazosin and FD-008, but was increased by yohimbine. The role of adrenergic mechanisms in GABAA and GABAB receptor-mediated analgesia is discussed.     

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.     

The serotonin 5-HT2 receptor system, but not the alpha 1-adrenergic receptor system, is involved in the estrogen-induced afternoon prolactin surge in the rat

Ketanserin (Ket), a new serotonergic (5-HT2) antagonist, has recently been shown to block the estrogen-induced afternoon PRL surge (Endocrinology 120: 2070-2077, 1987). It is not certain, however, whether the effect of Ket was due to its serotonergic or adrenergic receptor antagonistic property. Another 5-HT2 receptor antagonist, LY53857, which possesses no alpha 1-adrenergic receptor affinity, as well as an alpha 1-adrenergic receptor antagonist, prazosin, were used in this study to further clarify the mechanism of 5-HT in the control of PRL secretion. Adult female Sprague-Dawley rats ovariectomized for 2-3 weeks and given a single injection of polyestradiol phosphate were studied 6 days later. Ket, LY53857 and prazosin were examined singly or in combination and animals were injected twice on the sampling day at 1200 and 1300h, respectively. The dosages were as follows: Ket and LY53857, 3 mg/kg BW, ip and 2 mg/kg BW, sc; prazosin, 1 mg/kg BW, ip and 0.7 mg/kg BW, sc. Blood samples were drawn from indwelling intraatrial catheters throughout the afternoon PRL surge.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

Postnatal treatment of rats with adrenergic receptor agonists or antagonists influences differentiation of sexual behavior

The aim of the study was to investigate the possible role of the adrenergic system in development and differentiation of neural centers controlling sexual behavior in adulthood. For this purpose normal and androgenized female rats were treated with the alpha 1-receptor antagonist prazosin, the alpha 2-receptor agonist clonidine, or the alpha 2-receptor antagonist yohimbine-HCl throughout the first week of life. In adulthood all animals were ovariectomized and, after appropriate hormone-priming, they were tested for the capacity to display female and male sexual behavior patterns. Alteration of adrenergic transmission during the critical postnatal period for sexual differentiation of neural centers resulted in significant changes in the capacity to express female lordosis behavior in adulthood. In nonandrogenized animals clonidine significantly reduced the capacity for lordosis behavior. In androgenized animals clonidine had the opposite effect; it attenuated the inhibitory effect of testosterone propionate (TP) on differentiation of lordosis behavior. Prazosin, which was without effect in nonandrogenized animals, also attenuated the inhibitory effect of TP on differentiation of lordosis behavior. Yohimbine was without effect in androgenized and nonandrogenized animals. There was no influence of any of the adrenergic drugs on differentiation of male sexual behavior. In conclusion, differentiation of lordosis behavior seems to be mediated or modulated via adrenergic transmission. The defeminizing effect of testosterone postnatally on the differentiation of lordosis behavior seems to be expressed via alpha 1-adrenergic transmission, and diminished adrenergic activity during the postnatal period seems to protect the developing brain against this effect of testosterone.     

Differential effect of pertussis toxin on the affinity state for agonists of renal alpha 1- and alpha 2- adrenoceptors

The effect of pertussis toxin treatment on the guanine nucleotide-induced modulation of the affinity of renal alpha 1- and alpha 2-adrenergic receptors was investigated. Pretreatment of rats with pertussis toxin did not induce any change in the number of or affinity for antagonists of alpha 1- or alpha 2-receptors studied using [3H]prazosin and [3H]yohimbine, respectively. Guanyl-5'-yl imidodiphosphate induced an "up-shift" in the number of alpha 2-adrenergic receptors; this up-shift was not observed for alpha 1-adrenergic receptors. Pertussis toxin treatment decreased the affinity of epinephrine for the [3H]yohimbine-binding sites and reduced the ability of guanine nucleotides to modulate alpha 2-adrenoceptor agonist affinity. The regulation by guanine nucleotides of alpha 1-adrenoceptor affinity for agonists was not altered. These results suggest that the modulation of alpha 1- and alpha 2-adrenoceptors by guanine nucleotides is probably exerted through different molecular entities.     

Centrally administered neuropeptide Y enhances the hypothermia induced by peripheral administration of adrenoceptor antagonists

The distribution of neuropeptide Y in the brain includes extensive coexistence within adrenaline- and noradrenaline-containing neurons and many of its actions are often associated with adrenergic systems. Since neuropeptide Y immunoreactivity is particularly intense in the preoptic area, one of the principal sites for thermoregulation, we have tested the effects of neuropeptide Y on core temperature in normothermic rats, and rats rendered hypothermic by systemic treatment with adrenergic antagonists. In the normothermic rat, intracerebroventricular administration of 1 microgram of neuropeptide Y did not have a significant effect on core temperature. Intraperitoneal treatment with the alpha 1-adrenoceptor antagonist, prazosin, or the beta-adrenoceptor antagonist, propranolol, caused an immediate and significant hypothermia; the intracerebroventricular administration of 1 microgram of neuropeptide Y, 10 minutes after these drugs, strongly potentiated their hypothermic effect. Although intraperitoneal treatment with the alpha 2-adrenoceptor antagonist, idazoxan, had no hypothermic effect per se, the intracerebroventricular administration of NPY 10 minutes after this antagonist led to a significant decrease in core temperature.     

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.     

Norepinephrine down-regulates the activity of protein S on endothelial cells

The adrenergic agonist norepinephrine is shown to stimulate endothelium to induce protein S release and degradation, leading to diminished anti-coagulant activity and to down-regulation of protein S cell surface-binding sites. Norepinephrine-induced release of intracellular protein S was blocked by the alpha 1-adrenergic antagonist prazosin (10(-7) M) but not by the alpha-adrenergic antagonist propranolol (10(-6) M) or the alpha 2-adrenergic antagonist yohimbine (10(-5) M) indicating that this response resulted from the specific interaction of norepinephrine with a class of alpha 1-adrenergic receptors not previously observed on endothelium. Attenuation of norepinephrine-induced release of protein S by pertussis toxin in association with the ADP-ribosylation of a 41,000-D membrane protein indicates that this intracellular transduction pathway involves a regulatory G protein. The observation that protein S was released from endothelium in response to maneuvers which elevate intracellular calcium or activate protein kinase C suggests that the response may be mediated via intermediates generated through the hydrolysis of phosphoinositides. Morphologic studies were consistent with a mechanism in which norepinephrine causes exocytosis of vesicles containing protein S. In addition to release of protein S, norepinephrine also induced loss of endothelial cell protein S-binding sites, thereby blocking effective activated protein C-protein S-mediated factor Va inactivation on the cell surface. Norepinephrine-mediated endothelial cell stimulation thus results in loss of intracellular protein S and suppression of cell surface-binding sites, modulating the anti-coagulant protein C pathway on the vessel wall. These studies define a new relationship between an anti-coagulant mechanism and the autonomic nervous system, and indicate a potential role for an heretofore unrecognized class of alpha 1-adrenergic receptors in the regulation of endothelial cell physiology.