Relevance of seminal plasma nitric oxide levels and the efficacy of SSRI treatment on lifelong premature ejaculation.

The aim of this study was to determine the relevance of seminal plasma nitric oxide (NO) levels and the efficacy of selective serotonin reuptake inhibitor (SSRI) treatment on premature ejaculation. A total of 16 men (aged 32.18 ± 3.32) with lifelong premature ejaculation [intravaginal ejaculation latency time (IELT) <1 min] and 11 healthy men (control group) were included in this study. The healthy men formed Group 1, and the patients were randomly categorised into two groups. Group 2 patients received 20 mg day(-1) of paroxetine, and Group 3 patients received 50 mg day(-1) of sertraline for 4 weeks. Baseline and post-treatment findings were compared among the three groups. Mean baseline seminal NO levels in men with premature ejaculation were significantly higher than in the healthy control group (32.24 ± 5.61 µm l(-1) versus 19.71 ± 3.50 µm l(-1) , respectively) (P < 0.001). There was no significant difference between the sertraline and paroxetine groups in terms of IIEF scores, IELT scores and NO levels. At the end of the first month, the mean IELT scores of the paroxetine and sertraline groups showed a significant improvement compared with the baseline values (P < 0.001). After treatment with paroxetine and sertraline, NO levels dec-reased from baseline. Our study indicates that premature ejaculation is significantly related with a higher level of seminal NO. Baseline seminal plasma NO values obtained in patients with premature ejaculation were significantly higher than in the healthy control group. After treatment with SSRIs, decreased seminal NO may retard ejaculation. Further studies are needed to confirm this suggestion and the role of NO in the pathophysiology and treatment of premature ejaculation.

α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor.

BACKGROUND AND PURPOSE: High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α(1) -adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α(1) -adrenoceptors. EXPERIMENTAL APPROACH: Thoracic aorta was isolated from 12-14 week old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine were characterized in the absence and presence of the EGFR kinase inhibitors, AG1478 and DAPH, in intact and endothelium-denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, matrix metalloproteinase (MMP), extracellular signal-regulated kinase (ERK)1/2 and phosphatidyl inositol 3-kinase (PI3K). Phosphorylation of EGFR and ERK1/2 was measured after short-term phenylephrine or EGF stimulation in aorta segments in the presence of AG1478 and the PI3K inhibitor, wortmannin. KEY RESULTS: AG1478 and DAPH concentration dependently attenuated phenylephrine-induced contractile responses in intact or endothelium-denuded aortic rings. Inhibition of PI3K (wortmannin and LY294002) but not heparin or inhibitors of Src or MMP, prevented the effect of AG1478 on the responses to phenylephrine. Phenylephrine induced phosphorylation of EGFR, which was partially blocked by AG1478. Phenylephrine also increased phosphorylation of ERK1/2, time-dependently and was blocked by AG1478 and wortmannin. CONCLUSIONS AND IMPLICATIONS: Contractions of rat thoracic aorta mediated by α(1) -adrenoceptors involved transactivation of EGFR, mediated via a PI3K and ERK1/2 dependent pathway.

The effect of long-term resveratrol treatment on relaxation to estrogen in aortae from male and female rats: role of nitric oxide and superoxide.

Resveratrol, which is found in several foods, has vasorelaxing and estrogen-like activities. The aim of the present study was to determine whether the relaxation to estrogen is differently modified between male and female genders after long-term resveratrol treatment. To test this, we compared endothelium-dependent and -independent relaxations to estrogen in the aortae of control and resveratrol-treated male and female rats. Nitric oxide and superoxide levels were also evaluated to explain the mechanism of action of resveratrol. Concentration-response curves to estrogen (10(-10)-10(-4) M) were obtained in aortic rings with and without endothelium from control or long-term resveratrol-treated (50 mg/l in drinking water for 21 days) male and female rats. Estrogen produced mainly endothelium-dependent relaxation in aortic rings of rats, with a higher potency in females than males. Resveratrol treatment increased both endothelium-dependent and -independent relaxations to estrogen especially in aortae from males. The relaxations to estrogen in the aortae of resveratrol-treated rats were inhibited, almost to the same extent as those of control, by pretreatment with ICI 182,780 (10(-6) M), an estrogen receptor antagonist. In both genders, resveratrol treatment increased basal nitric oxide and nitrite/nitrate productions and decreased both basal and NAD(P)H-induced superoxide productions in the aortae. In addition, plasma estrogen levels were found decreased in long-term resveratrol-treated animals of both genders. The improvement in the relaxations to estrogen observed in resveratrol-treated animals could be related to elevated nitric oxide and/or decreased superoxide productions and possibly mediated by classical estrogen receptors. The modulating effect of resveratrol on estrogen responsiveness may differ between male and female.

