Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex.

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Biphasic regulation of the messenger ribonucleic acid coding for the estrogen receptor by cyclic adenosine 3':5'-monophosphate in tumor Leydig cells.

In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo cyclic AMP (8-Br-cAMP) and human chorionic gonadotropin in a mouse tumor Leydig cell line (MA-10 cells). When the MA-10 cells were cultured in the presence of the cAMP analogue for varying time periods, a transient increase in the level of ER mRNA was observed. Short time incubation (0-2 h) with 8-Br-cAMP enhanced the expression of ER mRNA (2-fold), whereas longer times of incubation (6 h) had the opposite effect (the level of ER mRNA was reduced by 60-70%). The inhibitory effect of 8-Br-cAMP on ER mRNA was not counteracted by aminoglutethimide, an inhibitor of steroidogenic enzymes, indicating that this effect is not mediated via steroids (progesterone). Treatment of 8-Br-cAMP for 6 h caused a concentration-dependent inhibition of ER mRNA with a half-maximal effect of approximately 150 microM. Increasing concentrations of human chorionic gonadotropin for 6 h was also associated with a biphasic effect on the ER mRNA level. Low concentrations (0.20-0.40 ng/ml) increased ER mRNA in the MA-10 cells whereas the highest concentration (20 ng/ml) caused a suppression of this mRNA. In contrast to the biphasic effects observed for the ER mRNA, the level of the regulatory subunit type II beta of the cAMP-dependent protein kinase (protein kinase A) was enhanced in a concentration-dependent manner by human chorionic gonadotropin. Furthermore, 8-Br-cAMP stimulated the mRNA for regulatory subunit type II beta (10- to 20-fold) by all concentrations examined (50-1000 microM). The observations reported here indicate that the expression of ER mRNA is regulated both by endogenously formed and exogenously added cAMP and that there may exist regulatory loops between the steroid and the cAMP/protein kinase A systems.

Kinetic properties of the C-terminal Src kinase, p50csk.

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu, Tyr) as substrate, phosphotransferase activity was linear for 7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na+, K+) and divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

The alpha-subunit mRNAs for Gs and Go2 are differentially regulated by protein kinase A and protein kinase C in rat Sertoli cells.

In the present study, we have examined regulatory effects of protein kinase A and protein kinase C activation by 8-CPTcAMP and TPA, respectively, on mRNAs for various G protein alpha-subunits and corresponding immunoreactive proteins in rat Sertoli cells. Gs alpha and Go alpha mRNA levels were transiently increased 1.5-fold and 4-fold, respectively, by 8-CPTcAMP in cultured Sertoli cells. This up-regulation of mRNAs for Gs alpha and Go alpha was also observed when Sertoli cells were incubated in the presence of FSH. When protein synthesis was inhibited by cycloheximide, the cAMP-mediated stimulation of Gs alpha mRNA was abolished, whereas Go alpha mRNA was superinduced to a 50- to 100-fold higher level than basal. Activation of protein kinase C with TPA had a strong, synergistic effect on cAMP-mediated stimulation of Gs alpha mRNA, whereas the cAMP-mediated stimulation of Go alpha mRNA was completely blocked. Surprisingly, changes in mRNA levels were not accompanied by any alterations in the levels of immunoreactive Gs alpha and Go alpha proteins.

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase.

The gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5'-flanking region (1.2 kb) and exon 1 of the human RII alpha gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5'-flanking region of the RII alpha gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5'-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII alpha cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Oyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46-54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII alpha cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

Biphasic response to 3',5'-cyclic adenosine monophosphate (cAMP) at the messenger ribonucleic acid level for a regulatory subunit of cAMP-dependent protein kinase.

In the present study we have examined the effect of long-term stimulation with (Bu)2cAMP on mRNA levels for the hormone responsive regulatory subunit (RII beta) of cAMP-dependent protein kinase in cultured rat Sertoli cells. The effects of the same treatment on two other mRNAs [androgen binding protein (ABP) and cellular retinol binding protein (cRBP)], shown to be regulated by cAMP, were examined simultaneously. The addition of (Bu)2cAMP (0.1 mM) to primary Sertoli cell cultures, for 14 and 24 h, caused a 50- to 60-fold stimulation in the steady state levels of mRNA for RII beta. During the same period of stimulation, we also observed a significant increase (2- to 3-fold) in the mRNA levels for ABP, and a 80% decrease in the mRNA levels for cRBP. Continued stimulation for 36 and 48 h was associated with a significant time-dependent decrease in the mRNA level for RII beta, in spite of the continuous presence of (Bu)2cAMP (0.1 mM) in the medium. This reduced response by long term stimulation with (Bu)2cAMP appears to be specific for RII beta, since mRNA for ABP remained elevated and mRNA for cRBP remained depressed during the entire period of cAMP stimulation. Our data demonstrate the presence of a biphasic type of regulation at the mRNA level, specific for the regulatory subunit RII beta of cAMP-dependent protein kinase. This response may be analogous to the desensitization mechanisms observed at other levels of the cAMP signalling pathway. For proteins constituting part of the signal transduction pathway this type of biphasic regulation, may be particularly important in maintaining homeostasis in the cell.

