Forskolin-stimulated Adenylate Cyclase Activity Research Articles

Effect of iron on renal tubular epithelial cells

Since iron has been implicated as a potential nephrotoxin, we examined the effect of iron on several aspects of cultured renal tubular epithelial cell biology. We found that exposure to 10(-4) M of either the ferrous or ferric form of iron impaired healing of denuded areas made within confluent monolayers of LLC-PK1 cells. This impairment required 30 to 80 hours of exposure to iron to occur and was also seen in another renal tubular epithelial cell line (MDCK cells). To delineate the potential mechanism(s) of this impairment, we examined the expression of a key integrin subunit involved in cell-matrix adhesion. Exposure of LLC-PK1 cells to 10(-4) M ferric citrate for 72 hours significantly decreased expression of the beta 1 integrin subunit as determined by flow cytometry. To determine if iron impairs another process that occurs at the basolateral surface, the effects of 72 hours of exposure to iron on adenylate cyclase activity were examined. Both ferric and ferrous citrate significantly enhanced vasopressin- and forskolin-stimulated adenylate cyclase activity. To examine if iron can regulate proliferation, the effect of iron on 3H-thymidine uptake was measured. We found that ferric citrate diminished proliferation and this decrease required the presence of either serum or transferrin. To ascertain if iron affected ultrastructure, we used transmission electron microscopy and found that iron accumulation within cells was much more apparent with ferric than ferrous citrate. Ferric iron induced mild-to-moderate cytopathic changes. These results indicate that iron is capable of inducing multiple changes in renal tubular epithelial function. The effect of iron to impair wound healing may be related to diminished expression of the beta 1 integrin subunit and perhaps to impaired proliferation.

Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality.

Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Both FGF-2 peptides also induced functional modifications of G-proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP-ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation.

Exposure to either gamma or a mixed neutron/gamma field irradiation modifies vasoactive intestinal peptide receptor characteristics in membranes isolated from pig jejunum.

The effect of acute whole body exposure to ionizing radiation was investigated on intestinal vasoactive intestinal peptide (VIP) receptors and adenylate cyclase activity in membranes isolated from pig jejunum. Pigs under light anaesthesia were exposed to a single dose (6 Gy) of gamma (gamma) or to mixed neutron/gamma field (ratio 1:1; neutron/gamma) irradiation. Seven days after irradiation, plasma-membranes were prepared from post mortem jejunal mucosal scrapings. Marker enzyme activities (sucrase, leucine aminopeptidase (LAP), Na,K-ATPase) were measured in each preparation. The characteristics (KD, Bmax) of VIP receptors were determined using 125I-labelled VIP. In addition VIP-sensitive adenylate cyclase activity was measured. Results showed that enzyme activities were reduced following both gamma (sucrase 67%; LAP 53%; Na/K-ATPase 29%; N = 7) and neutron/gamma (sucrase 53%; LAP 59%; Na/K-ATPase 68%; N = 5) compared with control values (N = 5). VIP receptor affinity was decreased following either type of irradiation (gamma or neutron/gamma P < 0.01) and receptor numbers increased. Both VIP- and forskolin-stimulated adenylate cyclase activities were reduced but the sensitivity of the enzyme remained the same for VIP (EC50 values (nmol dm-3)-control-1.27 +/- 0.35; gamma-2.18 +/- 0.41; neutron/gamma-1.91 +/- 0.28). In conclusion, exposure to either gamma or neutron/gamma irradiation attenuates intestinal enzyme activities and VIP receptor affinity but increases VIP receptor numbers.

Early alterations in the brown adipose tissue adenylate cyclase system of pre-obese Zucker rat fa/fa pups: decreased G-proteins and beta 3-adrenoceptor activities.

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5'-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5'-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.

Effects of tandospirone on second messenger systems and neurotransmitter release in the rat brain

1. 1. We studied the effects of tandospirone, a novel serotonin (5-HT) 1A receptor-related anxiolytic, on the intracellular second messenger systems and neurotransmitter release. 2. 2. Tandospirone inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes by activation of 5-HT 1A receptors and had high efficacy comparable to 5-HT 1A receptor agonists such as 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). 3. 3. Tandospirone suppressed carbachol-stimulated phosphatidyl-inositol metabolism (PI response), which was shown to be a 5-HT 1A receptor-mediated event. 4. 4. Tandospirone did not affect the release of 5-HT, norepinephrine (NE), dopamine (DA) and acetylcholine (ACh) from rat brain slice preparations. 5. 5. These findings suggested that tandospirone shows high agonistic efficacy on the postsynaptic 5-HT 1A receptors but does not affect the presynaptic autoreceptors located on nerve endings. The modulation of the second messenger system via postsynaptic 5-HT 1A receptors might be involved in the anxiolytic efficacy of tandospirone.

