Adenylyl Cyclase System Research Articles

Striatal specific orphan GPCRs crosstalk with Dopamine D2 receptors to regulate cAMP signaling

The striatum is a subcortical brain region that performs vital functions including movement control, regulation of attention and is a critical component of the reward system. Current drug targets have displayed limited efficacy in treating striatal‐related diseases including for substance‐use disorders, schizophrenia and Parkinson’s disease. G protein‐coupled receptors (GPCRs) are valuable drug targets with ~35% of FDA approved medicines targeting these receptors. However, nearly 120 GPCRs remain orphan receptors, whose endogenous ligands, G protein signaling, and therapeutic potential are unknown. We previously conducted a comprehensive RNAseq genome‐wide survey of human tissue gene expression and identified several orphan GPCRs selectively expressed in the human striatum, namely, GPR6, 52, 88, 101, 139 and 149. Here we elucidate striatal orphan receptors that modulate G protein‐mediated cAMP signaling and examine potential signaling crosstalk between orphan receptors and dopamine D2 receptors (D2R). Biochemical and CRISPR/Cas9 knockout studies in human embryonic kidney 293 (HEK293) cells confirmed GPR6, 52 and 101 increased cyclic adenosine monophosphate (cAMP) via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Notably, when expressed alone, GPR6, 52 and 101 profoundly increased basal levels of cAMP, indicating these receptors are constitutively active. We hypothesized that GPR6, 52, and 101 may set the basal tone for cAMP that would allow robust function of the Gi/o‐coupled dopamine D2R to inhibit adenylyl cyclase and decrease cAMP. When expressed alone in HEK293 cells and without any stimulation to the adenylyl cyclase system, the cAMP inhibition window for the D2R and agonist quinpirole was minimal and difficult to reliably detect (~1000 light counts/sec, Glosensor assay). However, when the D2R was co‐expressed with either GPR6, 52, or 101, the assay window to inhibit cAMP for quinpirole was magnified by over 200X (200,000–500,000 light counts/sec), with agonist potency unchanged. These studies suggest that these striatal specific human orphan receptors may establish dynamic basal cAMP levels and this may facilitate inhibition of cAMP via Gi/o‐coupled receptors, such as the D2R. Our findings also suggest GPR6, GPR52 and GPR101 are potential drug targets to modulate cAMP signaling in striatal specific neurons, which may be therapeutic for treating several neurological and neuropsychiatric diseases.Support or Funding InformationNIDA T32 DA07287 (DEF) and RSGPT18‐ PhRMA Foundation (JAA).

Decrease in the Basal and Luteinizing Hormone Receptor Agonist–Stimulated Testosterone Production in Aging Male Rats

In the course of aging, the steroidogenic function of the testes weakens and their gonadotropin sensitivity decreases. However, the underlying mechanisms are poorly understood. The goal of this work was to study the stimulating effects of human chorionic gonadotropin (hCG) and TP03, a low molecular weight agonist of luteinizing hormone (LH)/hCG receptor, on testosterone production and the expression of steroidogenic proteins in young (3-month-old) and aging (15-month-old) male rats and to investigate the activity of the adenylyl cyclase system in the membranes isolated from the rat testes. Treatment with hCG (100 IU/rat/day) and TP03 (15 mg/kg/day) was carried out for 3 days. In the testes of aging rats, adenylyl cyclase stimulation by gonadotropin and guanine nucleotide was decreased, indicating a weakening of the coupling of LH/hCG receptor and Gs protein, the main components of the adenylyl cyclase system regulating the steroidogenesis. In elderly rats, the blood testosterone levels and the expression of the Star, Cyp11a1, and Cyp17a1 genes, which encode the StAR protein and the steroidogenic enzymes cytochromes P450scc and P450-17α in the testes, were decreased. The stimulating effect of both hCG and TP03 on testosterone production diminished with age, although their effects on LH/hCG receptor are mediated by different mechanisms. In both young and aging rats, hCG treatment upregulated the expression of the genes encoding StAR, P450scc and dehydrogenase 3β-HSD; at the same time, Hsd17B expression in aging rats increased, and the expression of the genes encoding P450-17α and 17β-HSD decreased in young rats. In young rats, TP03 treatment upregulated Star and Cyp17a1 expression, and it increased Star and Hsd17B expression in aging rats. Thus, in the testes of aging rats, the coupling between the LH/hCG receptor and Gs protein and LH/hCG receptor sensitivity to agonists were weakened, which led to a decrease in hCG- and TP03-induced testosterone production and altered the basal and LH/hCG receptor agonist–stimulated expression levels of some steroidogenic protein genes.