Binding of a member of the NF1 family of transcription factors to two distinct cis-acting elements in the promoter and 5'-flanking region of the human cellular retinol binding protein 1 gene.

We studied the interaction of nuclear proteins with the 5'-flanking and promoter region of the human cellular retinol binding protein 1 (hCRBP1) gene and identified seven specific sequences that interacted with nuclear proteins from liver and prostate. Two of these sequences, footprint 1 (Fp1) and footprint 5 (Fp5), were highly homologous, sharing the core sequence GGCCAAC, which has a certain similarity to the consensus sequence for the NF1 binding site. Competition experiments in gel mobility shift assays and DNasel footprinting indicated that a common protein interacted with both elements. Immunological and biochemical data indicated that this protein belongs to the nuclear factor 1 (NF1) family of transcription factors. The ability of the Fp1 and Fp5 sequences to control gene expression was studied by transient transfections of several cell types. In the wild type promoter, both Fp1 and Fp5 acted as repressors of human (h) CRBP1 gene transcription. Once inserted upstream of the basal promoter from the heterologous p12 gene, the function of both Fp1 and Fp5 was reverted to that of transcriptional activators, although Fp5 exerted only moderate transcriptional activation of the chloramphenicol acetyl transferase (CAT) reporter gene. Hence, the position of these NF1 binding sites and the nature of the flanking sequences appear to direct their effect on transcription. Despite close sequence homology, a common core sequence, and a similar ability to bind nuclear proteins in vitro, these results indicate that Fp1 and Fp5 exert similar regulatory functions but to different levels in vivo. In conclusion, these results indicate that a member of the NF1 family plays a significant role in regulating CRBP1 gene expression.

Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent protein kinases.

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis.

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase.

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

Effects of beta blocking agents on the density of beta adrenoceptors and adenylate cyclase response in human myocardium: intrinsic sympathomimetic activity favours receptor upregulation.

The density of beta adrenoceptors, the relative number of beta 1 and beta 2 adrenoceptor subtypes, and adenylate cyclase activity were studied in preparations from atrial biopsy specimens of 32 patients with coronary heart disease. Six patients were not receiving beta blocking agents, whereas the others were treated with different beta blocking drugs (timolol, propranolol, pindolol, metoprolol, and atenolol). Clinical and haemodynamic variables were similar in the different groups of patients. Beta adrenoceptor density was 17% significantly lower in the non-treated group than in the groups treated with beta blocking drugs. Among these, the group treated with pindolol, a drug with intrinsic sympathomimetic activity, had receptor densities that were 38% significantly higher than those treated with other beta blocking drugs and 51% significantly higher than the non-treated group. The relative numbers of beta 1 and beta 2 adrenoceptor subtypes were very similar in the different groups (beta 1 receptors 75-80%, beta 2 receptors 20-25%). A significant increase in the ratios of terbutaline stimulated to basal and terbutaline stimulated to isoproterenol stimulated adenylate cyclase activities was found in patients treated with beta 1 selective blockers (metoprolol, atenolol), indicating that beta 1 selective drugs may improve beta 2 receptor-adenylate cyclase coupling. In contrast, pindolol caused a significant reduction in the ratio of terbutaline stimulated to isoproterenol stimulated adenylate cyclase activity, indicating that this drug may cause a reduction in beta 2 receptor-adenylate cyclase coupling efficacy. Thus treatment with beta blocking agents causes upregulation of human myocardial beta receptor density. Intrinsic sympathomimetic activity seems to favour receptor upregulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apparent lack of beta 2 adrenergic receptors in porcine myocardium.

