Forskolin-stimulated Adenylate Cyclase Research Articles

D2dopamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in rat striatum

Forskolin markedly stimulates striatal adenylate cyclase activity in a concentration-dependent manner, and at 10−4 M produces an approximate 40-fold increase in enzyme activity above basal levels. Dopamine (in the presence of 100 nM SCH 23390), bromocryptine and quinpirole (LY 171555) significantly inhibit both basal and forskolin-stimulated adenylate cyclase activity. There is a significant increase in the absolute but not in the percent inhibition of enzyme activity by dopaminergic agonists as a function of forskolin concentration. This inhibition is agonist-concentration dependent and antagonized by the D2 antagonist, spiperone. These results suggest that forskolin may be used as a tool for amplifying the abolute D2-receptor-mediated inhibition of adenylate cyclase in rat striatal homogenates.

Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D 2 receptor

The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3H]-ATP to [ 3H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity.

Forskolin activation of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica

Properties of forskolin activation of adenylate cyclase in the liver fluke Fasciola hepatica are described. Forskolin stimulated adenylate cyclase activity in cell-free fluke particles to levels more than 30-fold above the basal rate. This activation was not dependent on guanine nucleotides and, upon washing of the particles, was rapidly reversed. Forskolin potentiated the activation of adenylate cyclase by serotonin (5-HT) and lysergic acid diethylamide (LSD), resulting in both an increase in the maximal level of enzyme activity and a decrease in the apparent activation constant( K A ). The 5-HT antagonist 2-bromo-LSD did not inhibit enzyme activation by forskolin. Furthermore, forskolin had no effect on specific[ 3H]LSD binding to fluke particles. Activation of adenylate cyclase by sodium fluoride or guanine nucleotides was modified in a complex manner by forskolin with both stimulatory and inhibitory effects present. The results suggest that forskolin does not interact directly with the 5-HT receptor coupled to adenylate cyclase. Instead, it appears that forskolin effects are, at least in part, due to its ability to alter the interction between the regulatory and catalytic components of adenylate cyclase. Incubation of intact flukes with forskolin increased their cAMP levels 2- to 3-fold. The concentration dependence of this response was similar to that for forskolin activation of adenylate cyclase in fluke particles, with 300 μM forskolin giving the maximum response. Forskolin and other agents that increased fluke cAMP levels also stimulated fluke motility.

Steroidogenesis in isolated rat granulosa cells--changes during follicular maturation.

Steroid release was investigated in granulosa cells isolated from rat ovarian follicles at different maturation stages. Immature rats were treated with 10 IU of pregnant mare's serum gonadotrophin (PMSG) to induce follicular growth and maturation. Granulosa cells were isolated and subsequently incubated for 4 h in the absence or presence of follicle stimulating hormone (FSH), luteinizing hormone (LH) or forskolin. The concentrations of progesterone (P), 20 alpha-dihydroprogesterone (20 alpha DHP), testosterone (T) and oestradiol (E2) in the media were determined by radioimmunoassay. During maturation, basal release of T increased markedly at late dioestrus. At pro-oestrus, concomitant with a sharp rise in E2 and P release, T release returned to previously low levels. Addition of FSH or LH stimulated P release at late dioestrus and pro-oestrus but had no effect on E2 or T levels. Basal 20 alpha DHP levels remained low until a sharp rise at mid pro-oestrus. A stimulatory effect of gonadotrophins on 20 alpha DHP was first seen at this stage. The adenylate cyclase stimulator forskolin resembled FSH in all its effects. In summary, the present study demonstrates that maturing, but not preovulatory, granulosa cells have an increased capacity to release T during a relatively short period. It is suggested that this T peak could be of great importance for the further progress of follicular maturation.