Взаимосвязь между андрогенным дефицитом и ослаблением чувствительности аденилатциклазы к гонадотропинам в семенниках крыс со стрептозотоциновым диабетом различной степени тяжести

Type 1 diabetes mellitus (DM1) leads to dysfunctions of the male reproductive system, which is caused by a decrease in testosterone (T) synthesis by the testes. Currently, there is little studied how the severity of DM1 affects the T production, and what role in the development of androgen deficiency is played by a decrease in the sensitivity of the adenylyl cyclase system of Leydig cells to gonadotropins. The aim of the work was to study the basal and human chorionic gonadotropin(hCG)-stimulated T levels in male rats with severe, moderate and mild forms of DM1, as well as to study the regulation of the enzyme adenylyl cyclase (AC) by non-hormonal agents (forskolin, GppNHp) and hCG in the testicular membranes of diabetic rats. The severe (D-T) and moderate (D-S) forms of DM1 were caused by treatment of four-month-old rats with streptozotocin at the doses of 60 and 35 mg/kg, and the mild form (D-M) was induced by treatment of five-day-old rat pups with streptozotocin (75 mg/kg), which led to the development of diabetes at the age of 4 months. The values ​​of AUC(11.00-18.00) (the integrated area under the curve “concentration of T(nM) –time(h)”) in the D-T, D-S and D-M groups was 50.4±10.4, 75.7±21.2 and 105.9±9.3 arb.u. and were reduced by 69, 53 and 34% in comparison with control (161.1±18.6 arb.u., p<0.05). The AUC(11.00-16.00) for the T level stimulated by hCG (100 IU/rat) in the same groups were 115.4±30.4, 229.6±64.3 and 306.4±58.8 arb.u. and were reduced by 74, 48 and 31% in comparison with control (445.4±132.7 arb.u., p<0.05). In the testicular membranes of rats of the D-T group, the basal and the forskolin-, GppNHp- and hCG-stimulated AC activity were decreased by 34, 39, 54 and 68%, respectively. In the D-S and D-M groups, only the AC effects of GppNHp and hCG were decreased. Thus, the androgen deficiency in rats with DM1 and the steroidogenic response in the case of their treatment with hCG depend on the severity of DM1, and the key cause of the androgen deficiency is the impaired functional activity of the gonadotropin-sensitive adenylyl cyclase system in the testes of diabetic rats.

Cardiotoxicity of β-mimetic catecholamines during ontogenetic development — possible risks of antenatal therapy

Catecholamines are involved in the regulation of a wide variety of vital functions. The β-adrenergic receptor (β-AR) - adenylyl cyclase system has been identified early in embryogenesis before the heart has received adrenergic innervation. The structure of β-receptors in the immature myocardium is similar to that in adults; there are, however, significant quantitative developmental changes in the inotropic and chronotropic responsiveness. Information on the toxic effect of the β-AR agonists in the immature heart is surprisingly scarce, even though these agents are used in clinical practice both during pregnancy and in early postnatal development. Large doses of β-AR agonists induce malformations of the cardiovascular system; the type of change depends upon the time at which the β-AR agonist was administered during embryogenesis. During postnatal ontogeny, the cardiotoxicity of β-AR agonists increased from birth to adulthood. It seems likely that despite interspecies differences, developmental changes in the cardiac sensitivity to β-AR agonists may exist in all mammals, depending on the degree of maturation of the system involved in β-adrenergic signaling. All the existing data draw attention to the possible harmful consequences of the clinical use of β-AR agonists during early phases of cardiac development. Late effects of the early disturbances of the cardiac muscle cannot be excluded.