Identification and characterization of guanine nucleotide-sensitive melatonin receptors in chicken brain

Specific melatonin receptors present in crude membrane preparations of chicken forebrain were identified by specific binding activity of 2-[ 125I]iodomelatonin and melatonin-dependent inhibition of forskolin-stimulated adenylate cyclase activity by a pertussis toxin-sensitive mechanism. The membrane-bound melatonin receptors were successfully solubilized with digitonin, and the ligand binding activity was shown to be preserved in solubilized membranes when compared with that of crude membranes. In both fractions, melatonin receptors consisted of two binding components with picomolar and nanomolar affinity. The ligand binding activity of solubilized receptors was decreased after treatment with guanine nucleotides, and guanosine 5 - ́ (γ-thio)triphosphate (GTPγS) induced a transformation of the high affinity receptors to low affinity state. Moreover, [ 35S]GTPγS was indicated to bind to solubilized membranes in a melatonin-dependent fashion. These results demonstrate the existence of functional melatonin receptors-G proteins coupling in digitonin-solubilized membranes of chicken forebrain membranes.

Effects of lactation on the signal transduction systems regulating lipolysis in sheep subcutaneous and omental adipose tissue

The effect of lactation on the regulation of lipolysis by beta- and alpha 2-adrenergic agents and by adenosine has been investigated. When changes in adipocyte mean cell volume (which decreases with lactation) are allowed for, lactation increased the maximum response both to beta-adrenergic agents and to the adenosine analogue N6-phenylisopropyladenosine, but had no apparent effect on the responsiveness of the alpha 2-adrenergic system in both subcutaneous and omental adipocytes. For subcutaneous adipocytes, there was no significant change in the number of beta-adrenergic or alpha 2-adrenergic receptors, but the amount of Gs and the maximum (forskolin-stimulated) adenylate cyclase activity were increased by lactation. In contrast, in omental adipocytes, the number of beta- (but not alpha 2-) adrenergic receptors and the amount of Gs were increased, whereas forskolin-stimulated adenylate cyclase activity was unchanged by lactation. In both types of adipocyte, cyclic AMP phosphodiesterase and total protein kinase A activities were unchanged. Lactation had no effect on the number of adenosine receptors but increased the amounts of the Gi isoforms expressed in both types of adipocyte. These various adaptations differ markedly in a number of respects from those described previously in the rat. Lactation, then, while having a similar overall effect on the response to beta-adrenergic agonists of adipocytes, achieves this by depot-specific mechanisms. In contrast, changes in response to adenosine appear to involve the same mechanism in the two depots investigated.

Decreased Adenylate Cyclase Activity and Expression of G sα in Human Myocardium After Orthotopic Cardiac Transplantation

We studied several aspects of guanine nucleotide-stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained > or = 6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n = 12; P < .05), GTP gamma S-stimulated adenylate cyclase activity was decreased by 78% (n = 12; P < .05), Mn+2+forskolin-stimulated adenylate cyclase activity was decreased by 80% (n = 8; P < .05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n = 8, P < .05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs alpha protein was decreased by 61 +/- 12% (n = 8; P < .001). There was no change in the level of Gi alpha as assessed by pertussis toxin-catalyzed ADP-ribosylation (n = 4; P = NS). With the use of the quantitative polymerase chain reaction, a 50 +/- 10% (n = 6; P < .001) reduction in the steady-state level of Gs alpha mRNA was observed. There was no change in the level of mRNA for Gi-3 alpha. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide-stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs alpha protein and mRNA.

Tannin inhibits adenylate cyclase in airway epithelial cells

Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.

Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin

Rat galanin inhibits basal as well as forskolin-stimulated adenylate cyclase activity in rat ventral and dorsal hippocampus. The inhibition of adenylate cyclase activity, both basal and forskolin-stimulated, is characterised by IC 50 values being 250-fold lower in ventral hippocampus (IC 50 = 1.1 nM) compared to the dorsal hippocampus (IC 50 = 270 nM). The maximal inhibition of basal and forskolin-stimulated adenylate cyclase activity in both ventral and dorsal hippocampus in the presence of 10 μM rat galanin is 34–45%. The analysis of the binding data obtained with 125I-labelled Tyr 26-porcine galanin as a tracer reveals similar binding constants for rat galanin in both ventral and dorsal hippocampus with 4.8-fold higher concentration of galanin receptors in the ventral hippocampus. Putative galanin receptor subtype differences between the ventral and dorsal hippocampus have been noted by Hedlund et al. (Eur. J. Pharmacol., 224 (1992) 203–205). This study yields further confirmation for the existence of different galanin receptor subtypes or for differential coupling of galanin receptors to the adenylate cyclase in the dorsal versus ventral hippocampus.