Effect of Candida albicans septicemia on the cardiovascular function of rabbits.

Candida albicans is an opportunistic pathogen that causes life-threatening systemic infection in immunocompromised host. However, little is known about the effects of yeast on the cardiovascular functions. This study examined the effects of C. albicans septicemia on the heart and vessel functions and nitric oxide (NO) production in infected rabbits. Anaesthetized animals were challenged with intravenous C. albicans (6 x 10(8)/kg) or saline and the blood pressure of rabbits were measured over 5 h. After that response of the isolated thoracic aorta, right atrium and left papillary muscle were recorded. Blood pressure significantly decreased in the infected rabbits during the septicemia but in the control animals it was stable. The blood nitrite levels and NO-synthases (eNOS, iNOS) expression and tissue nitrite levels in the heart and aorta were similar in the both groups. In the aorta isolated from C. albicans-infected rabbits, acetylcholine-induced endothelium-dependent relaxation was decreased, but contractions induced by phenylephrine were potentiated. The NOS inhibitor, L-N(G)-nitro-arginine methyl ester (L-NAME)-induced contraction increase in the right atrium was depressed by the yeast-infection. In the heart and aorta, microscopic examination revealed no tissue invasion of C. albicans. These results indicate the ability of C. albicans-induced septicemia to destroy NO-related responses of the heart and aorta and may have important implications for functional damage to endothelium and the regulation of cardiovascular functions. In addition, NOS induction and NO over-production are not stimulated by systemic C. albicans infection, which would alter the host immune reaction and homeostasis.

Quasi-irreversible binding of agonist to beta-adrenoceptors and formation of non-dissociating receptor-G(s) complex in the absence of guanine nucleotides.

Here, we tested the hypothesis that receptor-G protein and agonist may form an irreversible complex in the absence of guanine nucleotides. We used the beta-adrenoceptor-G(s) system of guinea pig lung parenchymal membranes as a model. Two groups of membranes were used in the experiments: (1) washed with nucleotide-free buffer in the presence of isoproterenol (isoproterenol-treated), and (2) washed with buffer alone or with agonist+GDP (both were treated as control). Results were as follows: (1) the iodopindolol binding capacity of isoproterenol-treated membranes was reduced by about 30%. (2) No such reduction was observed in control membranes. (3) Addition of GDP to the isoproterenol-treated membranes completely restored the pindolol binding capacity. We interpreted this result as indicating irreversible agonist-receptor complex is formed when the receptor interacts with nucleotide-free G(salpha). (4) We observed a single peak of beta(2)-adrenoceptor activity in the control group by size-exclusion chromatography of the solubilized membranes. Inclusion of isoproterenol in the washing buffer led to an additional (heavier) peak of beta(2)-adrenoceptor activity. This peak disappeared when GDP was added to the detergent extract before high-pressure liquid chromatography (HPLC) analysis. Western blot analysis of these HPLC fractions showed that the agonist-induced heavier peak contained significantly more G(salpha) protein than did the other fractions. We interpreted this result as indicating that a practically irreversible complex of receptor and G protein is formed in the absence of GDP. We suggest that the tightly bound (nucleotide-free) receptor-G protein complex also contains the agonist, and that this complex can be reversed only by the addition of nucleotides. The implications of these results are also discussed.

Short-term effects of three continuous hormone replacement therapy regimens on platelet tritiated imipramine binding and mood scores: a prospective randomized trial.

