Hormone-sensitive Adenylyl Cyclase System Research Articles

The Functional State of Hormone-Sensitive Adenylyl Cyclase Signaling System in Diabetes Mellitus

Diabetes mellitus (DM) induces a large number of diseases of the nervous, cardiovascular, and some other systems of the organism. One of the main causes of the diseases is the changes in the functional activity of hormonal signaling systems which lead to the alterations and abnormalities of the cellular processes and contribute to triggering and developing many DM complications. The key role in the control of physiological and biochemical processes belongs to the adenylyl cyclase (AC) signaling system, sensitive to biogenic amines and polypeptide hormones. The review is devoted to the changes in the GPCR-G protein-AC system in the brain, heart, skeletal muscles, liver, and the adipose tissue in experimental and human DM of the types 1 and 2 and also to the role of the changes in AC signaling in the pathogenesis and etiology of DM and its complications. It is shown that the changes of the functional state of hormone-sensitive AC system are dependent to a large extent on the type and duration of DM and in experimental DM on the model of the disease. The degree of alterations and abnormalities of AC signaling pathways correlates very well with the severity of DM and its complications.

Alterations of Hormone-Sensitive Adenylyl Cyclase System in the Tissues of Rats with Long-Term Streptozotocin Diabetes and the Influence of Intranasal Insulin

One of the causes of complications in type 1 diabetes mellitus (T1DM) is the changes in adenylyl cyclase (AC) signaling system, identified on the early stages of the disease. However, the most significant disturbances in this system occur on the later stages of T1DM, which ultimately leads to severe complications, but functional state of the AC system in late T1DM is poorly understood. The aim of this work was to study alterations in AC system sensitive to biogenic amines and polypeptide hormones in the heart, brain, and testes of male rats with long-term, 7-month, streptozotocin T1DM and to assess the influence on them of 135-day therapy with intranasal insulin. It was shown that AC effects ofβ-adrenergic agonists in the heart, serotonin receptor agonists and PACAP-38 in the brain, chorionic gonadotropin and PACAP-38 in the testes, and somatostatin in all investigated tissues in long-term T1DM were drastically decreased. The treatment with intranasal insulin (0.48 IU/day) significantly restored these effects. The results were obtained suggesting that long-term T1DM induces significant alterations in hormone-sensitive AC system in the heart, brain, and testes that are much more pronounced, compared with short-term T1DM, and include a large number of hormonal regulations.

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.

Effects of thiols and blockers of sulfhydryl groups on negative regulation of the adenylyl cyclase signaling system by biogenic amines

Effect of a thiol-containing compound, β-mercaptoethanol, and of blockers of cysteine sulfhydryl groups (para-chlormercury benzoate andN-ethylmaleimide) on hormonal negative regulation of functional activity of the adenylyl cyclase system was studied in smooth muscles of the freshwater bivalve molluscAnodonta cygnea (agonist of s-adrenoreceptors—isoproterenol) and in the rat skeletal muscles (serotonin). It was found that in the presence of s-mercaptoethanol the inhibitory effects of isoproterenol (mollusc) and serotonin (rat) were preserved. At the same time, the blockers of SH-groups disturb essentially both the hormonal effects and their potentiation in the presence of Gpp[NH]p, this process being practically irreversible in the presence ofN-ethylmaleimide. Thus, it has been shown that the negative hormonal regulation in the muscle membranes of the mollusc and rat is dependent on the SH-group state in the protein components of the hormonesensitive adenylyl cyclase system.

In vitro bioassay for human serum follicle-stimulating hormone (FSH) based on L cells transfected with recombinant rat FSH receptor: validation of a model system.