Changes in Nociceptive Thresholds and Adenylyl Cyclase System Activity in Skeletal Muscles in Rats with Acute and Mild Type 1 Diabetes

Diabetic peripheral neuropathy (DPN) is one of the commonest complications of type 1 diabetes mellitus (DM1). The aims of the present work were to study the dynamics of the development of pain-type DPN and the functional status of the hormone-sensitive adenylyl cyclase signal system (ACSS) in the skeletal muscle of rats with models of acute and mild DM1 and to investigate the influences on these of insulin therapy using different routes of hormone administration – intranasal and peripheral. The nociceptive threshold in rats decreased in both models of DM1; the stimulatory effects of guanine nucleotides (GIDP) and adrenergic agonists (isoproterenol, BRL-37344) on adenylyl cyclase (AC) also decreased. The AC-stimulating effect of relaxin decreased in animals with acute DM1, while the change in mild DM1 was minor. Peripheral administration of insulin to rats with acute DM1 increased the nociceptive threshold and partially restored the AC effect of the β3 agonist BRL-37344. Intranasal administration of insulin to rats with mild DM1 also led to an increase in the nociceptive threshold and partially restored basal and BRL-37344-stimulated AC activity in the skeletal muscle of diabetic animals. Thus, skeletal muscle in rats with acute and mild DM1 showed impaired nociceptive sensitivity and ACSS function, which was partially restored by treatment of animals with insulin given peripherally (acute DM1) or intranasally (mild DM1).

The effect of prolonged metformin treatment on the activity of the adenylyl cyclase system and NO-synthase in the brain and myocardium of obese rats

The biguanide metformin, which is widely used for the treatment of type 2 diabetes, improves carbohydrate and lipid metabolism and has substantial cardioand neuroprotective effects. The positive effects of metformin on the activity of NO synthases catalyzing the synthesis of NO, a major vasodilator, and that of the hormone-sensitive adenylyl cyclase signaling system (ACSS) are believed to play an important role in the mediation of these effects. To prove this hypothesis, we initiated a study addressing the effect of prolonged metformin treatment of obese rats on metabolic parameters and the activity of ACSS and NO synthases in the myocardium and brain of the animals. Obesity was induced in Wistar rats by a high-fat diet, and the animals were subsequently treated with metformin for 2 months (the daily dose was 200 mg/kg). Metformin treatment induced a decrease in adipose tissue mass, total body mass, levels of insulin and glucose, and the insulin resistance index HOMA-IR, as well as improved glucose tolerance, and led to a decrease of the content of atherogenic forms of cholesterol. ACSS stimulation by agonists of β1/β2-adrenergic receptors (ARs) and enhancement of β3-AR signaling was detected in the myocardium of obese rats, while metformin treatment restored ACSS regulation by adrenergic agonists in the myocardium and normalized the balance between β-AR-signaling pathways. The stimulating effects of serotonin and agonists of type 4 melanocortin receptors on adenylyl cyclase, which are attenuated in obesity, were restored in the brain of rats treated with metformin. The treatment completely restored the total activity of NO synthases and the activity of endothelial NO synthase in the myocardium, which are reduced in obesity. Metformin treatment was also shown to induce hyperactivation of NO synthases in the brain and myocardium of healthy animals. It is proposed the effects of prolonged treatment of obese rats with metformin underlie the cardioand neuroprotective actions of this drug.

The influence of bromocryptine treatment on activity of the adenylyl cyclase system in the brain of rats with type 2 diabetes mellitus induced by high-fat diet.

The influence of bromocryptine treatment on activity of the adenylyl cyclase system in the brain of rats with type 2 diabetes mellitus induced by high-fat diet.