The aim of this study was to investigate the relative numbers of myocardial beta 1 and beta 2 receptors in pigs. Membrane particles from left ventricular porcine and mixed ventricular rat myocardium were examined for subtypes of beta adrenergic receptors with a radioligand binding technique using [125I]-cyanopindolol (ICYP) as trace, and the new highly beta 1 selective antagonist Sandoz 204 545 and the beta 2 selective antagonist ICI 118 551 for displacement. Radioligand displacement experiments were also performed using propranolol, isoprenaline and terbutaline. The displacement curves obtained with the subtype selective antagonists and agonist revealed biphasic inhibition of specific ICYP binding in rat preparations, indicating a beta 1/beta 2 ratio of approximately 2/1. In porcine preparations displacement of specific ICYP binding with all agents resulted in monophasic curves, thus sharply contrasting the rat preparations. Affinity constants of displacing drugs derived from these monophasic curves indicated that the specific binding site was a beta 1 receptor. No displacement compatible with beta 2 affinity was found. In the same rat preparations we found that adenylate cyclase activation and inhibition by beta receptor subtype specific agonists and antagonists were mediated by two receptor subtypes, whereas in the pig, adenylate cyclase activation and its inhibition seemed to occur via only one receptor subtype, the beta 1 adrenoceptor.

Effects of chronic amiodarone treatment on human myocardial beta adrenoceptor density and adenylate cyclase response.

STUDY OBJECTIVE: The aim of the study was to evaluate the effect of chronic treatment by amiodarone on beta adrenoceptor density and adenylate cyclase response in human myocardium. DESIGN: Density of beta 1 and beta 2 adrenoceptors was measured by radioligand binding assay. beta Adrenoceptor stimulated production of cAMP was measured by adenylate cyclase assay. EXPERIMENTAL MATERIAL: Right auricular tissue from five patients on chronic amiodarone treatment was compared with that from nine patients in similar clinical and haemodynamic state undergoing coronary bypass surgery. MEASUREMENTS AND MAIN RESULTS: beta 1 and beta 2 adrenoceptor subtypes were quantified using the highly beta 1 selective antagonist Sandoz 204 545. The total beta adrenoceptor density was 28% lower in the amiodarone treated group than in the controls (42.0 v 58.3 fmol.mg-1 protein, p less than 0.02), beta 1 adrenoceptors were 25% lower (33.1 v 44.3 fmol.mg-1 protein, p less than 0.02), and beta 2 adrenoceptors were 36% lower (8.9 v 14.0 fmol.mg-1 protein, p less than 0.02). The cAMP production following non-selective beta adrenoceptor stimulation (isoprenaline 5 mumol.litre-1) was reduced by 38% in the amiodarone treated group (14.2 to 8.7 pmol.min-1.mg-1 protein, p = 0.05). Terbutaline stimulated cAMP production was reduced by 49% (8.3 to 4.3 pmol.min-1.mg-1 protein, p = 0.03). Fluoride stimulated cAMP production was not significantly different (9.4 v 8.4 pmol.min-1.mg-1 protein, p = 0.15). CONCLUSIONS: Chronic treatment with amiodarone is associated with a non-selective downregulation of beta adrenoceptors. beta Adrenoceptor stimulated cAMP production was also reduced. The "beta blocking effect" of amiodarone is probably related to downregulation of beta adrenoceptors.

Specific non-beta-adrenergic binding sites for 125I-iodocyanopindolol in myocardial membrane preparations: a comparative study between human, rat, and porcine hearts.

STUDY OBJECTIVE: The aim was to investigate observed differences in beta adrenergic and apparent non-beta-adrenergic binding of (-)[125I]-iodocyanopindolol (125I-ICYP). DESIGN: Binding parameters for several beta adrenergic agonists and antagonists were examined in radioligand binding assay, using 125I-ICYP as radioligand, in membranes prepared from myocardial tissue. SUBJECTS: Human right auricular myocardium was obtained from patients undergoing open heart surgery. Ventricular myocardium was from Norwegian landrace pigs and Wistar rats. MEASUREMENTS AND MAIN RESULTS: Specific binding of 125I-ICYP was observed. This was only partially competed for with high affinity by isoprenaline, noradrenaline, adrenaline, and atenolol. Considerable interspecies variations in the magnitude of specific non-beta-adrenergic (NBA) binding of 125I-ICYP were shown. The equilibrium constant of dissociation (Kd) of the specific NBA binding sites for 125I-ICYP was 0.3-0.4 nmol.litre-1, and the binding capacities were 20, 106, and 192 fmol.mg-1 protein in rat, human, and porcine myocardium, respectively. The NBA sites were heat sensitive and destroyed by trypsin. Association to NBA sites occurred more rapidly than to beta adrenoceptors. Dissociation of bound 125I-ICYP from NBA sites and beta adrenoceptors at 30 degrees C revealed first order kinetics with t1/2 of 19 min from NBA, as compared to 120 min from beta adrenoceptors. In all three species the ligand specificity for NBA sites was very similar and various adrenergic agonists and antagonists competed with 125I-ICYP binding with the following potencies: timolol greater than propranolol greater than ICI 118 551 greater than pindolol greater than Sandoz 204 545 greater than terbutaline greater than noradrenaline and adrenaline much greater than isoprenaline and atenolol. Of agonists and antagonists for other receptor systems, only the serotoninergic 5-HT2 antagonist ritanserin could displace 125I-ICYP from the NBA sites with relatively high affinity. CONCLUSIONS: 125I-ICYP and several beta adrenoceptor antagonists interact specifically with receptor like proteins other than beta adrenoceptors, and remarkable interspecies difference in the levels of myocardial NBA sites was observed.