FSH plays a central role in normal reproductive function, i.e. control of follicular maturation in the female and initiation and maintenance of spermatogenesis in the male. The effects of FSH are mediated by its interaction with a specific receptor that belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors. Due to the microheterogeneity of gonadotropins, measurement of immunoreactivity does not necessarily reflect their bioactivity. Mutations in gonadotropin beta-subunits, which affect bioreactivity and/or immunoreactivity of gonadotropins, have been described as causes of infertility, thus highlighting the need for rapid and convenient methods to measure bioactivity. To establish a model system for recombinant in vitro bioassays for FSH that would obviate the use of live animals, we developed a strategy for efficient expression of the rat FSH receptor (FSHR) in L cells. A cell line, FSHR 7/12, was developed that bound [125I]FSH with high affinity (Kd 1.42 nM) and responded to human FSH with an increase in cAMP accumulation. Untreated human serum was found to have an unspecific inhibitory effect on cAMP formation. This effect could be thoroughly avoided by mild heating (10 min at 56 C) of serum samples before addition to cells without detectable loss of FSH immunoactivity or bioactivity. Studies on the hormone-sensitive adenylyl cyclase system of transformed FSHR 7/12 cells and of the parental Ltk- cells showed that the cellular response to FSH was highly specific. Using a parallel line assay design, FSHR 7/12 cells were used to validate a novel recombinant in vitro bioassay relying on intracellular cAMP accumulation as a readout system. Up to 10% of serum could be added to the incubation buffer without leading to nonparallelism to the standard curve. When 70 serum samples of male patients attending an infertility clinic were analyzed, the novel assay system displayed high sensitivity and a close correlation (r > 0.8; P < 0.01) to the established rat Sertoli cell aromatase bioassay and to a highly specific fluoroimmunoassay. When sera of 25 normal menstruating women were analyzed for FSH bioactivity at different stages of the menstrual cycle, a midcycle FSH peak followed by a decline in the late luteal phase could be discerned. The analysis of 26 serum samples of postmenopausal women revealed a close correlation between FSH values obtained by the novel in vitro bioassay and by a fluoroimmunoassay (r = 0.90; P < 0.01). Thus, the present in vitro bioassay represents a sensitive, rapid, and convenient model system to measure bioactive FSH in human serum.

In vitro bioassay for human serum follicle-stimulating hormone (FSH) based on L cells transfected with recombinant rat FSH receptor: validation of a model system

FSH plays a central role in normal reproductive function, i.e. control of follicular maturation in the female and initiation and maintenance of spermatogenesis in the male. The effects of FSH are mediated by its interaction with a specific receptor that belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors. Due to the microheterogeneity of gonadotropins, measurement of immunoreactivity does not necessarily reflect their bioactivity. Mutations in gonadotropin beta-subunits, which affect bioreactivity and/or immunoreactivity of gonadotropins, have been described as causes of infertility, thus highlighting the need for rapid and convenient methods to measure bioactivity. To establish a model system for recombinant in vitro bioassays for FSH that would obviate the use of live animals, we developed a strategy for efficient expression of the rat FSH receptor (FSHR) in L cells. A cell line, FSHR 7/12, was developed that bound [125I]FSH with high affinity (Kd 1.42 nM) and responded to human FSH with an increase in cAMP accumulation. Untreated human serum was found to have an unspecific inhibitory effect on cAMP formation. This effect could be thoroughly avoided by mild heating (10 min at 56 C) of serum samples before addition to cells without detectable loss of FSH immunoactivity or bioactivity. Studies on the hormone-sensitive adenylyl cyclase system of transformed FSHR 7/12 cells and of the parental Ltk- cells showed that the cellular response to FSH was highly specific. Using a parallel line assay design, FSHR 7/12 cells were used to validate a novel recombinant in vitro bioassay relying on intracellular cAMP accumulation as a readout system. Up to 10% of serum could be added to the incubation buffer without leading to nonparallelism to the standard curve. When 70 serum samples of male patients attending an infertility clinic were analyzed, the novel assay system displayed high sensitivity and a close correlation (r > 0.8; P < 0.01) to the established rat Sertoli cell aromatase bioassay and to a highly specific fluoroimmunoassay. When sera of 25 normal menstruating women were analyzed for FSH bioactivity at different stages of the menstrual cycle, a midcycle FSH peak followed by a decline in the late luteal phase could be discerned. The analysis of 26 serum samples of postmenopausal women revealed a close correlation between FSH values obtained by the novel in vitro bioassay and by a fluoroimmunoassay (r = 0.90; P < 0.01). Thus, the present in vitro bioassay represents a sensitive, rapid, and convenient model system to measure bioactive FSH in human serum.

A mechanism of catecholamine tolerance in congestive heart failure--alterations in the hormone sensitive adenylyl cyclase system of the heart.

