Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Expression of the human beta(3)-adrenergic receptor gene in SK-N-MC cells is under the control of a distal enhancer.

Mechanisms of transcriptional regulation of the human beta(3)-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses beta(3)- and beta(1)-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5'-flanking region of the human beta(3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act together to achieve full transcriptional activity. Mutational analysis, antibody supershift, and electrophoretic mobility shift assay competition experiments indicated that element A binds the transcription factor Sp1, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-MC and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positive cis-regulatory elements located 6.5 kb upstream from the translation start site may play an important role in transcriptional regulation of the human beta(3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-specific expression of the beta(3)-adrenergic receptor gene.

Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells.

Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.

Receptor-independent activators of heterotrimeric G-proteins.

Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif. Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves. Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1 defines a distinct member of the super family of ras related proteins. AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways. AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs. The GPR motifs are found in a number of proteins that interact with and regulate Galpha. Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways.

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. III. Coupling of alpha 2-adrenergic receptor subtypes in a cell type-specific manner.

A number of diverse signaling pathways can be activated by G-protein coupled receptors. However, the factors involved in selection of a particular transduction pathway by a single receptor are not well understood. We are attempting to address this issue utilizing the alpha 2-adrenergic receptor (alpha 2-AR) subfamily as a representative model system. In this report, we demonstrate that the cellular response mediated by an alpha 2-AR subtype is cell-specific and thus depends on its environment. Receptor coupling to adenylylcyclase was determined following stable expression of the rat alpha 2B- and alpha 2D-AR subtypes in three functionally distinct cell types (NIH-3T3 fibroblasts, DDT1 MF-2 smooth muscle cells, and the pheochromocytoma cell line PC-12). When the receptor subtype gene is expressed in NIH-3T3 and DDT1 MF-2 cells, receptor activation inhibits basal and forskolin-induced increases in cellular cAMP. However, in PC-12 transfectants the same receptor subtype actually increases basal cAMP and augments the effect of forskolin. Potentiation of the forskolin effect in PC-12 cells is insensitive to pertussis toxin but is blocked by loading the cells with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid) which minimizes changes in Ca2+i by calcium chelation. These data and the functional demonstration of a Ca2+/calmodulin-sensitive adenylylcyclase in PC-12 but not NIH-3T3 and DDT1 MF-2 cells, suggests that the cell-specific effects of epinephrine are due to receptor coupling to both different G-proteins and types of adenylylcyclase.

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. I. Coupling of alpha 2-adrenergic receptor subtypes to distinct G-proteins.

alpha 2-Adrenergic receptor (alpha 2-AR) subtypes couple to pertussis toxin (PT)-sensitive G-proteins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G-proteins with different affinity. To address this issue we stably expressed three alpha 2-AR subtypes, RNG alpha 2 (alpha 2B-AR), RG10 (alpha 2C-AR), and RG20 (alpha 2D-AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (Gi alpha 2, Gi alpha 3), and analyzed receptor/G-protein interactions by determining: 1) functional coupling to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In alpha 2D-AR transfectants expressing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 microM) inhibited forskolin-induced elevation of cellular cAMP by 26 +/- 4.8% and 72 +/- 6.2%, respectively. Similar results were obtained in alpha 2B-AR transfectants. However, in alpha 2C-AR transfectants (200 fmol/mg) the forskolin-induced elevation of cellular cAMP was not altered by agonist treatment. In alpha 2C-AR transfectants expressing higher receptor densities (650-1,200 fmol/mg), epinephrine inhibited the effect of forskolin by 30 +/- 3.2%. This difference in functional coupling among the alpha 2-AR subtypes is reflected at the receptor/G-protein interface. In membrane preparations of alpha 2B and alpha 2D-AR but not alpha 2C-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl-5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the alpha 2C-AR differs from the alpha 2B and alpha 2D-AR subtypes in its ability to recognize PT-sensitive G-proteins expressed in NIH-3T3 fibroblasts. The alpha 2C-AR may couple preferentially to PT-sensitive G-proteins (Gi1, Go1,2) not expressed in NIH-3T3 fibroblasts and thereby elicit different cellular responses.

