[Characteristics of alpha2-adrenoceptor mediated contractile response in isolated aortae of rats].

In order to investigate the characteristics of vascular alpha(2)-adrenoceptor (alpha(2)-AR) and its relation to alpha(1)-AR, isolated Wistar rat (10 weeks) aortae were used as a model for testing contractile function in vivo. It was found that both alpha(1) and alpha(2)-AR (mainly alpha(1)-AR) mediated the contractile response. While alpha(1)-AR enhanced the contractile response mediated by alpha(2)-AR, alpha(2)-AR had no effect on that mediated by alpha(1)-AR. When alpha(1)-AR was irreversibly blocked and the activity of alpha(2)-AR remained intact, the contractile response mediated by alpha(2)-AR was no longer observed. The contractile response only reappeared in the presence of KCl of the threshold level, and the extent of the maximum contraction decreased, compared with control (with alpha(1)-AR being intact). The results show that there exists a functional alpha(2)-AR in rat aorta, however, the contractile effect of which depends on the stimulation of alpha(1)-AR.

Cell proliferation and Ca(2+)-calmodulin dependent protein kinase activation mediated by alpha 1A- and alpha 1B-adrenergic receptor in HEK293 cells.

AIM: To examine the ability of alpha 1-AR subtypes on proliferation and Ca(2+)-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) activation in transfected human embryo kidney 293 (HEK293) cells. METHODS: pREP8/alpha 1A-AR, pREP4/alpha 1B-AR, and pREP9/alpha 1D-AR were transfected, respectively, into HEK293 cells by calcium phosphate precipitation. The expression of alpha 1-AR was detected by radioligand binding assays. DNA synthesis was measured by [3H]thymidine incorporation. CCDPK activity was determined by immunoprecipitation method and myelin basic protein was used as substrate. RESULTS: Three clonal HEK293 cell lines stably expressing alpha 1A- or alpha 1B- or alpha 1D-AR were chosen and characterized by radioligand binding assay with receptor densities of about 0.6 nmol.g-1. Treatment with norepinephrine (NE) in the presence of propranolol for 24 h increased DNA synthesis in HEK293/alpha 1A- or HEK293/alpha 1B-AR cells concentration-dependently, with EC50 values of 48.8 nmol.L-1 (95% confidence limits 9.7-246 nmol.L-1) and 8.4 nmol.L-1 (95% confidence limits 2.1-32.9 nmol.L-1), respectively. The increase of DNA synthesis induced by NE 10 mumol.L-1 was 201% +/- 28% and 269% +/- 44% of basal, and the activation of CCDPK was 171% +/- 84% and 292% +/- 92% of basal in HEK293/alpha 1A-AR and HEK293/alpha 1B-AR cells, respectively. Preincubation with prazosin completely abolished NE-induced CCDPK activation in HEK293/alpha 1A- and alpha 1B-AR cells. Those changes were not found in HEK293/alpha 1D-AR cells. CONCLUSION: The activation of alpha 1A- or alpha 1B-AR but not alpha 1D-AR induces cell proliferation.

[Antagonistic characterization of 1-(2,6-dimethylphenoxyl)-2-(3,4-dimethylphenyl ethylamino) propane hydrochloride on alpha 1-adrenoceptor].

AIM: To study the antagonistic effect of 1-(2,6-dimethylphenox)-2-(3,4-dimethylphenyl ethylamino) propane hydrochloride (DDPH) on alpha 1-adrenoceptor (AR). METHODS: Radioligand binding assay was used. Specific 125I-BE2254 (2-beta(4-hydroxyphenyl)-ethyl aminomethyl-tetralone) binding was measured by incubating membrane of rat cerebral cortex, spleen and the three cloned alpha 1-AR subtypes (alpha 1A, alpha 1B, alpha 1D) stably expressed in human embryonic kidney 293 cell preparation with a single concentration of 125I-BE2254 in the presence of 14 concentrations of DDPH. Equilibrium binding constant (KI) and Hill coefficients (nH) were determined from Hill plots. The -log value of the KI was expressed as pKI. Contractile responses of isolated rat aorta, renal artery ring and spleen were determined. The pA2 values for DDPH in competitively inhibiting NE-stimulated contraction of tissues were measured with the method of Ainlakshana and Schild. RESULTS: DDPH competitively inhibited binding of 125I-BE2254 to alpha 1-AR in a concentration-dependent manner. The pKI values for DDPH in rat cerebral cortex and spleen were 7.17 +/- 0.06 and 7.41 +/- 0.11, respectively, and the Hill efficiency values were not significantly different from unit. The pKI values for cloned alpha 1A, alpha 1B and alpha 1D-AR were 7.21 +/- 0.12, 6.88 +/- 0.04 and 7.26 +/- 0.06, respectively, and the Hill efficiency values were not significantly different from unit. Contractile studies showed that DDPH competitively antagonized the NE concentration-response curve with a pA2 values of 7.40 +/- 0.23 in aorta, 7.41 +/- 0.04 in renal artery and 7.63 +/- 0.07 in spleen and the slopes of schild plot were not significantly different from unit. The pKI values for DDPH in tissues and the cloned alpha 1A or alpha 1D-AR were shown to fit well in with the pA2 values in antagonizing NE-induced constriction in rat isolated aorta, renal artery and spleen. CONCLUSION: These results suggest that DDPH appear to be a non-subtype selective competitive antagonist for alpha 1-AR.