OBJECTIVE: To evaluate the effects of continuous hormone replacement therapy (HRT) regimens on platelet-tritiated ((3)H-) imipramine binding (Bmax) and mood. DESIGN: Prospective randomized study. SETTING: University hospital. PATIENT(S): Sixty postmenopausal patients. INTERVENTION(S): Randomization to 3 months of daily treatment with tibolone and conjugated equine estrogen (CEE).625 mg combined either with 2.5 or 5 mg of medroxyprogesterone acetate (MPA). The inclusion criteria-matched patients declined for HRT were prescribed daily alendronate. Pre- and posttreatment blood sampling for Bmax and mood evaluation with the Beck Depression Inventory (BDI) and the State-Trait Anxiety Inventory (STAI) were done. MAIN OUTCOME MEASURE(S): Pre- and posttreatment Bmax and mood scores. RESULT(S): As compared with baseline, both CEE+MPA regimens and tibolone significantly increased Bmax. The comparisons of percent change from baseline Bmax for the CEE+MPA and tibolone groups were similar. All three HRT regimens improved the BDI significantly, while there were no significant changes in the STAI. In the alendronate group, there were no significant changes in both pre- and posttreatment Bmax and mood scores. CONCLUSION(S): Continuous treatment with CEE+MPA and tibolone increases platelet (3)H-imipramine binding and improves mood. Mood-enhancing effects of tibolone may occur through the serotonergic system, as is the case with estrogen.

Spleen damage in endotoxaemic mice: the involvement of nitric oxide.

The association between Escherichia coli endotoxin-induced organ damage and nitric oxide-related mechanisms was investigated in the spleen of male Swiss albino mice (20-40 g) by using (1) Pt/Ir electrochemical sensor connected to an amperometric detection system (NO-501, InterMedical Co., Japan), (2) nitrotyrosine immunohistochemistry, (3) conventional light microscopy and (4) immunoblotting techniques in parallel. 1 h before endotoxin injection, animals were pretreated with either nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME, 20 mg kg(-1), i.p.) or inducible nitric oxide synthase expression inhibitor, dexamethasone (5 mg kg(-1), i.p.) or the inhibitor of murine inducible nitric oxide synthase in vivo, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 1 mg kg(-1), i.p.). 5 h after endotoxin treatment, electrochemically detected concentration of nitric oxide was significantly elevated (nM, endotoxin: 716.6 +/- 178.2, n = 10 vs saline: 209.4 +/- 127.8, n = 9, P = 0.0312, unpaired Student's t-test) and remained so throughout the 30 min monitorization period. Neither dexamethasone nor AMT blocked the endotoxin-induced overproduction of nitric oxide indicating that the enhanced inducible nitric oxide synthase activity cannot be the only explanation. When dexamethasone and L-NAME combination was used to block both the constitutive and the inducible isoforms, nitric oxide production was virtually abolished, indicating a significant contribution from the constitutive isoform of nitric oxide synthase. The results of nitrotyrosine immunohistochemistry and the conventional light microscopy were also in agreement with the amperometric method while immunoblotting revealed the expression of both the endothelial and the inducible isoforms of nitric oxide synthase were induced endotoxaemic animals. Thus, conclude that endotoxin-induced splenic damage in endotoxaemia can be explained by enhanced production of nitric oxide due to the induction of both endothelial and inducible nitric oxide synthases while causal relationship and the roles of other deleterious mediators such as oxygen-derived free radicals are yet to be established.

The effects of agonist stimulation and beta(2)-adrenergic receptor level on cellular distribution of gs(alpha) protein.