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. II. Preferential coupling of the alpha 2C-adrenergic receptor to the guanine nucleotide-binding protein, Go.

Cell to cell communication by many hormones and neurotransmitters involves three major entities: receptor (R), G-protein (G), and effector molecule (E). Plasticity in this system is conferred by the existence of each entity as isoforms or closely related subtypes that are expressed in a tissue-specific and developmentally regulated manner. Factors that determine signal specificity in this system are poorly understood. Such factors include the relative affinity and stoichiometry of R-G or G-E and the possible colocalization of R-G-E in cellular microdomains. Utilizing the alpha 2-adrenergic receptor (alpha 2-AR) system as a representative subfamily of this class of signal transducers, we determined the relative importance of these factors. By analysis of R-G coupling in mammalian cells cotransfected with alpha 2-AR genes and G alpha cDNA, we demonstrate preferential coupling between an alpha 2-AR subtype and Go. Our data implicate R-G affinity as an important determinant of signal transduction specificity and indicate that a critical level of Go alpha is required for coupling. This report indicates the utility of R-G cotransfection in mammalian cells as a "natural environment model" to characterize events occurring at the R-G and G-E interface.

Liver blood flow during haemorrhagic shock in the dog treated with phenoxybenzamine.

The liver blood flow has been extensively studied in hemorrhagic shock, but considerable disagreement exists as to the nature of hemodynamic changes and their controlling mechanism. The present investigation was undertaken in order to determine the effects of hemorrhage and phenoxybenzamine (PBZ) on the participation of hepatic artery (HAF) and portal vein flow (PVF) in total liver blood flow (LBF) changes. The dynamics of LBF (H2 washout method), HAF and PVF (electromagnetic flowmeter) during 3-hours posthemorrhagic hypotension (90 min. = 50-60 mmHg; 90 min. = 25-30 mmHg) and one-hour postretransfusion period were investigated on 20 mongrel dogs under chloralose anesthesia. All animals were divided into 2 groups (control and PBZ-treated--5 mg/kg b.w. 30 minutes following first bleeding). Half an hour following bleeding there occurred a significant decrease of LBF (P less than 0.001) in dogs of both experimental groups. This degree of decrease was due to equal decrease in the PVF and HAF. The infusion of PBZ caused a slight tendency towards increase of LBF, while the subsequent decrease in blood flow values during second hypotensive period in the treated dogs was not so pronounced as in the untreated dogs. Although retransfusion led to an increase of LBF, HAF and PVF in both groups, the restauration was significantly better in PBZ-treated animals. The degree of metabolic acidosis was more pronounced in the untreated dogs than in PBZ-treated.

Effect of phenylephrine and isoproterenol on mesenteric blood flow in experimental posthaemorrhagic hypotension.

In previous reports it was found that in mesenteric blood vessels exist profound alpha-adrenergic, and less profound beta-adrenergic receptors. Our experiments were done on 20 mongrel dogs, both sexes, weighing 6.5-22.5 kg, age from 1-2 years, which were divided into 2 groups: experimental (14 dogs) and control (6 dogs). Experimental group of animals passed through the posthemorrhagic hypotension (180 min. at 5.3 kPa). Aim of investigation was to clarify mesenteric vascular reactivity changes during posthemorrhagic hypotension. In order to reach this goal, phenylephrine and isoproterenol were injected alternatively in one hour intervals through cannulated superior mesenteric artery first proximal branch. Our results indicate that mesenteric blood flow responses to those drugs changed quantitatively, but never ceased. It is apparent from our experiments that those quantitative changes are due to beta 2 adrenoceptors changes during the posthemorrhagic hypotension.

Evaluation of agonist efficacy and receptor reserve for alpha 2D-adrenergic receptor regulated G protein activation in PC12 cell membranes.

