Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.     

Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways.

We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulin-mimetic bisperoxovanadium 1,10-phenanthroline (bpV(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin- and bpV(phen)-induced activation of the atypical protein kinase C (zeta/lambda) (PKC (zeta/lambda)) was reversed by wortmannin. Inhibition of PKC (zeta/lambda) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (zeta/lambda) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Calcium inhibition of cardiac adenylyl cyclase. Evidence for two distinct sites of inhibition

Increasing the free calcium concentration from 10(-8) M to 10(-4) M inhibited cardiac sarcolemmal adenylyl cyclase activated by the addition of 5 X 10(-4) M forskolin or 1 X 10(-4) M GTP or Gpp(NH)p. The calcium inhibition curve in the presence of all three activators was shallow and best fit by a two site model of high affinity (less than 1.0 microM) and low affinity (greater than 0.1 mM). Gpp(NH)p appeared to decrease the sensitivity of adenylyl cyclase to inhibition by calcium at the high affinity site. Similar inhibition constants were obtained with each of the activators. Calmodulin content of native freeze-thaw vesicles was 76.2 +/- 14.2 ng/mg. Treatment of the vesicles with 1 mM EGTA to remove calmodulin significantly reduced calmodulin content to 19.7 +/- 1.35 ng/mg. This treatment had no significant effect on the calcium inhibition profile. Increasing free calcium to 3 X 10(-6) M was shown to have no effect on the EC50 estimated for either Gpp(NH)p or forskolin but did slightly increase the EC50 estimated for Mg2+ in the presence of maximal concentrations of either activator. Nevertheless, maximally stimulating concentrations of Mg2+ were unable to overcome calcium inhibition. Pretreatment of sarcolemmal membranes with pertussis toxin was shown to have no significant effect on calcium inhibition of adenylyl cyclase. The results suggest that the overall inhibitory action of calcium was most likely calmodulin independent and involved a direct interaction with the catalytic subunit at two distinct sites of high and low affinity. At the low affinity site calcium most likely competes with Mg2+ for an allosteric divalent cation binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for delta opioid receptor subtypes regulating adenylyl cyclase activity in rat brain

Opioid agonists selective for mu- or delta opioid receptors inhibit adenyl yl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.     

Studies on the mechanism of follicle-stimulating hormone-induced desensitization of Sertoli cell adenylyl cyclase in vitro

When Sertoli cells were cultured in the presence of follicle-stimulating hormone (FSH), a time-and concentration-dependent desensitization of FSH-responsive adenylyl cyclase (AC) was observed. Maximal desensitization (80%) was attained after 6-9 h of incubation with FSH (10 micrograms/ml; NIH-FSH-S12). During 24 h of incubation the concentration of FSH causing a half-maximal desensitization was about 100 ng/ml. Removal of the hormone from the culture medium was associated with a gradual reappearance of the FSH response. Follicle-stimulating hormone-induced desensitization of Sertoli cell AC was specific for homologous hormone, since AC activation by isoproterenol was unaffected. Furthermore, AC activity of control and FSH-desensitized cells was equally activated by GTP and fluoride, showing that the interaction of the guanyl nucleotide regulatory (N) component with the catalytic subunit is not affected during FSH-induced desensitization. A loss in specific FSH binding was detected after 9 and 24 h of exposure to FSH, but not at shorter times of incubation. Desensitization of Sertoli cell AC to both FSH and isoproterenol stimulation could also be achieved by dibutyryl cyclic AMP (dbcAMP); however, a 30-40% desensitization required a high nucleotide concentration (1 mM) and a long incubation time (24 h). These results show that desensitization of Sertoli cell AC by FSH is associated with normal function of the N component, and precedes any significant loss in specific FSH binding sites. Furthermore, exogenous addition of dbcAMP (1 mM) did not cause the same effects on Sertoli cell AC as did FSH.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

On the mechanism of isoproterenol-induced desensitization of adenylate cyclase in cultured differentiated hepatocytes

The adenylate cyclase of cultured differentiated RL-PR-C hepatocytes is desensitized to 1-isoproterenol by exposure to this beta-agonist. Virtually complete desensitization occurred by 60 min (intact cells) or 30 min (isolated plasma membranes). Isoproterenol was maximally effective at 10 micrometers, although substantial desensitization occurred at isoproterenol concentrations as low as 10 nM. Protein synthesis was not required for desensitization. Recovery from desensitization under tissue culture conditions was only 25% complete by 24 h. Maximum desensitization was accompanied by only a modest 35% decrease in binding sites (as determined by binding assays with [3H]dihydroalprenolol), with no change in binding affinity. Adenylate cyclase desensitized to 1-isoproterenol responded normally to guanine nucleotides and to fluoride, suggesting that the regulatory and catalytic proteins were not the sites of the desensitization "defect'. Using N-ethylmaleiimide to inactive the regulatory and catalytic proteins, and dicyclohexylcarbodiimide to inactivate the beta-adrenergic receptor, of intact hepatocytes, various heterologous cell fusion hybrids were produced, and their adenylate cyclases tested for responsiveness to 1-isoproterenol; only hybrids containing "desensitized' receptor failed to respond to isoproterenol. These results suggest that the mechanism of desensitization to isoproterenol involves only the receptor component of the receptor-regulatory protein(s)-adenylate cyclase complex, and that the receptors are reduced in number and/or ability to interact with the regulatory protein as a result of the desensitization process.     

