Hormone inhibition of adenylyl cyclase. Differences in the mechanisms for inhibition by hormones and G protein beta gamma

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.     

Protective effects of insulin-like growth factor-I on the somatostatinergic system in the temporal cortex of beta-amyloid-treated rats

Insulin-like growth factor-I (IGF-I) has protective effects against beta-amyloid (Abeta)-induced neuronal cell death. Because alterations of the somatostatinergic system have been described in Alzheimer's disease, we investigated the effects of the Abeta peptide and the possible protective role of IGF-I on the somatostatinergic system of the rat temporal cortex and on cell death and phosphorylated (p)-Akt levels in this area. Abeta25-35 was administered intracerebroventricularly to male rats via an osmotic minipump over 14 days (300 pmol/day). Another group received a subcutaneous IGF-I infusion (50 microg/kg/day), concomitant with Abeta25-35 administration, whereas a third group received IGF-I alone. Abeta25-35 significantly decreased the somatostatin (SRIF)-like immunoreactive content and the SRIF receptor density, as a result of a decrease in the levels of the SRIF receptor subtype 2. The inhibitory effect of SRIF on adenylyl cyclase activity was significantly lower after Abeta25-35 infusion, whereas the levels of the inhibitory G protein subunit Gialpha1, Gialpha2 or Gialpha3 were unaltered. Cell death was increased and p-Akt levels decreased in Abeta25-35-treated animals. IGF-I administration increased immunoreactive IGF-I levels in the temporal cortex and restored all parameters affected by Abeta25-35 to baseline values. These findings suggest that IGF-I prevents the deleterious effect of Abeta25-35 on the somatostatinergic system.     

Vitamin E deficiency impairs the somatostatinergic receptor–effector system and leads to phosphotyrosine phosphatase overactivation and cell death in the rat hippocampus

Vitamin E plays an essential role in maintaining the structure and function of the nervous system, and its deficiency, commonly associated with fat malabsorption diseases, may reduce neuronal survival. We previously demonstrated that the somatostatinergic system, implicated in neuronal survival control, can be modulated by α-tocopherol in the rat dentate gyrus, increasing cyclic adenosine monophosphate response element binding protein phosphorylation. To gain a better understanding of the molecular actions of tocopherols and examine the link among vitamin E, somatostatin and neuronal survival, we have investigated the effects of a deficiency and subsequent administration of tocopherol on the somatostatin signaling pathway and neuronal survival in the rat hippocampus. No changes in somatostatin expression were detected in vitamin-E-deficient rats. These rats, however, showed a significant increase in the somatostatin receptor density and dissociation constant, which correlated with a significant increase in the protein levels of somatostatin receptors. Nevertheless, vitamin E deficiency impaired the ability of the somatostatin receptors to couple to the effectors adenylyl cyclase and phosphotyrosine phosphatase by diminishing Gi protein functionality. Furthermore, vitamin E deficiency significantly increased phosphotyrosine phosphatase activity and PTPη expression, as well as PKCδ activation, and decreased extracellular-signal-regulated kinase phosphorylation. All these changes were accompanied by an increase in neuronal cell death. Subsequent α-tocopherol administration partially or completely reversed all these values to control levels. Altogether, our results prove the importance of vitamin E homeostasis in the somatostatin receptor–effector system and suggest a possible mechanism by which this vitamin may regulate the neuronal cell survival in the adult hippocampus.     

Modulation of somatostatin receptors,somatostatin content and Gi proteins by substance P in the rat frontoparietal cortex and hippocampus