The receptor-coupling efficiency for epinephrine (EPI) stimulated heterotrimeric G protein activation was studied at the G protein level in membranes prepared from PC12 cells expressing cloned alpha 2D-adrenergic receptors (alpha 2D-AR). After pretreatment with different concentrations of N-ethoxycarbonyl-1,2-dihydroquinoline, which irreversibly inactivates alpha 2D-AR, the portion of alpha 2D-ARs remaining active (q) was estimated from EPI-stimulated [35S]GTP gamma S binding. This function-derived estimate was close to the actual remaining number of receptors, as determined in saturation-binding studies using the selective alpha 2-AR antagonist [3H]rauwolscine in the same membranes. The agonist dissociation constant (KA) derived from EPI-stimulated [35S]GTP gamma S binding via Furchgott analysis was similar to the EC50 of EPI in the same assay, but 40-fold lower than its Ki measured from EPI competition for [3H]rauwolscine-binding sites in the presence of GTP gamma S and Na+. The occupancy-response relationship, calculated using Ki rather than KA, was markedly nonlinear, consistent with the high expression of alpha 2D-AR in these membranes. A nonlinear occupancy-response relationship was more directly confirmed by measuring the maximal level (i.e., full occupancy level) of G protein activation at graded densities of alpha AD-AR after N-ethoxycarbonyl-1,2-dihydroquinoline treatment. Determination of the number of G-proteins activated per receptor yielded lower values at higher receptor densities, indicating that overexpression of receptors can reduce their efficiency. Our results indicate the potential utility of using GTP-binding studies to assess agonist efficacy at the G protein level under conditions where receptor occupation can also be directly measured.

Androgen regulation of thromboxane A2/prostaglandin H2 receptor expression in human erythroleukemia cells.

Thromboxane A2 (TxA2), a platelet aggregator and vasoconstrictor, has been implicated as a potential mediator of cardiovascular diseases. Abuse of androgenic steroids has been associated with thrombotic cardiovascular diseases. Human erythroleukemia (HEL) cells, a megakaryocyte-like cell line, express functional TxA2/prostaglandin H2 (PGH2) receptors with characteristics similar to those seen in platelets. This study characterized testosterone regulation of HEL cell TxA2/PGH2 receptors. TxA2/PGH2 receptor affinity (Kd) and density (Bmax) were determined via equilibrium binding experiments using the radiolabeled TxA2 mimetic (1S-[1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha])-7-(3-[3-hydroxy-4-(4'- iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-he ptenoic acid (125I-labeled BOP). Testosterone (200 nM) but not estradiol increased Bmax from 108 +/- 9 fmol/mg protein to 157 +/- 9 fmol/mg protein (n = 7 experiments; P < 0.01) without any significant change in Kd. Testosterone had no significant effect on alpha 2-adrenergic receptor density. The maximum increase in intracellular free calcium induced by the TxA2 agonists I-BOP or U-46619 was significantly (P < 0.005) greater in testosterone-treated cells compared with controls. Hydroxyflutamide (1 microM), an androgen-receptor antagonist, completely blocked the effect of testosterone (P < 0.01). Dihydrotestosterone, the active metabolite of testosterone, also increased Bmax in a concentration-dependent manner and was more potent than testosterone. The effect of testosterone to increase Bmax was significantly (P < 0.01) inhibited by coincubation with cycloheximide (0.1 microgram/ml) or actinomycin D (10 ng/ml). These results indicate that androgenic steroids regulate the expression of functional TxA2/PGH2 receptors in HEL cells. These findings may have relevance to cardiovascular disease.

Regulation of alpha 2-adrenergic receptor expression and signaling in pancreatic beta-cells.