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.     

Metabolic activity in primary cultures of fish hepatocytes

In aquatic toxicology, isolated liver cells from fish can be used as a tool to generate initial information on the hepatic metabolism of xenobiotics, and on the mechanisms of xenobiotic activation or deactivation. This isolation of teleost liver cells is achieved by enzymic dissociation, and monolayer cultures of fish hepatocytes in serum-free medium maintain good viability for 3-8 days. During in vitro culture, fish liver cells express stable levels of phase I and phase II enzymes, such as cytochrome P4501A or glutathione S-transferase, and the cells show an induction of biotransformation enzymes after exposure to xenobiotics. The xenobiotic metabolite pattern produced by fish hepatocytes in vitro is generally similar to that observed in vivo. Limitations to more-intensive application of cultured fish hepatocytes as a screen in aquatic hazard assessment are partly due to the rather limited scope of existing studies, i.e. the focus on one particular species (rainbow trout), and on one particular biotransformation enzyme (cytochrome P4501A), as well as a lack of comparative in vitro/in vivo studies.     

Gene transfer in primary cultures of human hepatocytes

Summary Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli β-galactosidase gene (β-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-l antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

The relationship between beta-adrenoceptor regulation and beta-adrenergic responsiveness in hepatocytes. Studies on acquisition, desensitization and resensitization of isoproterenol-sensitive adenylate cyclase in primary culture

The role of beta-adrenoceptor regulation in the mechanisms controlling beta-adrenergic responsiveness in hepatocytes was explored, using primary monolayer cultures. When plated in vitro, these cells gradually acquire a strong catecholamine-sensitive adenylate cyclase activity and an enhanced ability to bind the beta-adrenoceptor ligand [125I]iodocyanopindolol (125ICYP). Examination of the time course showed that the increase in the number of 125ICYP binding sites was detectable within 1-2 h of culturing and slightly preceded the elevation of isoproterenol-responsive activity. Then the responsiveness rose steeply and between about 5-24 h it closely followed the increase in beta-receptor binding. Addition of isoproterenol (10 microM) to cells after 20 h of culturing caused a rapid homologous desensitization of the adenylate cyclase (50% after about 5 min). This was paralleled by a down-regulation of beta-adrenoceptors measured both in membrane particles and in total cell lysates. Removal of isoproterenol led to a resensitization of the adenylate cyclase, which was rapid and protein-synthesis-independent after a brief (10-min) desensitization, or slow and cycloheximide-sensitive after prolonged (4-h) exposure to the agonist. In both cases an up-regulation of the 125ICYP binding paralleled the recovery from refractoriness. In contrast, no concurring changes in 125ICYP binding were measured when the beta-adrenoceptor-linked adenylate cyclase activity was enhanced by pretreatment with pertussin toxin (islet-activating protein, IAP) or was desensitized by exposure of the cells to glucagon or 8-bromo-cAMP; however, these modulations of the adenylate cyclase were nonselective, since the pretreatments with IAP, glucagon or 8-bromo-cAMP affected both isoproterenol-sensitive and glucagon-sensitive activities. The results suggest that, in hepatocytes, regulation at the beta-adrenoceptor level is a major determinant for both short-term and long-term selective changes of the beta-adrenergic responsiveness.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase

We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.     

The recycling of apolipoprotein E in primary cultures of mouse hepatocytes. Evidence for a physiologic connection to high density lipoprotein metabolism

Internalization of apoE-containing very low density protein (VLDL) by hepatocytes in vivo and in vitro leads to apoE recycling and resecretion. Because of the role of apoE in VLDL metabolism, apoE recycling may influence lipoprotein assembly or remnant uptake. However, apoE is also a HDL protein, and apoE recycling may be related to reverse cholesterol transport. To investigate apoE recycling, apoE(-/-) mouse hepatocytes were incubated (pulsed) with wild-type mouse lipoproteins, and cells and media were collected at chase periods up to 24 h. When cells were pulsed with VLDL, apoE was resecreted within 30 min. Although the mass of apoE in the media decreased with time, it could be detected up to 24 h after the pulse. Intact intracellular apoE was also detectable 24 h after the pulse. ApoE was also resecreted when cells were pulsed with HDL. When apoA-I was included in the chase media after a pulse with VLDL, apoE resecretion increased 4-fold. Furthermore, human apoE was resecreted from wild-type mouse hepatocytes after a pulse with human VLDL. Finally, apoE was resecreted from mouse peritoneal macrophages after pulsing with VLDL. We conclude that 1) HDL apoE recycles in a quantitatively comparable fashion to VLDL apoE; 2) apoE recycling can be modulated by extracellular apoA-I but is not affected by endogenous apoE; and 3) recycling occurs in macrophages as well as in hepatocytes, suggesting that the process is not cell-specific.