It is well known that failing hearts show diminished responsiveness (desensitization) to catecholamines. In this study, 2 different animal models were used to investigate the alterations in the hormone sensitive cardiac adenylyl cyclase system in a congestive heart failure. In the first model, cardiomyopathic Syrian hamsters (BIO53.58), we found reduced activity of the catalytic protein of adenylyl cyclase; this reduction was more prominent in an older animals (28-week-old vs 16-week-old). At both ages, the amount of inhibitory GTP-binding protein (Gi) was markedly increased in BIO53.58 compared to control healthy hamsters. Moreover the increased Gi was shown to be fully functional in the inhibitory pathway of the adenylyl cyclase system. In the second model, chronically norepinephrine-infused rats, we found a decrease in beta-adrenergic receptor density at an early stage of injection (3 days), while the activity of catalytic protein decreased beyond 14 days of injection, and the amount of Gi increased after 7 days of injection. These results suggest that increased plasma catecholamine concentrations in the setting of congestive heart failure might be a major trigger for qualitative and quantitative alterations observed in various components of cardiac adenylyl cyclase system, and that GTP-binding proteins and catalytic protein of adenylyl cyclase are involved in the mechanism of desensitization especially after chronic in vivo stimulation of adrenergic receptors.

Cross-regulation between G-protein-mediated pathways. Activation of the inhibitory pathway of adenylylcylclase increases the expression of beta 2-adrenergic receptors

Cross-regulation from the stimulatory (Gs alpha)-mediated) to the inhibitory (Gi alpha-mediated) pathways controlling adenylylcyclase has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). The extent to which cross-regulation occurs from inhibitory to stimulatory pathways for adenylylcyclase was explored. Persistent activation of the inhibitory pathway of adenylylcyclase by the A1-adenosine receptor agonist (-)-N6 (R-phenylisopropyl) adenosine (PIA) in hamster smooth muscle DDT1 MF-2 cells enhanced the stimulatory pathway of adenylylcyclase and its activation by the beta 2-adrenergic receptor agonist isoproterenol. PIA treatment (48 h) of cells increased isoproterenol-stimulated adenylylcyclase by 2-fold. In addition, the ED50 for stimulation of adenylylcyclase by isoproterenol decreased 50-fold to approximately 1 nM. Persistent activation of cells with PIA increased beta 2-adrenergic receptor number in a time- and dose-dependent manner. The steady-state levels of beta 2-adrenergic receptors (radioligand binding and immunoblotting) and receptor mRNA levels increased by more than 70%, while the half-life of the receptor (24 h) was unaltered. Both A1-adenosine receptor binding and Gi alpha 2 levels declined by half in cells persistently activated with PIA. Although Gi alpha 2 mRNA levels and the relative rate of synthesis of Gi alpha 2 protein upon persistent activation of the inhibitory pathway were found to increase, a decrease in the half-life of Gi alpha 2 from approximately 75 h in naive cells to approximately 40 in cells provides the basis for the decline in Gi alpha 2 levels. The steady-state level of mRNA and half-life of Gs alpha protein were unaltered in persistently activated cells. Thus, activation of the inhibitory pathway of adenylylcyclase cross-regulates the stimulatory, hormone-sensitive adenylylcyclase system by: (i) up-regulating beta 2-adrenergic receptors and enhancing the activation of the stimulatory adenylylcyclase pathway and (ii) down-regulating elements of the inhibitory adenylylcyclase pathway (Gi alpha 2 and A1-adenosine receptor binding).

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.

Altered action of glucagon on human liver in type 2 (non-insulin-dependent) diabetes mellitus.

Glucagon may play a role in the metabolic derangements of overt Type 2 (non-insulin-dependent) diabetes mellitus. We therefore have evaluated the early steps in glucagon action by investigating the hormone-sensitive adenylyl cyclase system in liver membranes from seven Type 2 diabetic patients with fasting hyperglycaemia and two-fold elevations in plasma glucagon. The comparison was made with seven control subjects matched for age, sex and body weight. Glucagon receptor binding was almost identical in the two groups. There were, however, marked alterations in the adenylyl cyclase activity in membranes from the diabetic patients. This activity was reduced by 35-50% when compared to control activity. Basal cyclase activity, as well as the activity after stimulation with glucagon or with agents (i.e., sodium fluoride and forskolin) that act beyond the glucagon receptor, was significantly decreased (p less than 0.05, p less than 0.001 respectively). In conclusion, uncontrolled Type 2 diabetes in associated with an over-all loss of responsiveness of the hormone-sensitive adenylyl cyclase in human liver, which apparently results from post-receptor alterations. This change may provide a mechanism for reducing the effect of hyperglycagonaemia in Type 2 diabetes mellitus.