Peptide 612–627 of thyrotropin receptor and its modified analogs as regulators of adenylyl cyclase in rat thyroid gland

The specific activity of the thyroid gland is regulated by thyroid-stimulating hormone (TSH) via TSH receptor (TSHR). This receptor is coupled with various types of G proteins, including Gs proteins, through which TSH stimulates activity of enzyme adenylyl cyclase (AC). Since the use of TSH in medicine is restricted, selective regulators of TSHR with activity of agonists and antagonists are being developed. One approach to their creation is development of peptides corresponding to the functionally important TSHR regions that are located in its cytoplasmic loops and are involved in binding and activation of G proteins. We synthesized peptide corresponding to the C-terminal region 612–627 of the third cytoplasmic loop of TSHR and its derivatives modified by residues of palmitic acid (from the N- or C-termini) or by polylysine dendrimer (from the N-terminus) and we studied their effect on the basal and TSH-stimulated AC activity in membrane fractions isolated from the rat thyroid gland. The most active was peptide 612–627-K(Pal)A modified by palmitate from the C-terminus, where the hydrophobic transmembrane region is located in TSHR. At the micromolar concentrations, it increased AC activity and reduced the AC-stimulating TSH effect. The action of 612–627-K(Pal)A was directed to its homologous TSHR, which is indicated by the following facts: the ingibition of Gs proteins, the transductory component of the AC system, by treatment of the membranes with cholera toxin blocked the AC effect of peptide and this effect was not revealed in the tissues where TSHR are absent, the peptide did not influence the stimulating AC effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer had a much lower ability to activate AC in the thyroid gland than 612–627-K(Pal)A, whereas the peptide modified by palmitate from the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable with 612–627-K(Pal)A by the ability to inhibit the AC action of TSH, but it also decreased (although to the lesser degree) the AC effects of other hormones, which indicates its low receptor specificity. Thereby, the obtained data indicate the high efficiency of the peptide 612–627-K(Pal)A as a regulator of TSHR and the possibilities of creation of drugs on its basis for regulation of thyroid-gland functions in the case of pathology.

Attenuation of inhibitory influence of hormones on adenylyl cyclase systems in the myocardium and brain of obese and type 2 diabetic rats as affected by the intranasal insulin treatment

The functional state of adenylyl cyclase signaling system (ACSS) and its regulation by the hormones and the inhibitor of adenylyl cyclase (AC), somatostatine (SST), in the brain and myocardium and by 5-nonyloxytryptamine (5-NOT) in the brain of rats of different ages (2- and 7-month-old) with experimental obesity and combination of obesity and type 2 diabetes mellitus (DM2), as well as the effect of the long-term treatment with intranasally administered insulin (II) on ACSS were studied. It was shown that the basal AC activity in obese and DM2 rats increases in the myocardium and, to a lesser extent, in the brain and decreases under the II treatment. The AC stimulating effect of forskolin decreases in the myocardium, but not in the brain of obese and DM2 rats. The II treatment recovers the AC stimulating effect of forskolin in 7-month-old animals, but has little effect in 5-month-old rats. In obesity as well as under the II treatment, the basal AC activity and its stimulation by forskolin change insignificantly. The AC inhibitory effects of 5-NOT and, particularly, SST are strongly attenuated in the investigated pathology, supposedly due to a reduction in the functional activity of Gi-proteins. The treatment of obesity and its combination with DM2 recovers, completely or partially, the AC inhibitory effects of hormones, most of all in the brain. Since the ACSS dysfunctions are causal to metabolic syndrome and DM2, their elimination by the II treatment promises an effective approach to combat these pathologies and their CNS and cardiovascular complications.