Beta-adrenoceptor density and relative number of beta-adrenoceptor subtypes in biopsies from human right atrial, left ventricular, and right ventricular myocard.

In the present study we estimated the relative number of beta-adrenoceptors in membrane preparations from right atrial and left ventricular biopsies from 8 patients, as well as from right ventricular biopsy from one patient. Determinations of relative number of beta adrenoceptor subtypes (beta1, beta2) were also performed. Estimations were performed in a radioligand assay, measuring specific binding of [125I]-cyanopindolol (CYP). Inhibition of specific [125I] CYP binding was studied by adding propranolol (non-selective antagonist), atenolol (beta1 selective antagonist) and ICI 118551 (beta2 selective antagonist) to the preparations. Some individual variance in total number of receptors was found. Per mg protein, the number of binding sites were higher in atrial preparations (mean 71.4 +/- 14.4 fmol X mg-1) than in left ventricular preparations (mean 30.2 +/- 7.1 fmol X mg-1). Receptor number in the right ventricular preparation was 49.5 +/- 2.6 fmol X mg-1. Approximately 25% of beta adrenoceptors were of beta2 subtype in preparations from both right atrial and left ventricular biopsies, as well as from the right ventricular biopsy. Thus, there are regional differences in the quantity of beta adrenoceptors in human myocard whereas the relative proportion of beta1 and beta2 receptors appears to be fairly constant.

Beta adrenoceptor density and adenylate cyclase response in right atrial and left ventricular myocardium of patients with mitral valve disease.

Beta adrenoceptor density, measured by radioligand binding techniques, is reportedly much higher in atrial than in ventricular myocardium of patients with mitral valve disease. In the present study adenylate cyclase activity, both basal and in response to beta adrenergic agonists and sodium fluoride, in biopsy preparations from these same patients was significantly lower in atrial than in ventricular tissue when stimulated with either isoproterenol, noradrenaline, isoproterenol combined with terbutaline, or sodium fluoride. Terbutaline stimulated and basal adenylate cyclase activity was not significantly different in the two cardiac regions. The ratios of receptor density to isoproterenol stimulated and to sodium fluoride stimulated adenylate cyclase activity were 4-5 times higher in atrial than in ventricular biopsy specimens. Thus ventricular beta receptors, although present in comparatively low concentrations, are coupled to considerably more catalytic moieties of the receptor-adenylate cyclase complex than their atrial counterparts. The reason for this is probably a relative lack of coupling proteins (N components) in atrial tissue. A weak positive correlation between receptor density and isoproterenol stimulated adenylate cyclase activity was found in atrial but not in ventricular tissue. This may indicate individual variation in receptor-adenylate cyclase coupling. Furthermore, no correlation was found between atrial and ventricular values for any variable. One reason for this may be the different haemodynamic stresses in the two cardiac chambers.

Hypertrophic cardiomyopathy characterised by beta-adrenoceptor density, relative amount of beta-adrenoceptor subtypes and adenylate cyclase activity.