Vasopressin inhibits cyclic AMP accumulation and adenylate cyclase activity in cerebral preparations

Arginine-vasopressin (AVP) and lysine vasopressin (LVP) elicited a dose-dependent inhibition of noradrenaline-sensitive cyclic AMP accumulation in rat cerebral cortical slices, and of forskolin-stimulated adenylate cyclase activity in a rat cerebral cortical membrane preparation. In both cases LVP was more potent than AVP, and exerted half-maximal effects at concentrations similar to those found effective in binding studies on rat hippocampal membranes. In hippocampal slices, AVP did not affect, cyclic AMP accumulation at low concentrations but potentiated the effect of noradrenaline at higher concentrations. In caudate membranes, AVP inhibited dopamine-stimulated adenylate cyclase with a similar dose-dependency to that seen for forskolin activation in cortex membranes.

Forskolin stimulation of adenylate cyclase in human thyroid membranes.

Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with half-maximal stimulation occurring at 5-10 microM. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p- (above 1 microM). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.

Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat.

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.

Forskolin-induced elevation of rat jejunal cyclic AMP levels and stimulation of active glucose transport in vitro.

The adenylate cyclase stimulator forskolin enhanced both mucosal cyclic AMP levels and D-aldohexose transport in rat jejunum in vitro in a concentration-dependent manner. With mucosal cyclic AMP elevated to 400% of basal values, active D-glucose transport was at 200% of control values. Transport parameters, as determined in a three compartment model using a dual label method, indicate a) increased "uphill" glucose transport at the brush border membrane with higher intracellular accumulation, b) unchanged serosal to mucosal glucose fluxes and passive permeabilities. Transport stimulating effects of forskolin were also present with D-galactose in the rat. In mouse jejunum active transport of D-glucose and 3-O-methylglucose was increased by forskolin 10 mumol/l. Phlorizin inhibited D-glucose transport as well as L-glucose transport in the rat were not changed by the persisting cyclic AMP elevation induced by forskolin. The results show a positive correlation of active intestinal hexose transport with a modest elevation of mucosal cyclic AMP. In accordance to current models of cyclic AMP-induced changes in intestinal fluid absorption, rat jejunal net fluid transport was reduced by 40% with 20 mumol/l forskolin.

Forskolin-stimulated adenylate cyclase activity in rat cerebral cortex following chronic treatment with psychotropic drugs.

Rats were treated with lithium, imipramine, reserpine, and lithium combined with imipramine or reserpine. Lithium was given in the diet (40 mmol/kg) resulting in a serum-Li+ level of 0.5-0.6 mmol/l. Other drugs were dissolved in 0.9% saline and given intraperitoneally once or twice daily. After 3 weeks of treatment, forskolin-stimulated adenylate cyclase activity was measured in cerebral cortex homogenates. Reserpine did not affect the forskolin stimulation, while both imipramine and lithium caused a decrease in this activity. The combined treatments lithium-imipramine and lithium-reserpine also exhibited a clear decrease in forskolin stimulation, but the effect of concomitant lithium and imipramine treatment did not differ from the effect seen after any of the treatments alone. The unstimulated activity was unaltered by all treatments. The inhibition of lithium and imipramine on the forskolin stimulation indicates an interference of these two drugs with the forskolin-mediated activation of the adenylate cyclase.

Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors.

It has been proposed elsewhere [Meeker, R.B. & Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319] that muscarinic cholinergic receptor-mediated attenuation of cAMP accumulation occurs through activation of phosphodiesterase in 1321N1 human astrocytoma cells. Pertussis toxin, which ADP-ribosylates the guanine nucleotide regulatory protein involved in receptor-mediated inhibition of adenylate cyclase (Ni), has been utilized to further differentiate between the mechanism of cholinergic regulation of cAMP metabolism in 1321N1 cells and the mechanism involving inhibition of adenylate cyclase in other tissues. Muscarinic receptor-mediated regulation of cAMP accumulation in NG108-15 neuroblastoma-glioma cells occurs through inhibition of adenylate cyclase. Pretreatment of these cells with pertussis toxin completely blocked the capacity of carbachol to attenuate cAMP accumulation. In contrast, concentrations of pertussis toxin two to three orders of magnitude higher than those effective in NG108-15 cells had no effect on muscarinic receptor-mediated attentuation of cAMP accumulation in 1321N1 cells. In addition, no effect of pertussis toxin was observed either on the control rate or the carbachol-stimulated rate of cAMP degradation measured directly in intact 1321N1 cells. A 41,000 Mr protein previously proposed to be the alpha subunit of Ni was labeled during incubation of a plasma membrane fraction from 1321N1 cells with [32P]NAD and pertussis toxin. Pertussis toxin is apparently active in 1321N1 cells, since this protein substrate was not labeled in plasma membrane preparations from cells previously incubated with toxin. Functional activity of Ni was demonstrated by the observation that guanosine 5'-[gamma-thio]triphosphate- and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity occurred in cell-free preparations from 1321N1 cells. The inhibitory activity of these guanine nucleotides was lost in membrane preparations from pertussis toxin-treated cells. The data suggest that adenylate cyclase is not involved in cholinergic action in 1321N1 cells and, furthermore, Ni is not involved in muscarinic receptor-mediated activation of phosphodiesterase in these cells. Thus, pertussis toxin can be used to differentiate between two mechanisms of cholinergic regulation of cAMP metabolism.