This study examines the effects of adrenergic ligands, cholera toxin, forskolin, and varying levels of beta(2) adrenergic receptors (beta(2)AR) on the cellular distribution of Gs(alpha) subunits in CHO cells. Localization of Gs(alpha) was evaluated by confocal microscopy and beta(2)AR-mediated signalling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0.2 pmol/mg protein beta(2)ARs (WT18), the localization of Gs(alpha) subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gs(alpha) so that Gs(alpha) appeared as intense spots throughout the plasma membrane as well as the cytoplasm. Exposure to a neutral beta(2)AR antagonist, alprenolol, prevented the ISO-stimulated Gs(alpha) translocation from peripheral to inner cytoplasm. In cells expressing high level of beta(2)ARs (8.2 pmol/mg) (WT4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive correlation between receptor expression and receptor-mediated signalling. Basal Gs(alpha) distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gs(alpha) significantly when tested in WT4 clone. No difference in the cellular level of Gs(alpha) protein between WT18 and WT4 clones was detected. Alprenolol did not affect the distribution of Gs(alpha) in WT4 clone. ICI 118,551, a negative beta(2)AR antagonist, altered Gs(alpha) distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that observed in unstimulated WT18 clone. Taken together, these data suggest that: (1) enhanced beta(2)AR-Gs(alpha) coupling induced by agonist stimulation or by increased expression of beta(2)ARs remodel the cellular distribution of Gs(alpha); (2) the alteration in Gs(alpha) distribution induced by beta(2)AR overexpression provides evidence for agonist-independent interaction of beta(2)AR and Gs(alpha), that can be inhibited by a negative antagonist but not by a neutral antagonist; and (3) forskolin influences the activity state of Gs(alpha) that displays a Gs(alpha) distribution pattern comparable to that observed when Gs(alpha) is activated via beta(2)AR stimulation or directly by cholera toxin.

Dietary selenium and vitamin E intakes alter beta-adrenergic response of L-type Ca-current and beta-adrenoceptor-adenylate cyclase coupling in rat heart.

Previously we have shown that both insufficient (combined with vitamin E deficiency) and excess intake of selenium (Se) impairs isoproterenol (ISO)-induced contractions of rat papillary muscle. In the present study, we used patch-clamp and biochemical techniques to investigate mechanisms of this effect in rats fed a Se- and vitamin E-deficient, a Se-excess or a normal diet. Whole-cell configuration of patch-clamp technique was used to investigate L-type Ca(2+) currents (I(Ca,L)) and their regulation by beta-adrenergic receptor stimulation in enzymatically isolated single rat ventricular myocytes. Alteration of Se and vitamin E intake did not affect peak I(Ca,L), but the threshold potential of activation was significantly different among groups. Maximal I(Ca,L) responses to ISO were depressed in both experimental groups, but the EC(50) values were not affected. In the Se-deficient group, basal, ISO- or forskolin-induced adenylate cyclase (AC) activity, measured in cardiac membrane preparations, was reduced when compared to the control, whereas 5' guanylyimidodphosphate (GppNHp) stimulated activity was unaffected. Decreased beta-adrenoceptor density and reduced GppNHp-induced affinity shift in ISO binding were also observed in the deficient group. No such differences were present in the excess group. These results suggest that combined Se and vitamin E deficiency interferes with beta-adrenoceptor-AC coupling, whereas excess intake of Se does not affect it. Thus, in the deficient group, the impairment of I(Ca) responses to ISO may be a result of a defect in beta-adrenoceptor-AC pathway. Impairment of I(Ca) response in the excess group, however, appears to have a different underlying mechanism.

Inverse agonist properties of dopaminergic antagonists at the D(1A) dopamine receptor: uncoupling of the D(1A) dopamine receptor from G(s) protein.