Clozapine effects on adenylyl cyclase activity and serotonin type 1A receptors in human brain post-mortem

Although the pharmacological profile of the atypical antipsychotic clozapine has been extensively studied in animal models, little information is available on its effects in the human brain. In particular, much interest is focused on the understanding of clozapine activity on serotonin (5-HT) neurotransmission, particularly on 5-HT receptor of type 1A (5-HT(1A)) that seems to play a pivotal role in the control of the 5-HT system. The present work, therefore, aimed at evaluating the effects of clozapine and its major metabolite, norclozapine, on the modulation of adenylyl cyclase (AC) velocity via 5-HT(1A) receptors in human post-mortem brain regions, in particular the prefrontal cortex, hippocampus and raphe nuclei. Concomitantly, the ability of the two compounds to displace the specific binding of the 5-HT(1A) receptor agonist [³H]-8-hydroxy-(2-di-N-propylamino) tetralin ([³H]-8-OH-DPAT) was evaluated in the same brain areas. The results showed that both clozapine and norclozapine, although with a 20-fold lower affinity, displaced [³H]8-OH-DPAT binding in all of the brain regions analysed, suggesting their interaction with 5-HT(1A) receptors. At the same time, clozapine and, to a lesser extent, norclozapine were found to inhibit the forskolin (FK)-stimulated AC system, while decreasing cyclic adenosine monophosphate (cAMP) concentrations in the hippocampus only. The receptor characterisation of the clozapine effect on AC observed in the hippocampus by the use of antagonists showed a mixed profile, involving not only the 5-HT(1A) receptor but also a muscarinic (M) receptor subtype, most likely the M₄ one. These findings, while considering all the limitations due to the use of post-mortem tissues, are strongly suggestive of a region-dependent pharmacological action of clozapine in the human brain that may explain its peculiar clinical effects and open up research towards novel targets for future antipsychotic drugs.

β-adrenergic receptor responsiveness in aging heart and clinical implications.

Elderly healthy individuals have a reduced exercise tolerance and a decreased left ventricle inotropic reserve related to increased vascular afterload, arterial-ventricular load mismatching, physical deconditioning and impaired autonomic regulation (the so called “β-adrenergic desensitization”). Adrenergic responsiveness is altered with aging and the age-related changes are limited to the β-adrenergic receptor density reduction and to the β-adrenoceptor-G-protein(s)-adenylyl cyclase system abnormalities, while the type and level of abnormalities change with species and tissues. Epidemiological studies have shown an high incidence and prevalence of heart failure in the elderly and a great body of evidence correlate the changes of β-adrenergic system with heart failure pathogenesis. In particular it is well known that: (a) levels of cathecolamines are directly correlated with mortality and functional status in heart failure, (b) β1-adrenergic receptor subtype is down-regulated in heart failure, (c) heart failure-dependent cardiac adrenergic responsiveness reduction is related to changes in G proteins activity. In this review we focus on the cardiovascular β-adrenergic changes involvement in the aging process and on similarities and differences between aging heart and heart failure.

Relationship between the changes in peripheral thresholds of nociception and activity of the adenylyl cyclase system in the skeletal muscles of rats with streptozotocin-induced diabetes

Relationship between the changes in peripheral thresholds of nociception and activity of the adenylyl cyclase system in the skeletal muscles of rats with streptozotocin-induced diabetes

The effect of long-term diabetes mellitus induced by treatment with streptozotocin in 6-week-old rats on functional activity of the adenylyl cyclase system