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin.

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.

Dopaminergic inhibition of adenylate cyclase correlates with high affinity agonist binding to anterior pituitary D 2 dopamine receptors

Dopamine (DA) and the dopaminergic agonists n-propylnorapomorphine (NPA), 2-amino-6,7-dihydroxytetrahydronaphthalene (ADTN) and apomorphine (APO) inhibit forskolin-stimulated adenylate cyclase activity in a dose-dependent fashion by more than 40% in membrane preparations of the porcine anterior pituitary gland. These agonists exhibit apparent dissociation constants that follow an expected dopaminergic order of potency (NPA > ADTN ⩾ APO > DA). The inhibition is dependent on guanine nucleotides and is reversible by dopaminergic antagonists (spiroperidol > (+)-butaclamol ⪢ (−)-butaclamol). The potencies of these agonists in inhibiting forskolin-stimulated adenylate cyclase activity correlate with the agonist dissociation constants ( K H) for binding to the high affinity receptor state (R H) in porcine anterior pituitary membranes (De Lean et al., Mol. Pharmacol. 1982, 22, 290–297) and the EC 50 for inhibition of prolactin release from rat anterior pituitary cells in culture (Caron et al. J. Biol. Chem. 1978, 253, 2244–2253). Furthermore, the intrinsic activities of dopamine and the other agonists for inhibition of forskolin-stimulated adenylate cyclase are similar and correlate well with the ability of these agents to induce a comparable proportion (50%) of the receptor in a high affinity state. Together these data provide additional support for the physiological relevance of the high affinity agonist binding state of the D 2 receptor in mediating the decrease in prolactin secretion via attenuation of adenylate cyclase.

Forskolin stimulates adenylate cyclase activity, cyclic AMP accumulation, and adrenocorticotropin secretion from mouse anterior pituitary tumor cells.

The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/ D16 -16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nucleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.

Forskolin increases cellular cyclic adenosine monophosphate content and parathyroid hormone release in dispersed bovine parathyroid cells

We studied the effects of the positive-inotropic and hypotensive agent forskolin on parathyroid hormone (PTH) release, cAMP accumulation, and adenylate cyclase activity in dispersed bovine parathyroid cells. Forskolin stimulated PTH release 1.7 to 3.7-fold at 10 −5 mol/L and increased cAMP content maximally 37.6-fold at 10 −4 mol/L. Forskolin induced a maximal increase in cAMP accumulation by 5 minutes of incubation. The rate of PTH release in forskolin-treated cells also reached maximal levels at five to ten minutes incubation. There was a dose-dependent increase in PTH release and cAMP content over the range of 10 −7 to 10 −4 M forskolin with half-maximal stimulation at 10 −7 to 10 −6 mol/L for PTH release and between 10 −5 and 10 −4 mol/L for cAMP content. PTH release and cAMP accumulation were inhibited by increasing extracellular calcium concentrations in both control cells and those incubated with 10 −5 mol/L forskolin. Forskolin stimulated adenylate cyclase activity in lysates from parathyroid cells 15.7-fold with half-maximal activation between 10 −6 and 10 −5 mol/L forskolin. Forskolin-stimulated (10 −5 mol/L) and basal adenylate cyclase activities declined in parallel with increasing calcium concentrations. In contrast, phosphodiesterase activity was unaffected by forskolin at 10 −6 to 10 −4 mol/L. Forskolin, therefore, by directly activating adenylate cyclase, enhances cAMP accumulation and PTH release in dispersed bovine parathyroid cells. As with other agents activating cAMP accumulation in this system, these effects are inhibited by elevated calcium concentrations. Thus, the activation of PTH secretion by cAMP and the modulation of these processes by calcium are independent of the locus at which the increase in cAMP takes place.