The interaction of dopaminergic antagonists with the D(1A) dopamine receptor was assessed in PC2 cells that transiently express this receptor. The maximal binding and dissociation constants for the D(1A) dopamine receptor, using the ligand [(125)I]SCH23982 were 0.38 +/- 0.09 nM and 1 to 4 pmol/mg, respectively, when assessed 48 h after transfection with cDNA encoding the rat D(1A) receptor. Basal adenylyl cyclase activity increased 50 to 60% in membranes of transfected PC2 cells compared with control membranes. The dopaminergic antagonists clozapine, cis-flupenthixol, (+)-butaclamol, haloperidol, chlorpromazine, and fluphenazine inhibited constitutive adenylyl cyclase activity in membranes of cells expressing the D(1A) receptor. SCH23390, a selective D(1) dopamine receptor antagonist, and (-)-butaclamol did not alter basal cyclase activity, whereas dopamine increased enzyme activity in membranes expressing the D(1A) dopamine receptor. The coupling of D(1A) receptors with G(s) proteins was examined by immunoprecipitation of membrane G(salpha) followed by immunoblotting with a D(1A) dopamine receptor monoclonal antibody. Clozapine, cis-flupenthixol, (+)-butaclamol, haloperidol, and fluphenazine but not SCH23390 or (-)-butaclamol decreased D(1A) receptor-G(salpha) coupling by 70 to 80%, and SCH23390 was able to prevent the receptor-G(salpha) uncoupling induced by haloperidol or clozapine. These results indicate that some dopaminergic antagonists suppress basal signal transduction and behave as inverse agonists at the D(1A) dopamine receptor. This action of the dopamine receptor antagonists may contribute to their antidopaminergic properties that seem to underlie their clinical actions as antipsychotic drugs.

Ligand efficacy and affinity in an interacting 7TM receptor model.

Quantitative understanding of the activation of G protein-coupled receptors is based mostly on some theoretical models that describe the interaction between ligand and protein partners and the activation process of the receptor. All of these models provide different definitions for observable affinity or efficacy. However, the property common to such parameters defined in the context of these models is that they are always independent of the concentration of the receptor molecule. This is based on the assumption that receptors do not interact with each other appreciably. In this article, experimental evidence for which this assumption does not seem to apply is discussed and an oligomerization model for seven-transmembrane-domain receptors that explains the relationship between receptor concentration, apparent affinity and efficacy is provided.

Decrease in apparent alpha1-adrenoceptor-G protein coupling during maturation in rat aorta.

We have previously shown that the adrenoceptor agonist norepinephrine (NE) is more potent in eliciting contraction in aortas from 1-month-old Fischer 344 rats than it is in older animals. In the present study, we examined alpha1-adrenoceptor-guanine nucleotide regulatory binding protein (G protein) coupling in aortic membranes in order to investigate the mechanism for the age-dependent reduced responsiveness of aorta to NE. We used the guanosine 5'(betagamma-imido)triphosphate (Gpp[NH]p)-induced shift in agonist binding affinity as a measure of the efficiency of alpha11-adrenoceptor-G protein coupling. The binding of NE was assessed by measuring the displacement of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl] tetralone ([125]-HEAT) by NE in aortic membranes. In 1-, 6-, and 24-month-old rat aortas, two apparent binding sites were detected in the competition isotherms for NE. This heterogeneous binding pattern was independent of Gpp(NH)p at all ages, and is likely to be due to a heterogeneous receptor population (alpha(1a), alpha(1b), and alpha(1d) subtypes). In 1-month-old rats, the high affinity binding of NE to alpha,-adrenoceptors was sensitive to Gpp(NH)p, indicating a significant interaction between the receptor and G protein. This Gpp(NH)p-sensitive high affinity binding was not observed in aortas from 6- or 24-month-old animals. Despite the lack of Gpp(NH)p-sensitive high affinity binding of agonist in 6- or 24-month-old aortas, NE was still able to induce maximal contraction in these aortas, albeit, with a relatively low potency. A partial reduction in alpha1-adrenoceptor-G protein coupling between 1 and 6 months of age can explain the observed decrease in ago- nist potency and the loss of Gpp(NH)p-sensitive high affinity binding of NE. This phenomenon can be explained as a reduction of allosteric coupling between the bindings of ligand and G protein to the receptor, that has been formulated in the ternary complex model. Computer simulation using the simple ternary complex model shows that manipulating the reciprocal coupling factor alone can lead to a loss of Gpp(NH)p-sensitive high affinity agonist binding, along with a reduction in agonist potency for contraction without altering the maximal response. Thus, a change in the relative expression of different alpha,-adrenoceptor subtypes, which we have previously observed in the aorta, and which possess diverse intrinsic allosteric couplings, may be speculated to be the mechanism for the apparent reduction of alpha,-adrenoceptor-G protein coupling during maturation.