Activation of alpha 2-adrenergic receptors (alpha 2-AR) in pancreatic beta-cells inhibits insulin secretion in response to various stimuli, and acute or long-term regulation of alpha 2-AR receptor-mediated effects may influence the tissue response to glucose dishomeostasis. As an initial approach to this issue, we determined the effect of various metabolic and hormonal treatments on alpha 2-AR expression and coupling in the pancreatic beta-cell lines HIT-T15 and RIN-5AH. Radioligand binding studies ([3H]RX-821002) and RNA blot analysis indicate that both pancreatic beta-cell lines express the alpha 2A/D-AR subtype [for HIT-T15 the maximum binding (Bmax) = 113 +/- 28; for RIN-5AH Bmax = 93 +/- 18 fmol/mg of cellular protein]. Treatment of HIT-T15 or RIN-5AH cells with glucocorticoids [dexamethasone, hydrocortisone, or prednisolone (1 microM)] increased alpha 2-AR mRNA level and receptor protein density three- to fivefold. The glucocorticoid-induced increase in receptor density in HIT-T15 cells was associated with 1) an increase in the amount of receptors coupled to G protein as determined by analysis of high-affinity 5'-guanylyl imidodiphosphate-sensitive binding of [3H]UK-14304, a selective alpha 2-AR agonist, and 2) a greater inhibition of forskolin-induced elevation of cellular adenosine 3',5'-cyclic monophosphate after receptor activation. Receptor density in HIT-T15 cells was not altered by different growth conditions, insulin (1 microM), phorbol 12-myristate 13-acetate (1 microM), or the sex steroids testosterone and progesterone (1 microM). These data indicate that glucocorticoids upregulate alpha 2-AR expression and signaling in pancreatic beta-cells. Such regulation may operate in a cell-specific manner, allowing discrete modulation of tissue responses to glucose dishomeostasis.

[Angiotensin converting enzyme activity in the blood of dogs with posthemorrhagic hypotension]. / Aktivnost enzima konvertora angiotenzina u plazmi pasa u posthemoragicnoj hipotenziji.

In 1984 and 1985 a questionnaire was applied to 5611 railway transport workers in Croatia. Out of them, those satisfying the criteria of the Geneva Convention (1980) of the International Working Group for the Early Detection of Colorectal Carcinoma (3379) were supplied with the Hemdetect test. Of these 3379 workers, 2428 (71.8%) returned the "consumed" test after three days: in 114 of them the Hemdetect test was positive. Since in this way 5 patients with colorectal carcinoma (4.85%), 17 with polyps (16.5%), and 3 with polyposis (2.92%) were detected, it is maintained that the Hemdetect test is very useful, in fact unavoidable in attempts at the secondary prevention of colorectal carcinoma and the lesions proceeding it. Although the 34.95% of so-called false positive results obtained by the Hemdetect test diminish its significance because of a reduced specificity, it should not be excluded from practical work on the secondary prevention of colorectal carcinoma--it should in fact be put ahead of other tests, because it is simple, easily applied, readily acceptable by examinees, and less costly than the use of immunochemical methods.

Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR (89% homology) and is also capable of mediating adenylylcyclase inhibition as determined following its stable expression in NIH-3T3 fibroblasts. However, in contrast to RG20, [3H] rauwolscine exhibits a KD of 2 nM for the C-10 receptor, and the potency order for competing ligands is rauwolscine greater than or equal to yohimbine greater than idazoxan greater than phentolamine greater than prazosin. RG20 and C-10 are also distinguished by their affinity for SKF-10478 (RG20 Ki = 531 nM, C-10 Ki = 101 nM), a compound that may functionally distinguish pre- and postsynaptic alpha 2-AR. These data suggest that RG20 represents a fourth alpha 2-AR subtype distinct from the known alpha 2A-C receptor subtypes.

[Early prognosis of free venous autograft in common bile duct reconstruction. Experimental study--first phase]. / Rana prognoza slobodnog autovenskog grafta kod rekonstrukcije duktus holedohusa. Eksperimentalni rad--prva faza istrazivanja.