Characterization of subtype of alpha 1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed.

AIM: To characterize the subtype of alpha 1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed. METHODS: The potencies (pA2 values determined by Schild plot) of alpha 1-adrenoceptor-selective antagonists were determined by isolated vasoconstrictive experiment. The pKi values were determined by 125I-BE 2254 binding from the cloned alpha 1A-, alpha 1B-, and alpha 1D-adrenoceptor, stably expressed in human embryonic kidney (HEK) 293 cells. RESULTS: The pA2 values for alpha 1A-adrenoceptor-selective antagonists, RS-17053, WB 4101, 5-methyl-urapidil, and the alpha 1D-adrenoceptor-selective antagonist, BMY 7378, were 8.98 +/- 0.28, 9.16 +/- 0.20, 8.69 +/- 0.02, and 6.03 +/- 0.26, respectively, with the slope not different from unity. The pA2 values of the above antagonists correlated well with the binding pKi values only for alpha 1A-adrenoceptors (r = 0.97), but not for alpha 1B-adrenoceptors (r = 0.52) and alpha 1D-adrenoceptors (r = 0.04). The concentration-vasopressor response curve for norepinephrine was not affected by pretreatment with chloroethylclonidine (Chl) 50 mumol.L-1 for 30 min. CONCLUSION: Only alpha 1A-adrenoceptors mediate the norepinephrine-induced vasopressor response in perfused rat mesenteric vascular bed.

Tyrosine kinase participates in alpha 1A-adrenoceptor-mediated increase of intracellular calcium in human embryo kidney 293 cells.

AIM: To determine the role of protein-tyrosine kinase (PTK) in alpha 1A-adrenoceptor-mediated increase of [Ca2+]i (intracellular calcium) in human embryo kidney (HEK) 293 cells expressed alpha 1A-adrenoceptor. METHODS: Effects of two PTK inhibitors: genistein and tyrphostin, were investigated on the increase of [Ca2+]i by using Fura-2, The activity of PTK was measured and the accumulation of [3H] InsPs were observed. RESULTS: Norepinephrine stimulated a rapid increase in [Ca2+]i to (371 +/- 31) nmol.L-1 in HEK 293 cells. Norepinephrine-induced increase of [Ca2+]i was inhibited by the tyrosine kinase inhibitors quercetin and tyrphostin by 23.8% and 21.4%, respectively, but the accumulation of [3H]InsPs induced by norepinephrine was not. The activity of the plasma-associated tyrosine kinase was increased to (1.73 +/- 0.72)-fold over the control by norepinephrine 10 mumol.L-1. The norepinephrine-activated PTK was inhibited by calphostin C and depletion of intra- and extra-cellular Ca2+. CONCLUSION: The PTK participates in mobilization of Ca2+ mediated by alpha 1A-adrenoceptors in HEK 293 cell lines.

[Modulatory action of alpha 2-adrenoceptor on calcitonin gene-related peptide release from rat mesenteric arterial bed].

The release of calcitonin gene-related peptide (CGRP) from peripheral terminals of sensory nerves was modulated via multiple mechanisms. In the present study, pharmacological agents were used to investigate the modulatory action of alpha 2-adrenoceptor on endotoxin-induced CGRP release from isolated perfused rat mesenteric arterial bed. The results showed that UK14304 (3 x 10(-6) mol/L), a potent alpha 2-adrenoceptor agonist, significantly inhibited both basal and endotoxin-induced CGRP release by 22%-42%, while specific antagonist of alpha 2-adrenoceptor yohimbine (10(-5) mol/L) blocked the effect of UK14304 completely. The data suggest that presynaptic alpha 2-adrenoceptor has an inhibitory effect on basal and endotoxin-induced CGRP release. Dysfunction of alpha 2-adrenoceptor in the late stage of endotoxic shock may be involved in the excess release of CGRP from the peripheral nerves.