Heterologous desensitization of the rat tail artery contraction and inositol phosphate accumulation after in vitro exposure to phenylephrine is mediated by decreased levels of Galphaq and Galphai.

Desensitization of alpha-1 adrenoceptor (alpha1AR)-mediated responses in aortic smooth muscle after exposure to catecholamines or alpha1AR agonists has been widely demonstrated. To determine whether exposure to an alpha1AR agonist results in desensitization of alpha1AR-mediated responses in a resistance artery, rat tail artery rings were exposed to 7.5 or 75 microM phenylephrine (PE) for 22 hr in vitro. Norepinephrine-stimulated contraction was significantly reduced in PE-exposed tail artery rings. Contractions mediated by the alpha2AR agonists, clonidine and UK 14,304, and by serotonin were also reduced in PE-treated tail artery rings. However, the contractile responses to KCl and ionomycin remained unchanged. Norepinephrine-, PE-, endothelin- and serotonin-stimulated inositol phosphate accumulations were reduced in PE-exposed tail artery rings, whereas KCl- and ionomycin-stimulated inositol phosphate accumulation remained unchanged. The density of membrane alpha1ARs, measured by specific [125I]2-([beta-(4-hydroxyphenyl)ethyl]aminomethyl)-1-etralone binding was not changed in PE-desensitized tail arteries. Further studies were performed to examine if alterations in receptor/G protein interaction accompanies arterial desensitization. In these studies receptor-stimulated increases in [35S]GTPgammaS binding to G proteins was assessed in membranes obtained from vehicle (control) and PE-treated tail arteries. In control membranes alpha1AR stimulation increased [35S]GTPgammaS binding to Galphaq and Galphai proteins, whereas the alpha2AR agonist UK14,304 activated [35S]GTPgammaS binding to Galphai exclusively. Both PE- and UK14, 304-induced responses were reduced in membranes from tail arteries that were exposed to either 7.5 or 75 microM PE for 22 hr. Western blot analyses of G protein alpha and beta subunits demonstrated that Galphaq and Galphai protein levels were decreased in PE-exposed tail artery membranes. These data show that the reduced transmembrane signaling for the alpha1AR in tail artery after in vitro PE exposure is associated with decreases in Galphaq and Galphai protein levels. The reduction in these Galpha proteins also appears to mediate the loss of function of alpha2AR and perhaps of other G protein-coupled receptors.

An efficacy-dependent effect of cardiac overexpression of beta2-adrenoceptor on ligand affinity in transgenic mice.

In previous studies, it was shown that the overexpression of beta2-adrenoceptor (beta2AR) in the hearts of transgenic mice (Tg) leads to agonist-independent activation of adenylate cyclase and enhanced myocardial function. Here, we measured the physical coupling of beta2AR and Gs by evaluating the coimmunoprecipitation of beta2AR and Gs and the ligand binding properties of beta2AR in the hearts of Tg mice to investigate the details of the interaction among ligand, receptor, and G protein. The following results were obtained: (i) coimmunoprecipitation of beta2AR and Gs was increased in the absence of agonist in Tg mice compared with the control animals. This demonstrates directly the increased interaction between unliganded beta2AR and Gs, which is consistent with increased background cAMP production and cardiac function in the hearts of Tg mice. (ii) Guanosine-5'-(beta,gamma-imido)triphosphate abolished the association of beta2AR/Gs in the immunoprecipitate. (iii) The affinities for ligands that show agonist (isoproterenol, clenbuterol, and dobutamine), neutral antagonist (alprenolol and timolol), and negative antagonist (propranolol and ICI 118551) activities in this experimental system were increased, not changed and decreased, respectively, in Tg mice compared with the controls. (iv) This efficacy-dependent alteration in ligand affinities was still observed in the presence of a guanosine-5'-(beta,gamma-imido)triphosphate concentration that abolishes beta2AR/Gs coupling. This suggests that the altered beta2AR binding affinities in Tg mice are not due to the increased interaction between beta2AR and Gs. These data cannot be explained by using ternary, quinternary, two-state extended ternary, or cubic ternary complex models. We therefore discuss the results using a "two-state polymerization model" that includes an isomerization step for the conversion of receptor between an inactive and an active form (denoted as R and R*, respectively) and a polymerization of the active state (R*n). The simplest form of this model (i.e., noncooperative dimerization of the receptor) is found to be consistent with the experimental data.

