alpha1-adrenergic receptor subtypes in human peripheral blood lymphocytes.
(1/2042)
We investigated the expression of alpha1-adrenergic receptor subtypes in intact human peripheral blood lymphocytes using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding assay techniques combined with antibodies against the three subtypes of alpha1-adrenergic receptors (alpha1A, alpha1B, and alpha1D). RT-PCR amplified in peripheral blood lymphocytes a 348-bp alpha1A-adrenergic receptor fragment, a 689-bp alpha1B-adrenergic receptor fragment, and a 540-bp alpha1D-adrenergic receptor fragment. Radioligand binding assay with [3H]prazosin as radioligand revealed a high-affinity binding with a dissociation constant value of 0. 65+/-0.05 nmol/L and a maximum density of binding sites of 175. 3+/-20.5 fmol/10(6) cells. The pharmacological profile of [3H]prazosin binding to human peripheral blood lymphocytes was consistent with the labeling of alpha1-adrenergic receptors. Antibodies against alpha1A-, alpha1B-, and alpha1D-receptor subtypes decreased [3H]prazosin binding to a different extent. This indicates that human peripheral blood lymphocytes express the three alpha1-adrenergic receptor subtypes. Of the three different alpha1-adrenergic receptor subtypes, the alpha1B is the most represented and the alpha1D, the least. Future studies should clarify the functional relevance of alpha1-adrenergic receptors expressed by peripheral blood lymphocytes. The identification of these sites may represent a step for evaluating whether they represent a marker of alpha1-adrenergic receptors in cardiovascular disorders or for assessing responses to drug treatment on these receptors. (+info)
Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing alpha1d-adrenoceptors.
(2/2042)
In rat-1 fibroblasts stably expressing alpha1d-adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) alpha1d-adrenoceptors exhibit spontaneous ligand-independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist-stimulated alpha1d-adrenoceptor activity. (+info)
Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis.
(3/2042)
Changes in alpha1-adrenoceptor (alpha1AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [3H]prazosin binding studies show that the density of alpha1AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that alpha1AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of alpha1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of alpha1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of alpha1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of alpha1bAR gene paralleled with the elevations in the alpha1bAR mRNA abundance and alpha1AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of alpha1bAR gene coincided with the declines in the alpha1bAR mRNA abundance and the alpha1AR protein level in the rat liver. These observations indicate that the altered expression of alpha1AR genes in the rat liver during the progression of sepsis was regulated transcriptionally. (+info)
Facilitation and depression of ATP and noradrenaline release from sympathetic nerves of rat tail artery.
(4/2042)
1. Excitatory junction currents (EJCs) were used to measure ATP release; noradrenaline (NA) oxidation currents and fractional overflow of labelled NA, [3H]NA, were used to monitor the release of endogenous and exogenous NA, respectively, from post-ganglionic sympathetic nerves of rat tail artery. 2. During nerve stimulation with 100 pulses at 5-20 Hz the EJCs initially grew in size (maximally by 23 %, at 2-10 Hz), and then depressed, maximally by 68 % at 20 Hz. 3. The peak amplitude of NA oxidation currents in response to nerve stimulation with 100 pulses at 2-20 Hz grew in size with frequency, while the area was independent of frequency and roughly constant. 4. The size of the NA oxidation currents evoked by nerve stimulation with 4-100 pulses at 20 Hz grew linearly with train length between pulses 4-16. Between pulses 20-100 there was a train length-dependent depression of the signal. 5. Fractional overflow of [3H]NA in response to nerve stimulation with 5-100 pulses at 20 Hz behaved similarly to the EJCs. It initially grew roughly linearly between pulses 5-25, and then showed a dramatic depression similar to that of the EJCs. 6. The alpha2-adrenoceptor antagonists rauwolscine and yohimbine increased the overflow of [3H]NA and the amplitude of NA oxidation currents, but not that of the EJCs. 7. It is concluded that during high-frequency stimulation (i) the release of ATP and NA is first briefly facilitated then markedly depressed, (ii) facilitation and depression of the two transmitters are similar in magnitude and time course, and (iii) alpha2-adrenoceptor antagonists differentially modify EJCs and the NA signals. The results obtained in the absence of drugs are compatible with the hypothesis that ATP and NA are released in parallel, while the effects of alpha2-adrenoceptor antagonists seem to suggest dissociated release. (+info)
Homologous regulation of the alpha2C-adrenoceptor subtype in human hepatocarcinoma, HepG2.