[Pathways of signal transduction of intracellular Ca2+ increase mediated by three subtypes of alpha 1-adrenoceptor in HEK293 cells].

In HEK293 cell lines in which alpha 1a-, alpha 1b- or alpha 1d-adrenoceptor subtypes were stably expressed, fluorescence intensities due to traces of Ca2+ were investigated using fura-2/AM with the aim of exploring signal transduction pathways of intracellular Ca2+ increase mediated by these receptor subtypes. Pertussis toxin had no effect on [Ca2+]i increase mediated by all three subtypes of alpha 1-adrenoceptors. However, U-73122 and PMA could inhibited the [Ca2+]i increase induced by NE, which was not affected by Foskolin and Rp-cAMPs. Pretreatment with calphostin C abolished the [Ca2+]i response to PMA. Quercetin and tyrphostin inhibited maximal [Ca2+]i increase but had no effect on the plateau phase in all the three transfected cells. In the HEK293 cells, the phosphatide-Ca2+ signalling system mediated by the 3 subtypes of alpha 1-AR was brought into play by activation of phospholiphase C via coupling with PTX-insensitive G proteins. PKC system inhibited both mobilization of internal Ca2+ stores and the Ca2+ influx across the plasma membrane. Tyrosine kinase, but not cAMP system, might participate in such Ca2+ signalling.

[Signal transduction of alpha 1-adrenoceptors].

alpha 1-adrenoceptors activate multiple signal transduction pathways. In addition to the classic inositol phosphates-Ca2+ system and diacylglycerol-protein kinase C system, the phospholipase A2-arachidonic acid system, the tyrosine kinase phosphorylation system, the adenylate cyclase-cAMP system etc. were also involved. These signal transduction cascades interact complicatedly with each other as a network. The activation of one signal system can initiate, potentiate or inhibit other systems. They play pivotal role in diversified physiological and pathophysiological processes. Different alpha 1-adrenoceptor subtypes activate different signal transduction pathways. In some experiments, however, the differences are due to the different tissues or cells used.

Tyrosine kinases participate in alpha 1A-adrenoceptor-mediated vasoconstriction in perfused rat hindlimb.

AIM: To determine whether or not tyrosine kinase is involved in the signal transduction of alpha 1A-adrenoceptors. METHODS: Effects of various pharmacological probes on norepinephrine (NE)-induced vasopressor responses were determined in the perfused rat hindlimb. RESULTS: The putative tyrosine kinase inhibitors, genistein, and tyrphostin, significantly inhibited the vasopressor responses induced by NE but not that induced by KCl. The protein-tyrosine-phosphatase inhibitor, sodium orthovanadate, selectively potentiated the vasopressor responses induced by NE. Neither genistein nor tyrphostin had effect on the contraction elicited by phorbol 12-myristate 13-acetate. In contrast, both genistein and tyrphostin attenuated the vasopressor responses evoked by NaF. CONCLUSION: The genistein- and tyrphostin-sensitive tyrosine kinases participate in alpha 1A-adrenoceptor-mediated vasoconstriction in perfused rat hindlimb.

No functional beta 3-adrenergic receptors expressed in rat skeletal muscle cells.

AIM: To determine the functional role of beta 3-adrenoceptors (beta 3-AR) in rat skeletal muscle cells. METHODS: Nonselective beta-AR agonist isoprenaline (isoproterenol, Iso), beta 3-AR agonist CGP12177A which is a beta 1-/beta 2-AR antagonist and selective beta 3-AR antagonist SR59230A on cAMP accumulation was studied in primary cultured rat skeletal muscle cells. RESULTS: Iso stimulated cAMP accumulation in a concentration-dependent manner with EC50 of 1.51 nmol.L-1 and propranolol inhibited cAMP accumulation stimulated by Iso with KB of 3.47 nmol.L-1. CGP12177A had no effect on cAMP accumulation but inhibited cAMP production induced by Iso. SR59230A 10 nmol.L-1 did not inhibit cAMP production induced by Iso. CONCLUSION: The functional beta 3-AR are not present or at least not coupled to adenylyl cyclase activity in skeletal muscle cells.

[Comparison of functional alpha 1-adrenoceptor subtypes in aortae between 12 month- and 10 week-old rats].