The total quantity of beta-adrenoceptors and the relative amount of beta 1 and beta 2 receptor subtypes were determined in heart biopsies of 10 patients with various heart diseases and 5 patients suffering from hypertrophic cardiomyopathy (HOCM). In membrane particle preparations from the same patients we also examined the activity of the adenylate cyclase (AC), and its response to isoprenaline, terbutaline, histamine and sodium fluoride (NaF). The high affinity ligand [125I] (1)-cyanopindolol (CYP) was used in the binding assays, and the highly beta 2-selective antagonist ICI 118 551 for the determination of beta-adrenoceptor subtypes. No differences were found in total beta-adrenoceptor density between patients with HOCM and "controls" (27.6 +/- 14.2 vs 26.5 +/- 10.7 fmol . mg-1 protein). The relative amounts of beta 1 and beta 2 receptor subtypes were similar, patients with HOCM had 82.1 +/- 4.9% of beta 1 and 14.4 +/- 3.9% of the beta 2 receptor subtype, compared with 76.3 +/- 11.5% of beta 1 and 20.7 +/- 11.0% of the beta 2 subtype in the "control" patients. Both absolute activity of AC (pmol . mg-1 protein . min) as well as the relative responses to the various stimulators were not significantly different between the two groups. Thus, this study does not support the hypothesis that HOCM is a disorder with altered beta-adrenoceptor number or adenylate cyclase response to adrenergic agonists. Furthermore, HOCM is not associated with altered response of the AC system to histamine or NaF.

Additive effects of IL-2 and protein kinase A type I antagonist on function of T cells from HIV-infected patients on HAART.

OBJECTIVE: To explore the basis for a possible immunomodulatory combination therapy with IL-2 and agents inhibiting protein kinase A (PKA) type I. DESIGN: Highly active antiretroviral therapy (HAART) has dramatically improved HIV therapy, but fails to eradicate the virus, and the persistence of HIV-associated immunodeficiency demonstrates the need for additional immunomodulating therapies. We have previously shown that hyperactivation of PKA type I inhibits the function of HIV-infected patient T cells. The separate and combined effect of a PKA type I-selective antagonist (Rp-8-Br-cAMPS) and Interleukin (IL)-2 on the function of T cells from HIV-infected patients on HAART was examined. METHODS: The effect of Rp-8-Br-cAMPS on anti-CD3 stimulated proliferation and IL-2 production and the combined effect with exogenous IL-2 was studied in vitro with cells from 13 HIV-infected patients on HAART and six uninfected controls. RESULTS: The PKA type I-selective antagonist improved cell proliferation (median 1.5-fold, maximal 2.8-fold) and IL-2 production (median 1.5-fold, maximal 2.4-fold) in T cells from HIV-infected patients on HAART, but not in controls. The addition of IL-2 enhanced proliferation of T cells from HIV-infected patients (approximately 1.9-fold) and that of controls (approximately 1.4-fold), but IL-2 had no effect at the concentrations produced by treatment with PKA type I antagonist. However, the combined effect of IL-2 and PKA type I antagonist was additive and resulted in a further increase in T-cell proliferation (median 2.5-fold, maximal 5.8-fold), reaching levels comparable with those of uninfected controls in most of the patients. CONCLUSION: Our findings suggest a basis for a novel strategy in treatment of HIV infection by combining IL-2 therapy and treatment modalities counteracting PKA type I activity with HAART.

Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat.

In the present work the molecular mechanisms of glucagon-induced desensitization of adenylate cyclase in cultured Sertoli cells have been studied in both whole cells and in a cell-free system. 1) Pretreatment of both whole Sertoli cells and membranes with glucagon induces a time- and dose-dependent desensitization of adenylate cyclase response that is primarily homologous and similar in the two systems. The component of heterologous desensitization, estimated by the reduced responsiveness to other hormones and NaF, accounted for only 12-20% loss of glucagon-responsive adenylate cyclase activity (P less than 0.01). 2) Glucagon-induced desensitization is ATP-dependent. Half maximal desensitization was achieved between 0.1 and 0.2 mM of ATP. 3) The typical time lag in the maximal activation of adenylate cyclase by GMPP(NH)P in the absence of hormone reappeared upon desensitization in spite of the presence of glucagon. The lag, however, was eliminated by FSH, showing that the homologous desensitization is due to a receptor-specific alteration. 4) The heterologous component of glucagon-induced desensitization is largely cAMP/protein kinase dependent. cAMP/protein kinase-induced desensitization was heterologous and caused approximately 30% loss of both hormonal and fluoride-stimulated enzyme activity. 5) Glucagon-induced desensitization is not due to altered activity of Ni since it proceeded equally well in membranes of cells pretreated with pertussis toxin (100 ng/ml) which eliminates Ni-mediated effects. It is concluded that glucagon induces both homologous and heterologous desensitization of the Sertoli cell adenylate cyclase. The locus of homologous desensitization appears to be at the level of the receptor and most probably involves a cAMP-independent phosphorylation reaction, whereas the heterologous desensitization appears to be cAMP-mediated and at least involves impaired functional activity of the Ns component.