Desensitization of norepinephrine receptor function is associated with G protein uncoupling in the rat aorta.

Infusion of norepinephrine (NE) in rats results in desensitization of NE-mediated aortic contraction and a reduction (55% at 1 and 10 microM) in NE-stimulated vascular inositol phosphate accumulations. The functional responses to angiotensin II (ANG II) were also reduced in the tissues of NE-infused animals. alpha 1-Adrenoceptor number determined by 2-[beta-(4-hydroxy-3-[125I]iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) binding and levels of G alpha or G beta proteins measured by immunoblot analyses were not changed in the aortic membranes of NE-infused animals. To determine whether desensitization is associated with receptor-G protein uncoupling, agonist-stimulated palmitoylation of G alpha proteins was measured. NE infusion decreased phenylephrine (1 microM)-stimulated [3H]palmitate incorporation into Gq alpha, Gs alpha, and Gi alpha proteins and ANG II (10 microM)-stimulated palmitoylation of Gq alpha and Gi alpha in aortic membranes. Phenylephrine- and ANG II-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) binding to Gq alpha was also decreased in the aortas of NE-infused animals. These results show that an infusion of NE causes heterologous desensitization of the contractile and inositol phosphate accumulation responses in the rat aorta and that these changes are mediated by an uncoupling of receptors from their G proteins.

Role of G alpha q or G alpha o proteins in alpha 1-adrenoceptor subtype-mediated responses in Fischer 344 rat aorta.

Previous studies showed that alpha-adrenoceptor (AR) stimulation with norepinephrine is more potent at eliciting contraction in aortas from 1-month-old Fischer 344 rats than from older rats and that this response is mediated by alpha 1b- and alpha 1d-AR subtypes in 1-month-old rats. We examined the G proteins responsible for alpha 1-AR-mediated contractile response and inositol phosphate accumulation in the aortas of 1-month-old Fischer 344 rats. Pertussis toxin (PTX) treatment (2.5 micrograms/ml for 4 hr) of aortic rings partially inhibited phenylephrine (PHE)-stimulated contraction and inositol phosphate accumulation, suggesting the involvement of PTX-sensitive and -insensitive G proteins. Specific antisera directed against G alpha q and G alpha o but not G alpha s and G alpha i precipitated specific alpha 1-AR binding sites labeled with 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]tetralone. The number of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]tetralone binding sites precipitated by G alpha proteins was increased by activating membrane alpha 1-ARs with PHE. Moreover, PHE stimulated the palmitoylation of G alpha q and G alpha o, and this response was blocked by the alpha 1-AR antagonist prazosin. Characterization of the alpha 1-AR subtypes that couple to G proteins indicates that although aortic alpha 1a-, alpha 1b-, and alpha 1d-ARs were associated with G alpha q, alpha 1b-AR was also linked to G alpha o. These results suggest that alpha 1-ARs mediate the contractile response in rat aorta by coupling to both Gq protein and the PTX-sensitive G(o) protein.

Cardiovascular hypertrophy and increased vascular contractile responsiveness following repeated cocaine administration in rabbits.

The effects of repeated cocaine administration on contractile responses were studied in adult rabbits. Repeated cocaine exposure caused a significant increase in the maximal response of the aorta to the agonists norepinephrine and serotonin as well as the receptor- independent stimulus KCl when compared to the saline controls. Cocaine exposure caused a significant increase in the wet weights of both heart and aorta. When the contraction was normalized to the wet weight of the aorta there was no difference between rabbits administered cocaine and saline. Acute cocaine administration caused a time-dependent increase in immunoreactivity of the proto-oncogene c-Fos in the aorta. These results show that repeated cocaine administration leads to the development of cardiovascular hypertrophy.