In type 1 diabetes mellitus (DM), changes occurring in the adenylyl cyclase signaling system (ACSS) are one of the key causes of complications of the disease. Since type 1 DM has been most often diagnosed in childhood and adolescence, the study of changes in ACSS in the early development of the disease is a genuine problem. For this, we developed a prolonged model of type 1 DM, which was induced by treatment of 6-week-old rats with moderate doses of streptozotocin (1.5M-DM), and studied the functional state of ACSS in the brain, myocardium, and testes of rats with this model of the disease 7 months after its start. The 1.5M-DM model was compared with the model that was induced by streptozotocin treatment of adult, 5-month-old animals (5M-DM). It was shown that, in 1.5M-DM, in the tissues of diabetic rats, the functional activity of ACSS sensitive to biogenic amines and polypeptide hormones was significantly changed. In rats with 1.5M-DM, the adenylate cyclase (AC) inhibitory effects of somatostatin (in all studied tissues), noradrenaline (in the myocardium and the brain), and agonists of type 1 serotonin receptor (in the brain) were weakened to the greatest degree. In the brain, the AC-stimulating effects of relaxin, isoproterenol, and agonists of Gs-protein-coupled serotonin receptors also decreased; in the myocardium, the corresponding effects of GppNHp, relaxin, and β-adrenergic agonists declined; and, in the testes, the AC effects of GppNHp and chorionic gonadotropin declined. When comparing the 1.5M-DM and 5M-DM models, the most pronounced differences between them were found in the effect of DM on hormonal regulation of ACSS in the brain, this being true both for AC-stimulating effects of dopamine and PACAP-38 and for AC-inhibiting effects of bromocriptine and somatostatin. These results indicate significant changes in hormonal regulation of the nervous, cardiovascular, and reproductive systems in rats with early induction of type 1 DM, in some cases more severe changes as compared with late model of 5M-DM. These changes may be the basis for development of diabetic cardiomyopathy, cognitive deficiency, and hypogonadotropic states, which are often detected in children and adolescents with type 1 DM.

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.

Effect of intranasal insulin and serotonin on functional activity of the adenylyl cyclase system in myocardium, ovary, and uterus of rats with prolonged neonatal model of diabetes mellitus

Disturbances in hormonal signaling systems, in the adenylyl cyclase system (ACS) in particular, occur at early stages of diabetes mellitus (DM) and are one of the key causes of its complications. Since there is a correlation between the severity of DM and of disturbances in the ACS, the study of the ACS activity can be used to monitor DM and its complications and to evaluate effectiveness of their treatment. Comparatively recently, for treatment of the type 2 DM, there began to be used the intranasal insulin (I-I) and drugs increasing brain serotonin level, which effectively restore CNS functions. However, mechanisms of their action on peripheral tissues and organs with DM remain to be not understood. The goal of this work was to study effects. of I-I and intranasal serotonin (I-S) on the ACS functional activity in myocardium, ovary, and uterus of rats with a neonatal model of the type 2 DM. In tissues of diabetic rats there were revealed changes in regulation of adenylyl cyclase (AC) by guanine nucleotides and hormones that both stimulated and inhibited this enzyme, such changes being characterized by the receptor and tissue specificity. In diabetic rats, I-I restored the AC stimulating effects of isoproterenol in the myocardium, guanine nucleotides and gonadotropin in ovaries and relaxin in uterus, as well as the AC inhibitory effects of somatostatin in all tissues, and of noradrenaline in myocardium. Treatment with IS led to a partial restoration of the AC-inhibitory effect of noradrenalin in the diabetic myocardium, but did not affect regulation of AC by other hormones. These data indicate that I-I normalizes the ACS functional activity in myocardium and in tissues of the reproductive system of female rats with neonatal DM, whereas the effect of I-S on in the studied tissues is less pronounced. These results are necessary to be taken into account at development and optimization of strategy of use of I-I and I-S for treatment of DM and of its complications.

Regulation of adenylyl cyclase signaling system by insulin, biogenic amines and glucagon at their separate and combined action in muscle membranes of mollusc Anodonta cygnea