Forskolin stimulates adenylate cyclase in human colonic crypts: Interaction with VIP

Forskolin in the 10 −8−10 −4 M concentration range (ED 50 2 μM) strongly stimulated the cyclic AMP production of epithelial crypts isolated from the human colon. At a maximal dose, production increased up to 500 and 700 times the basal cyclic AMP levels at 15 and 37°C, respectively. Forskolin was thus much more efficient than VIP, which is the physiological regulator of this system. Forskolin (ED 50 7 μM) also stimulated colonic membrane adenylate cyclase. The stimulation was immediate, did not require guanyl nucleotides and was inhibited by calcium (10 −5−10 −3 M). In the concentration range between 10 −9 and 10 −5 M (ED 50 0.04 μM), forskolin strongly potentiated the stimulation of adenylate cyclase by VIP. We conclude that: (1) forskolin exerts a double dose-dependent action on cyclic AMP production in human colonic crypts, i.e. direct activation of basal adenylate cyclase activity and potentiation of VIP effect; (2) forskolin may be a unique pharmacological tool to investigate the cyclic AMP-dependent processes in human intestine.

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release.

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes.

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Forskolin stimulates adenylate cyclase activity, adenosine 3',5'-monophosphate production and peptide release from the intermediate lobe of the rat pituitary gland.

Forskolin stimulates adenylate cyclase activity in a cell-free homogenate of the intermediate lobe (IL) of the rat pituitary gland. L-Isoproterenol, a beta-adrenergic agonist, enhances and apomorphine, a D-2 agonist, inhibits the forskolin-stimulated adenylate cyclase activity. Guanosine triphosphate is obligatory for the dopaminergic attenuation of the forskolin-enhanced enzyme activity. When tested upon enzymatically dispersed IL cells, forskolin enhances the release of both cAMP and immunoreactive alpha MSH-like peptides. These effects of forskolin upon the IL are discussed within the context of a previously published working hypothesis that cAMP in some way triggers the calcium-dependent release of hormones from the IL.

Adenylate cyclase in the maturing human reticulocyte: selective loss of the catalytic unit, but not of the receptor-cyclase coupling protein.

Hormone sensitive adenylate cyclase consists of at least three components; hormone receptor, a catalytic unit, and the N-protein, which couples the receptor to the catalytic unit. Human erythrocytes have very low adenylate cyclase activity, but do have N-protein activity. We have investigated the fate of the catalytic unit and the N protein during maturation of human reticulocytes. We compared red blood cell membranes derived from nine patients with reticulocytosis averaging 12% to erythrocyte membranes from eight normal subjects. The catalytic unit was assayed by stimulation of adenylate cyclase with forskolin, and the N-protein activity by an in vitro complementation assay. Forskolin stimulated adenylate cyclase activity was ten times higher in membranes derived from patients with reticulocytosis as compared with those of normal subjects. N-protein activity was only slightly (12%) and insignificantly (P = 0.08) higher in the reticulocytosis patients membranes. We conclude that during maturation of the human erythrocyte there is a differential loss of the catalytic unit of adenylate cyclase.

Transient inhibition on calcium mobilization from cultured mouse calvarial bones by the adenylate cyclase stimulator forskolin.

Transient inhibition on calcium mobilization from cultured mouse calvarial bones by the adenylate cyclase stimulator forskolin.