Experiments were carried out on 25 grown-up dogs of both sex in general anesthesia. The supraduodenal part of the choledochus was excised and the reconstruction of the defect with a free venous autograft was made by an end to end anastomosis without use of a transitory or permanent prosthesis. The experimental animals were observed for 60 days, during which period they were checked clinically, biochemically and radiologically. After this period the animals were sacrificed and surgical peroperative control and sampling of material for histological study were made. Out of 25 experimental animals 18 (72%) survived a period of 60 days and 7 dogs died. On autopsy it was found that the cause of death in all animals was biliary peritonitis, but in only one case due necrosis of the graft. The values of bilirubin concentration at the end of the experiment showed almost normal level, but the values of the alkaline phosphate activity and transminase showed significant increased values. Intravenous biligraphy showed significant excretion of the contrast material, freely passing the graft, which was dilated due to stenotic changes of the distal anastomosis. After sacrificing the animals we found: the graft increased length, diameter, thickness wall and stenosis of the distal anastomosis. By histological exploration we found in the liver the signs of the biliary stasis and in the grafts substitution of its endothelium by biliary epithelium. Free venous autograft used as a substitute in the reconstruction of the bile duct which survived 60 days postoperatively, remained transient but with changes in the sense of elongation and dilatation caused by the stenosis od distal anastomosis. Histologically the grafts were entirely bridged by biliary epithelium.

Determinants of alpha 2-adrenergic receptor activation of G proteins: evidence for a precoupled receptor/G protein state.

The ability of agonist-occupied alpha 2D-adrenergic receptors to activate G proteins was measured in membranes from PC-12 cells stably expressing the cloned receptor, using guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding as an endpoint. Epinephrine (EPI) stimulated [35S]GTP gamma S binding in a Mg(2+)-dependent manner, showing both micromolar and millimolar cation affinities. Prior treatment of cells with pertussis toxin completely eliminated the EPI stimulation. The presence of GDP decreased basal [35S]GTP gamma S binding and increased the proportion of EPI-stimulated binding. Increasing concentrations of Na+ also reduced basal [35S]GTP gamma S binding but had less effect on EPI-stimulated binding, such that the agonist response was proportionately greater at higher Na+ levels. In saturation binding studies with [35S]GTP gamma S only low affinity binding was observed in the presence of 100 mM Na+, whereas in the absence of Na+ a high affinity component was also present, indicating a Na(+)-regulated receptor/G protein interaction. EPI induced high affinity [35S]GTP gamma S binding in the presence of Na+ and increased the affinity of the high affinity component under Na(+)-free conditions. The selective alpha 2-adrenergic antagonist rauwolscine produced rightward shifts of EPI dose-response curves and decreased the basal level of [35-S]GTP gamma S binding across the same range of concentrations. The extent of decrease was dependent upon the alpha 2-adrenergic receptor expression level, indicating that alpha 2-adrenergic receptors contribute to basal G protein activation in the absence of agonist. The ability of rauwolscine to decrease basal [35S]GTP gamma S binding was diminished as the level of Na+ was increased, suggesting that both agents act to reduce receptor/G protein interaction, by distinctive mechanisms. alpha 2-Adrenergic receptor antagonists reduced basal G protein activation with a rank order for maximal effectiveness that was different from their receptor binding affinities. These results support the existence of precoupling between alpha 2D-adrenergic receptors and G proteins; coupling can be diminished by both Na+ and antagonists, whereas agonists increase the efficiency of receptor/G protein coupling.

Dual modulation of calcium channel current via recombinant alpha2-adrenoceptors in pheochromocytoma (PC-12) cells.

The ability of recombinant rat alpha2D-and alpha2B-adrenoceptors expressed in nerve-growth-factor-differentiated pheochromocytoma PC-12 cells to modulate Ca2+ currents, recorded by the whole-cell patch-clamp technique, has been studied. Ca2+ currents in different cells were either reversibly reduced or increased by dexmedetomidine, an alpha2-adrenergic agonist, in a concentration-dependent manner. Pertussis toxin pretreatment reduced the number of cells that showed an inhibitory response and reduced the magnitude of inhibition. In cells expressing the alpha2B-adrenoceptor, pertussis toxin increased the proportion of cells from which a stimulatory effect on Ca2+ currents could be recorded. The magnitude of the inhibitory responses was unaffected but the stimulatory responses were considerably reduced by the dihydropyridine Ca2+ channel blocker nifedipine (5 microM). All effects of dexmedetomidine were reversible upon wash-out and inhibited by the antagonist rauwolscine. The results support the idea that modulation of voltage-dependent Ca2+ channels in transfected PC-12 cells is mediated by activation of recombinant alpha2D- and alpha2B-adrenoceptors. This receptor activation predominantly causes inhibition of dihydropyridine-insensitive Ca2+ channels via pertussis-toxin-sensitive G proteins. Additionally receptor activation can also lead to stimulation of dihydropyridine-sensitive Ca2+ channels via pertussis-toxin-insensitive mechanisms.