(5/2042)
1. Previous studies of the regulation of the alpha2C-adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human alpha2C-adrenoceptor in the hepatocarcinoma cell-line, HepG2; a model which expresses this subtype spontaneously. 2. Short-period treatment of the cells with UK14304 provoked neither a diminution of the potency of the alpha2-agonist to inhibit forskolin-induced cyclic AMP-accumulation nor a change in the degree of receptor coupling to G-proteins. 3. Long-period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose-dependent (EC50 = 190 +/- 45 nM), rapid (t1/2 = 4.2 h) and reversible. Receptor down-regulation was also observed with clonidine or (-)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of alpha2-antagonists. 4. Conversely to that observed with alpha2-agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression. 5. The observed alterations of receptor density are not the reflection of changes at the alpha2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half-life demonstrated that down-regulation by alpha2-agonists and up-regulation by alpha2-antagonists, with inverse-agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation. 6. In conclusion, our data show that alpha2C-adrenoceptor does not undergo desensitization but is down-regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the alpha2C4 gene, but not with observations made in OK cells. Inversely, down-regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain alpha2-antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild-type alpha2-adrenoceptor. (+info)
Effects of heptanol on the neurogenic and myogenic contractions of the guinea-pig vas deferens.
(6/2042)
1. The effects of the putative gap junction uncoupler, 1-heptanol, on the neurogenic and myogenic contractile responses of guinea-pig vas deferens were studied in vitro. 2. Superfusion of 2.0 mM heptanol for 20-30 min produced the following reversible changes in the biphasic neurogenic contractile response (8 trials): (i) suppression of both phases; (ii) delayed development of both the first as well as the second phase, accompanied by complete temporal separation of the two phases; (iii) prominent oscillations of force during the second (noradrenergic) phase only. 3. To eliminate prejunctional effects of heptanol, myogenic contractions were evoked by field stimulation of the vas in the presence of suramin (200 microM) and prazosin (1 microM). Heptanol (2.0 mM) abolished these contractions reversibly. 4. These results show that (i) heptanol inhibits both excitatory junction potential (EJP)-dependent and non EJP-dependent contractions of the vas; (ii) a postjunctional site of action of heptanol, probably intercellular uncoupling of smooth muscle cells, contributes to the inhibition of contraction. (+info)
Nitric oxide mediates sympathetic vasoconstriction at supraspinal, spinal, and synaptic levels.
(7/2042)
The purposes of this study were to investigate the level of the sympathetic nervous system in which nitric oxide (NO) mediates regional sympathetic vasoconstriction and to determine whether neural mechanisms are involved in vasoconstriction after NO inhibition. Ganglionic blockade (hexamethonium), alpha1-receptor blockade (prazosin), and spinal section at T1 were used to study sympathetic involvement. NO was blocked with Nomega-nitro-L-arginine methyl ester (L-NAME). Regional blood flow in the mesenteric and renal arteries and terminal aorta was monitored by electromagnetic flowmetry in conscious rats. L-NAME (3-5 mg/kg iv) increased arterial pressure and peripheral resistance. Ganglionic blockade (25 mg/kg iv) significantly reduced the increase in resistance in the mesentery and kidney in intact and spinal-sectioned rats. Ganglionic blockade significantly decreased hindquarter resistance in intact rats but not in spinal-sectioned rats. Prazosin (200 micrograms/kg iv) significantly reduced the increased hindquarter resistance. We concluded that NO suppresses sympathetic vasoconstriction in the mesentery and kidney at the spinal level, whereas hindquarter tone is mediated at supraspinal and synaptic levels. (+info)
Facilitatory beta2-adrenoceptors on cholinergic and adrenergic nerve endings of the guinea pig trachea.
(8/2042)
Using electrical field stimulation of epithelium-denuded intact guinea pig tracheal tube preparations, we studied the presence and role of prejunctional beta2-adrenoceptors by measuring evoked endogenous acetylcholine (ACh) and norepinephrine (NE) release directly. Analysis of ACh and NE was through two HPLC systems with electrochemical detection. Electrical field stimulation (150 mA, 0.8 ms, 16 Hz, 5 min, biphasic pulses) released 29.1 +/- 2.5 pmol ACh/g tissue and 70.2 +/- 6.2 pmol NE/g tissue. Preincubation for 15 min with the selective beta2-adrenoceptor agonist fenoterol (1 microM) increased both ACh and NE overflow to 178 +/- 28 (P < 0.01) and 165 +/- 12% (P < 0.01), respectively, of control values, increases that were abolished completely by the selective beta2-adrenoceptor antagonist ICI-118551 (1 microM). Further experiments with increasing fenoterol concentrations (0.1-100 microM) and different preincubation periods (1, 5, and 15 min) showed a strong and concentration-dependent facilitation of NE release, with maximum response levels decreasing (from nearly 5-fold to only 2.5-fold of control value) with increasing agonist contact time. In contrast, sensitivity of facilitatory beta2-adrenoceptors on cholinergic nerves to fenoterol gradually increased when the incubation period was prolonged; in addition, a bell-shaped concentration-response relationship was found at 15 min of preincubation. Fenoterol concentration-response relationships (15-min agonist preincubation) in the presence of atropine and yohimbine (1 microM each) were similar in the case of NE release, but in the case of ACh release, the bell shape was lost. The results indicate a differential capacity and response time profile of facilitatory prejunctional beta2-adrenoceptors on adrenergic and cholinergic nerve terminals in the guinea pig trachea and suggest that the receptors on adrenergic nerves are more susceptible to desensitization. (+info)