The expression of alpha 1 adrenoceptor subtypes changes with age in the rat aorta.

Previous studies showed that alpha 1 adrenoceptor-mediated contractile responses change with age in the rat aorta, becoming more sensitive to Ca++ channel blockers and less sensitive to chlorethylclonidine (CEC), suggesting a change in the alpha 1 adrenoceptor subtypes that are present. In this study, alpha 1 adrenoceptor density and alpha 1 adrenoceptor subtypes were measured in the Fischer 344 rat aorta during aging. Aortic alpha 1 adrenoceptor densities, determined by saturation binding of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl] tetralone ([125I]-HEAT), were 47, 41 and 45 fmol/mg protein in 1-,6- and 24-month-old rats, respectively. The noncompetitive antagonist CEC completely blocked [125I]-HEAT binding in aortas from 1-month-old rats but inhibited binding only partially in aortas from older rats. Two binding sites were detected for norepinephrine and for WB4101 in all ages. The low-affinity constants for WB4101 (31-51 nM) were consistent with those for the alpha 1b adrenoceptor subtype, and this binding site decreased with age. The high-affinity constant for WB4101 (1.4 nM) in 1-month-old aorta was consistent with that for alpha 1d adrenoceptor subtype, whereas the high-affinity constants (0.03 nM) in 6- and 24-month-old aortas were consistent with those for the alpha 1a adrenoceptor subtype. At least three alpha 1 adrenoceptor subtypes appear to be colocalized in the rat aorta, so the binding affinities may reflect binding to more than one subtype. This makes it difficult to identify denfinitively the subtypes based on their radioligand binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenoceptor-G alpha S coupling decreases with age in rat aorta.

beta-Adrenoceptor (beta AR) responsiveness, receptor density, receptor-G protein coupling, and the possible role of membrane fluidity in receptor-G protein coupling were investigated in the rat aorta with age. The beta AR agonist isoproterenol (ISO) produced relaxation of KCl-induced aortic contractions by 97%, 21%, and 0% in aortae from 1- 6-, and 24-month-old Fischer 344 rats, respectively. Forskolin completely relaxed the contractions at all ages. beta AR density was determined in aortic membranes by saturation binding of 125I-cyanopindolol (125I-CYP). beta AR density was 76, 52, and 47 fmol/mg of protein in 1-, 6-, and 24-month-old rats, respectively. To investigate beta AR coupling to G proteins, displacement by ISO of 125I-CYP binding was determined in aortic membranes in the presence and absence of the GTP analog guanosine-5'-(beta gamma-imido)triphosphate [Gpp(NH)p] (0.1 mM). The effect of Gpp(NH)p on the ISO displacement curve for 125I-CYP binding was greatest in 1-month-old rats and decreased markedly with age. In 1-month-old aorta, in the absence of Gpp(NH)p the ISO displacement curve was biphasic and two affinity constants were determined (KH - 0.061 microM and KL = 2.4 microM). In the presence of Gpp(NH)p the ISO displacement curve was monophasic (Kd - 0.72 microM). In 6-month-old aorta, whereas an effect of Gpp(NH)p on the ISO displacement curve could still be observed [in the absence of Gpp(NH)p, KH = 0.2 microM and KL = 3.5 microM; in the presence of Gpp(NH)p, Kd - 0.83 microM], the affinity constant for high affinity agonist binding and the percentage of receptors with high affinity for agonist were decreased significantly. In 24-month-old aorta there was no effect of Gpp(NH)p on the ISO displacement curve and a single affinity constant was detected [0.7 microM and 0.8 microM in the presence and absence of Gpp(NH)p, respectively]. The presence of two affinity constants for ISO in 1- and 6-month-old aorta in the absence of Gpp(NH)p and single affinity constants in the presence of Gpp(NH)p presumably represent the G protein-coupled and uncoupled states of the beta ARs, which are not observed in 24-month-old aorta. The ability of the beta AR to form the high affinity nucleotide-sensitive complex with the agonist was restored by treatment of the membranes with cis-vaccenic acid, which increases the fluidity of the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)