In smooth muscles of mollusc Anodonta cygnea, hormones produce regulatory effects on the adenylyl cyclase (AC) signaling system via receptors of the serpentine (biogenic amine, isoproterenol, glucagon) and of tyrosine kinase (insulin) types. Intracellular mechanisms of their action are interconnected. Use of hormones, their antagonists, and pertussis toxin at the combined action of insulin and biogenic amines or of glucagon on the AC activity allows revealing possible intersection points in mechanisms of their action. The combined effect of insulin and serotonin or of glucagon leads to a decrease of stimulation of AC by these hormones, whereas at action of insulin and isoproterenol the AC-stimulatory effect of insulin is blocked, while the AC-inhibitory effect of isoproterenol is preserved both in the presence and in the absence of the non-hydrolyzed GTP analog - guanylylimidodiphosphate (GppNHp). Specific blocking of the AC-stimulatory serotonin effect by cyproheptadine - an antagonist of serotonin receptors - did not affect stimulation of AC by insulin. Beta-adrenoblockers (propranolol and alprenolol) interfered with inhibition of the AC activity by isoproterenol, but did not change the AC stimulation by insulin. Pertussis toxin blocked the AC-inhibitory effect of isoproterenol and attenuated the AC-stimulatory effect of insulin. Thus, in muscles of the mollusc Anodonta cygnea there have been revealed negative interrelations between the AC system, which are realized at the combined effect of insulin and serotonin or of glucagon, probably at the level of receptor of the serpentine type (serotonin, glucagon), while at action of insulin and isoproterenol - at the level of interaction of G1 protein and AC.

Hormonal Sensitivity of Adenylyl Cyclase in the Myocardium, Brain and Testes of 18-month-old Non-Diabetic and Diabetic Rats

The alterations and abnormalities in hormone -sensitive adenylyl cyclase (AC) system occur at early stages of the type 2 diabetes mellitus (T2DM) and at later stages of the disease they are, according to our vi ew, one of the factors causing T2DM complications. To study the changes in AC system likely to be involved in the development of these complications one needs a very long model of T2DM. Purpose: The aim of this work was the study of AC system in the myocardium, brain and testes of three-, eight-and 18-month-old male rats with experimental T2DM compared with control animals of the same age.

Failure of hCG/LH receptors to stimulate the transmembrane effector adenylyl cyclase in human endometrium

The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively; n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p tions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC; however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.

Regulatory properties of adenylyl and guanylyl cyclase in human spermatozoa

Completion of the process of maturation of spermatozoa (capacitation) occurs in the female genital tract. As a result, spermatozoa acquire hypermobility and the capability for the acrosomal reaction, which determine their fertility. There exist data that participating in these processes are the adenylyl cyclase and guanylyl cyclase signaling systems that are present in human and mammalian spermatozoa. The goal of the present work to characterize these systems in the human ejaculate spermatozoa (ES) and in the human fertile spermatozoa (FS) isolated by the density gradient centrifugation. In the FS homogenate the basal activity of adenylyl cyclase (AC) activity was significantly higher that in ES: 47 +/- 5 vs 28 +/- 3 pmol cAMP/min per mg of protein, respectively. At the same time, the AC-stimulating effects of non-hormonal activators of AC forms (NaHCO3, Mn2+, forskolin, and non-hydrolysable GTP analogue - GppNHp) were lower as compared with ES. The AC activity in ES were stimulated by isoproterenol, serotonin, PACAP-38, and, to the letter degree, by noradrenalin and adenosine. Among the AC-inhibiting hormones, adenosine alone decreased the enzyme activity. In FS, on the contrary, the AC-inhibiting effects of adenosine, noradrenaline, and serotonin were clearly expressed, whereas the stimulatory effects of these hormones were attenuated or absent. The basal activity of guanylyl cyclase (GC) in the ES and FS homogenates amounted to 27 +/- 3 and to 21 +/- 2 pmol cGMP for 1 min per 1 mg protein, respectively, and was significantly increased in the presence of 10 mM Mn2+. The GC-stimulating effects of natriuretic peptides - atrial natriuretic peptide (ANP) and the C-type natriuretic peptide (CNP)--activators of the GC receptor forms, were in ES significantly higher than in FS, the ANP effect being more pronounced as compared with CNP. The obtained data indicate multiplicity of the cAMP- and cGMP-dependent signal cascades regulating fertility of human spermatozoa. It has been established that sensitivity of AC and GC to hormones in the total ES pool and in the supermobile FS fraction isolated by centrifugation differs essentially, which is to be taken into account when using FS for auxiliary reproductive technologies.

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.