The distribution of alpha 1-adrenoceptor and its subtypes in isolated aortae was compared between 12 month- and 10 week-old Wistar rats by determinating vasoconstrictor responses. In the 12 month-old rats, compared with the 10 week-old rats, (1) the maximal contraction induced by norepinephrine (NE) was reduced, without significant alteration of pD2 value; (2) the inhibitory effect of chlorethylclonidine (an irreversible antagonist for alpha 1B and alpha 1D subtype) on NE-induced contraction was weaker; (3) using NE as an agonist, the pA2 value for WB4101 (an alpha 1A- and alpha 1D-selective antagonist) was not changed, but the pA2 value for BMY7378 (an alpha 1D-selective antagonist) was decreased, while the pA2 value for sertindole (an alpha 1A-selective antagonist) was increased. Thus, differently from the 10 week-old rats in which only alpha 1D-adrenoceptors mediate NE-induced contraction in aortae, both alpha 1A- and alpha 1D-adrenoceptors (mainly alpha 1A) mediate the response in the 12 month-old rats.

[Sertindole, a novel alpha 1A-adrenoceptor selective antagonist].

The antagonism effect of sertindole on alpha 1-AR subtypes was studied by combining radiologand binding assays in three cloned alpha 1-AR subtypes stably expressed in human embryonic kidney 293 cells and contractile response experiment in isolated rat blood vessels. The results showed that the affinity for sertindole in the cloned alpha 1A-AR (pKI 8.90 +/- 0.17) was 69-fold (pKI 7.06 +/- 0.09) and 132-fold (pKI 6.78 +/- 0.07) higher than that for the cloned alpha 1B- and alpha 1D-AR, respectively. The pA2 values for sertindole in antagonizing NE-induced vasoconstriction in isolated rat aorta and renal artery were shown to fit well to the pKI values on cloned alpha 1D- and alpha 1A-AR, respectively. Pretreatment of membrane preparations with sertindole for 30 min significantly reduced the maximal binding capacities. (Bmax) of 125 IBE2254 to the three cloned alpha 1-AR subtypes without alteration of affinities (KD values). In the presence of sertindole, the Bmax of 125IBE2254 binding to the cloned alpha 1-ARs were not significantly changed, while the KD values were significantly increased. Thus, sertindole is a selective irreversible competitive alpha 1-AR antagonist with alpha 1A subtype.

[Alteration of cardiac beta-adrenoceptors and subtypes with increasing age].

The alteration of cardiac beta-adrenoceptor (beta-AR) and subtypes in Wistar rats of 48 and 10 weeks old were investigated by radioligand binding assay and functional determination of isolated perfused left atria. The results indicated that: (1) The density of cardiac total beta-AR was downregulated about 28% in 48 weeks old rats as compared with rats of 10 weeks old for both beta 1- and beta 2-AR; (2) The sensitivity of beta-AR and subtypes to isoproterenol in isolated perfused atria was significantly lowered in rats of 48 weeks old; (3) The beta-AR mediated contractile effect of 10 weeks old rat hearts was mainly attributed to beta 1 type, but equally to both beta 1 and beta 2 types in 48 weeks old rat hearts; (4) The two receptor subtypes showed mutually potentiation effect in 10 weeks old rat hearts when stimulated simultaneously, but only additive in 48 weeks old rat hearts.

Chimeric dopamine D2/angiotensin AT1 receptors: role of the length of third intracellular loop of D2 receptors in conferring specificity of receptor binding and G-protein coupling.

AIM: To define roles of the third intracellular loop (IL3) length of G-protein coupled receptors in conferring the specificity for receptor binding and G-protein coupling. METHODS: By polymerase chain reaction (PCR), the IL3 of D2 receptor was replaced with the counter part of AT1 receptor which has the shortest loop among all G-protein coupled receptors. D2/AT1 receptor cDNA was then stably transfected into Chinese hamster ovary cells and a clone with high level expression was obtained for receptor binding and agonist-induced phosphatidylinositols (PI) turnover experiments. RESULTS: Comparing to the D2 receptor, D2/AT1 chimeric receptor had lower affinities for all D2 receptor antagonists tested (spiperone, haloperidol, (+)-butaclamol, chlopromazine, clozapine, trifluoperdazine) and different affinity profiles to agonists (apomorphine, dopamine, quinpirole, bromocriptine). But the chimeric receptor failed to couple to G-protein and subsequent stimulation of PI turnover. CONCLUSION: The length of IL3 of D2 receptor participates defining recpetor binding sites conformation, and structure beyond IL3 may affect receptor G-protein coupling.