Substance P (SP) and somatostatin (SRIF) are widely spread throughout the CNS where they play a role as neurotransmitters and/or neuromodulators. A colocalization of both neuropeptides has been demonstrated in several rat brain areas and SP receptors have been detected in rat cortical and hippocampal somatostatinergic cells. The present study was thus undertaken to determine whether SP could modulate SRIF signaling pathways in the rat frontoparietal cortex and hippocampus. A single intraperitoneal injection of SP (50, 250 or 500 micro g/kg) induced an increase in the density of SRIF receptors in membranes from the rat frontoparietal cortex at 24 h of its administration, with no change in the hippocampus. The functionality of the SRIF receptors was next investigated. Western blot analysis of Gi proteins demonstrated a significant decrease in Gialpha1 levels in frontoparietal cortical membranes from rats treated acutely (24 h) with 250 micro g/kg of SP, which correlated with a decrease in functional Gi activity, as assessed by use of the non-hydrolyzable GTP analog 5'-guanylylimidodiphosphate. SRIF-mediated inhibition of basal or forskolin-stimulated adenylyl cyclase activity was also significantly lower in the frontoparietal cortex of the SP-treated group, with no alterations in the catalytic subunit of the enzyme. SRIF-like immunoreactivity content was increased in the frontoparietal cortex after acute (24 h) SP administration (250 or 500 micro g/kg) as well as in the hippocampus in response to 7 days of SP (250 micro g/kg) administration. All these SP-mediated effects were prevented by pretreatment with the NK1 receptor antagonist RP-67580. Although the physiologic significance of these results are unknown, the increase in SRIF receptor density together with the desensitization of the SRIF inhibitory signaling pathway might be a mechanism to potentiate the stimulatory pathway of SRIF, inducing a preferential coupling of the receptors to PLC.     

Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase.

We previously reported the cloning of two distinct somatostatin receptor (SSTR) subtypes, SSTR1 and SSTR2. Although both SSTR1 and SSTR2 bound somatostatin specifically and with high affinity, neither was coupled to adenylyl cyclase, a major cellular effector of somatostatin's actions. Here we report the cloning and functional characterization of a third member of the SSTR family. Human SSTR3 is a protein of 418 amino acids and has 45% and 46% identity with human SSTR1 and SSTR2, respectively. RNA blotting studies showed that SSTR3 mRNA could be readily detected in brain and pancreatic islets. The pharmacological properties of human SSTR3 were characterized by transiently expressing the human SSTR3 gene in COS-1 cells. Membranes from cells expressing human SSTR3 bound the somatostatin agonist [125I]CGP 23996 specifically and with high affinity, with a rank order of potency of somatostatin-28 = CGP 23996 > somatostatin-14 > SMS-201-995. Studies using cells transiently coexpressing the human dopamine D1 receptor and human SSTR3 showed that somatostatin was able to inhibit dopamine-stimulated cAMP formation in a dose-dependent manner, indicating that SSTR3 was functionally coupled to adenylyl cyclase. These results indicate that the diverse biological effects of somatostatin are mediated by a family of receptor with distinct, but overlapping, tissue distributions, unique pharmacological properties, and potentially different functions.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Somatostatin-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.     

Adaptive increase in adenylyl cyclase activity in NG108-15 and S49 cells induced by chronic treatment with inhibitory drugs is not due to a decrease in cyclic AMP concentrations.

NG108-15 neuroblastoma x glioma hybrid cells and S49 lymphoma cells exhibit an enhancement in adenylyl cyclase activity after chronic treatment with receptor agonists that acutely inhibit the enzyme. Using agonists that activate five distinct inhibitory receptors in NG108-15 cells, we have found that there is a correlation between the extent of acute inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation and efficacy for induction of enhanced PGE1 stimulation of cAMP accumulation after chronic treatment and withdrawal. Chronic treatment with dideoxyadenosine, which acutely inhibits adenylyl cyclase activity by a mechanism independent or cell surface receptors or pertussis toxin-sensitive G proteins, did not induce enhanced PGE1 stimulation of cAMP accumulation in NG108-15 cells or forskolin stimulation of cAMP accumulation in S49 cells. While control basal cAMP concentrations were acutely decreased by carbachol in NG108-15 cells and by somatostatin in S49 cells, when the cAMP concentrations were maintained above the control basal values with a phosphodiesterase inhibitor, chronic treatment with these inhibitory drugs nonetheless resulted in enhanced cAMP responses in both NG108-15 and S49 cells. These results provide evidence that the initial decrement in cAMP concentrations caused by inhibitory drug is not the requisite signal for inducing the subsequent sensitization of adenylyl cyclase in NG108-15 and S49 cells but that activation of a pertussis toxin-sensitive G protein is involved in the development of this important adaptation.     

Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation

Abstract: To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre-and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7β-forskolin) increased enzyme activity ˜ 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3 R -ACPD) inhibited enzyme activity as did glutamate and quisqualate. l -Amino-4-phosphobutanoate ( l -AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist l -2-amino-3-phosphopropionic acid ( l -AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3 R -ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3 R -ACPD, quisqualate, and l -AP4 increased adenylyl cyclase activity. In the SN, l -AP3 was ineffective in antagonizing any agonist-induced increases (1S,3 R -ACPD, l -AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.     

Somatostatin inhibits follicle-stimulating hormone-induced adenylyl cyclase activity and proliferation in immature porcine Sertoli cell via sst2 receptor

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [(3)H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [(3)H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.     

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.     

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Study of molecular mechanisms of the relaxin action on adenylyl cyclase signaling system using synthetic peptides derived from the relaxin receptor LGR7

The peptide hormone relaxin in dose-dependent manner stimulates adenylyl cyclase activity in the rat tissues (brain striatum, heart and skeletal muscles) and the muscle tissues of invertebrates--bivalve mollusk Anodonta cygnea and earthworm Lumbricus terrestris. Adenylyl cyclase stimulating effect of the hormone is most expressed in striatum and heart muscles of rats. For identification of the type ofrelaxin receptors, participating in the realization of this effect of the hormone, the peptides 619-629, 619-629-Lys(Palm) and 615-629 derived from the primary structure of C-terminal region of the third intracellular loop of the relaxin receptor of type 1 (LGR7), were synthesized by us for the first time. It is shown that peptide: 619-629-Lys(Palm) and 615-629 in competitive manner inhibit the stimulation of the adenylyl cyclase by relaxin in brain striatum and heart muscle of rats. At the same time, these peptides do not change stimulating effect of the hormone in the skeletal muscles of rat and in the muscles of invertebrates. Thus, the peptide action on adenylyl cyclase effect of relaxin is tissue- and species-specific. These data, on the one hand, demonstrate participation of receptor LGR7 in realization of adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle of rats and, on the other, give evidence for existence of another adenylyl cyclase signaling mechanisms of relaxin action in the skeletal muscles and the muscle of invertebrates, which do not involve LGR7 receptor. The adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle was decreased in the presence of C-terminal peptides 385-394 of alpha(s)-subunit of mammalian G protein and was blocked by treatment of the membranes with cholera toxin. On the basis of data obtained the following conclusions were made: (i) in striatum and heart muscle the relaxin stimulates adenylyl cyclase through LGR7 receptors functionally coupled with Gs protein, and (ii) the coupling between hormoneactivated relaxin receptor LGR7 and Gs protein is realized via the interaction of C-terminal part of receptor third intracellular loop and C-terminal segment of Gs protein alpha-subunit.     

Multiple pertusis toxin substrates as candidates for regulatory G proteins of adenylate cyclase coupled to the somatostatin receptor in primary rat astrocytes

The involvement of G proteins in receptor mediated astroglial cAMP formation was studied. Isoproterenol or prostaglandin E2 stimulated adenylate cyclase of primary astroglial cells was inhibited by somatostatin. Preincubation, of cells with increasing concentrations of islet activating protein (IAP) diminished somatostatin inhibition of adenylate cyclase. At an IAP concentration of 50 ng/ml somatostatin inhibition was completely abolished. Studies on IAP catalyzed³²P-ADP-ribosylation of astroglial cell particulate material revealed an incorporation of radiolabel into three polypeptides in the molecular weight range of 41,000–39,000 Dalton. Pretreatment of intact cells with IAP reduced radiolabeling of this molecular species in a concentration dependent manner. No further radiolabeling above background level was detectable after pretreatment of cultures with 10 ng IAP/ml or more. At present, the occurrence of at least three IAP substrates (G proteins) does not permit an identification of the somatostatin receptor coupled G protein. Rather, the finding reveals that astrocytes are endowed with multiple variants of GTP binding proteins likely to be coupled to different receptors.Abbreviations cAMP cyclic adenosine monophosphate - DPBS Dulbecco's phosphate buffered saline - DTT dithiothreitol - IAP islet activating protein=pertussis toxin - PG prostaglandin     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

The dopamine D(2) receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.