The 3'-untranslated region of the alpha2C-adrenergic receptor mRNA impedes translation of the receptor message.

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

Adenylyl cyclase isoforms and vasopressin enhancement of agonist-stimulated cAMP in vascular smooth muscle cells.

The influence of arginine vasopressin (AVP) on agonist-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in vascular smooth muscle cells (VSMC) cultured from rat thoracic aorta. Incubation of VSMC with AVP for 60 s produced a 2- to 2.5-fold enhancement of isoproterenol-induced cAMP formation. AVP also increased cAMP stimulation by the prostaglandin I2 analogue iloprost. The effect of AVP to enhance agonist-stimulated cAMP formation was completely inhibited in cells pretreated with a selective antagonist of V1 vasopressin receptors but was not affected by blockade of V2 receptors. Inhibition of protein kinase C activation failed to alter the action of AVP to potentiate cAMP stimulation, but treatment of cells with calmodulin antagonists significantly attenuated this effect of the peptide. Moreover, depletion of Ca2+ stores with thapsigargin decreased AVP enhancement of isoproterenol-stimulated cAMP by > 70%. The action of AVP to increase cAMP stimulation was also demonstrated in freshly isolated strips of rat aorta where treatment with peptide produced a twofold increase in isoproterenol-stimulated cAMP formation. RNA blot analysis indicated expression in VSMC of mRNA encoding type III adenylyl cyclase, a Ca(2+)-calmodulin-sensitive isoform of the effector. Furthermore, when detergent-solubilized membrane extract was subjected to calmodulin affinity chromatography, a peak of adenylyl cyclase activity was identified which had affinity for calmodulin matrix in the presence of Ca2+. The results indicate that AVP activates V1 receptors in VSMC to enhance agonist-stimulated cAMP formation by a Ca(2+)-calmodulin-dependent mechanism and suggest that type III adenylyl cyclase may provide a focal point in the VSMC for cross talk between constrictor and dilator pathways.

[Reconstruction of the common bile duct with a free venous autograft (experimental work - preliminary communication)]. / Rekonstrukcija duktus holedohusa slobodnim autovenskim graftom (eksperimentalni rad - preliminarno saopstenje).

In seven dogs, the authors performed a resection of the supraduodenal part of the main bile duct, 15-2 cm. in length. The arising defect was bridged over with a free autovenous graft taken from the exterior jugular vein (T-T anastomosis), without using a temporary or permanent prosthesis. The animals were followed-up 60 days and the sacrificed. Three dogs perished: the first one on the third day due to intoxication, the second dog on the thirteenth day because of anastomosis' disruption and consequent biliary peritonitis, and the third one on the eighteenth day of unknown causes. In the immediate postoperative course the values of the bilirubin and alkaline phosphatase were increased while those of transaminase were not significantly altered. These values began to normalize during the third week. An intravenous biliography was made on the sixtieth day showing a normal filling of the gallbladder, with an orderly visualization of hepato-choledochus and its correct transit, but with stenosis at the distal anastomosis. Macroscopically, the graft appeared slightly enlarged in length and diameter due to the mentioned stenosis. Histologically, the graft took on an appearance similar to the main bile duct, while its endothelium was completely replaced by biliary epithelium. An answer about the viability of this autovenous graft to serve as a substituent of a normal common bile duct is to be expected with further experimental work.