Novel Benzodioxan Derivative, 5-{3-[((2S)-1,4-Benzodioxan-2-ylmethyl)amino]propoxy}-l,3-benzodioxole HC1 (MKC-242), with a Highly Potent and Selective Agonist Activity at Rat Central Serotonin1A Receptors

The present study characterizes the neurochemical profile of the newly synthesized compound 5-{3-[((2S)-l,4-benzodioxan-2-ylmethyl)amino]propoxy}-l,3-benzodioxole HC1 (MKC-242). In in vitro experiments, MKC-242 had high affinity for serotonin1A (5-HT1A) receptors (Ki: 0.35 nM) and moderate affinity for α1-adrenoceptors (Ki: 21 nM), whereas it had no appreciable affinity for any other neurotransmitter recognition sites studied and 5-HT transporter. MKC-242 (0.3-3.0mg/kg, s.c.; 1-l0 mg/kg, p.o.) caused presynaptic 5-HT1A-receptor-mediated responses (decreases in 5-HT turnover and 5-HT release) and postsynaptic 5-HT1A-receptor-mediated responses (hypothermia, an increase in serum corticosterone level and 5-HT1A behavioral syndrome). The effects of MKC-242 on decarboxylase inhibitor-induced 5-hydroxytryptophan accumulation and rectal temperature were blocked by the 5-HT1A-receptor antagonist N-rert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpropanamide. The comparative studies on the in vivo responses induced by MKC-242 and the 5-HT1A-receptor full agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) showed that MKC-242 and 8-OH-DPAT had similar efficacy at presynaptic 5-HT1A receptors, whereas the former had less efficacy than the latter at postsynaptic 5-HT1A receptors. Furthermore, MKC-242 partially inhibited forskolin-stimulated adenylate cyclase activity in hippocampal membranes. These findings suggest that MKC-242 acts as a full and partial agonist at pre- and postsynaptic 5-HT1A receptors, respectively, in the central nervous system.

Functional coupling of human D2, D3, and D4 dopamine receptors in HEK293 cells.

The D2 dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3 and D4 dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3 and D4 receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3 or D4 receptors resulted in the inhibition of forskolin-stimulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4 receptor than the D3 or D4 receptors, although only 6-fold higher than the D2 receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4 dopamine receptor mRNA in human brain regions.

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Equilibrium binding assays were conducted on intact cells with 125I Bolton-Hunter labeled NPY (125I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu31, Pro34)NPY (a Y1 receptor ligand, Y1RL), NPY13-36 (a Y2 receptor ligand, Y2RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (Kd) value of 39.00 +/- 12.84 nM and a maximal binding (Bmax) of 489.89 +/- 155.49 fmol/10(6) cells (mean +/- SE, n = 6). NPY, Y1RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC50 (nM) values of 4.06 +/- 1.66 (n = 4), 2.94 +/- 0.75 (n = 5), and 18.36 +/- 10.36 (n = 3), respectively. APP and Y2RL were unable to compete with 125I-NPY in the concentration range 0.001-1 microM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3',5'-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 +/- 6.28%, n = 5 and an IC50 of 5.96 +/- 1.87 nM. The Y1RL produced a comparable response (IC50 = 5.35 +/- 1.39 nM, n = 4; maximal inhibition of 61.05 +/- 13.03%) and Y2LR had no detectable effect at a similar concentration range. The results demonstrate the existence of a Y1 receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.

Serotonin inhibition of adenylate cyclase in human platelet membranes; relation to 5-HT-1A receptor-mediated activity

Serotonin inhibited both basal and forskolin-stimulated adenylate cyclase activity in human platelet membranes by approx. 30%, with an EC 50 of 54 nM. Addition of NaCl to the assay medium reduced the degree of inhibition. 5-carboxamidotryptamine (5-CT) behaved as a full agonist in this system ( EC 50 of 5.4nM) and BMY 7378 as a partial agonist (inducing 19% inhibition); the putative 5-HT-1a receptor agonists metergoline, spiroxatrine and MDL 73005 were inactive. The 5-HT-1a receptor antagonists metitepin and NAN-190 behaved as antagonists with K b or K i values of 11.2 and 1.17 nM, respectively. Spiperone behaved as a partial antagonist only. Epinephrine and 5-HT produced convergent, non-additive inhibition of both basal and forskolin-stimulated cyclase.

Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3.

Galanin inhibits adenylate cyclase activity and insulin secretion and modulates ion channels in pancreatic beta-cells through pertussis-toxin-sensitive G-protein(s). Antibodies directed against the C-terminal region of specific G-protein alpha-subunits were used to determine which G-protein(s) couple galanin receptors to inhibition of adenylate cyclase in the rat insulinoma cell line RINm5F. Preincubation of membranes with EC antibody (anti-alpha i3) decreased the inhibition of forskolin-stimulated adenylate cyclase activity by galanin (100 nM) by 45% compared with control IgG (P < 0.05) whereas preincubation with AS (anti-alpha i1, alpha i2) or GO (anti-alpha o) antibodies had no significant effect. To confirm these results, RINm5F cells were exposed intermittently over a 4-day period to phosphorothioate oligodeoxynucleotides that were either sense or antisense to alpha i1, alpha i2, alpha i3 or alpha o. Oligodeoxynucleotides antisense to alpha i2, alpha i3 and alpha o specifically decreased the levels of the targeted alpha-subunit in membranes. alpha i1 was undetectable in these cells. Inhibition of adenylate cyclase activity by galanin was largely abolished in membranes from cells exposed to the oligodeoxynucleotide antisense to alpha i3, whereas all other oligodeoxynucleotides had no significant effect on this pathway. Indirect immunofluorescence and immunoblotting of specific membrane fractions with EC antibody show significant localization of alpha i3 to intracellular membrane compartments. These results suggest that Gi3 is the G protein that couples galanin receptors to inhibition of adenylate cyclase activity in RINm5F cells.

Cellular and molecular alterations in the beta adrenergic system with cardiomyopathy induced by tachycardia.

The aim was to examine the relationship between changes in myocyte function to changes in protein and mRNA content of components of the beta adrenergic system with tachycardia induced cardiomyopathy. Contractile function and beta adrenergic responsiveness were measured in isolated myocytes from control pigs (n = 6) and in pigs subjected to three weeks of pacing induced supraventricular tachycardia (n = 6). beta Receptor density and affinity, the relative content of the stimulatory (Gs) and inhibitory (Gi) subunits of the G protein complex, and adenylate cyclase activity were determined from sarcolemmal preparations. In order to determine whether these changes were accompanied by alterations in steady state mRNA levels for specific components of the beta adrenergic system, mRNA content for the beta 1 adrenergic receptor and the G alpha s and G alpha i2 subunits of the G protein complex was measured. Chronic supraventricular tachycardia caused a 36% increase in left ventricular end diastolic dimension and a 61% decrease in left ventricular fractional shortening compared to controls. The velocity of isolated myocyte shortening was 50% lower in myocytes from hearts with tachycardia cardiomyopathy than in control myocytes. In the presence of 50 nM isoprenaline or 2 microM forskolin, the velocity of myocyte shortening was 65% lower in the myopathic myocytes than in the controls. With the development of tachycardic cardiomyopathy, beta adrenergic receptor density fell by 25% with no change in affinity, Gs decreased by 35%, and Gi increased by over 50% compared to controls. Basal adenylate cyclase activity and isoprenaline and forskolin stimulated adenylate cyclase activity fell by over 50% with supraventricular tachycardia compared to controls. The relative content of G alpha i2 mRNA increased threefold with the development of tachycardic cardiomyopathy with no change in the relative abundance of mRNA for the beta 1 receptor or G alpha s when compared with controls. The changes in myocyte beta adrenergic responsiveness with the development of tachycardic cardiomyopathy are due to alterations in cellular mechanisms (decreased beta receptor and Gs density, increased Gi) and in molecular mechanisms (increased Gi mRNA content).

β-Adrenoceptor-G protein-Adenylate Cyclase Complex in Rat Hearts with Ischemic Heart Failure Produced by Coronary Artery Ligation

We examined the role of the myocardial β-adrenoceptor-G protein-adenylate cyclase complex in 10-week-old Wistar rats with ischemic heart failure produced by ligating the left coronary artery (l) and in sham-operated control rats (C). We determined the number of β-adrenoceptors (B max), the dissociation constant (K d) using a binding assay and adenylate cyclase activity. Levels of mRNA encoding for the α subunit of the stimulatory guanine nucleotide-binding protein (Gsα) and the α subunit of the inhibitory guanine nucleotide-binding protein (Giα) were measured by Northern blot analysis. The amounts of Gsα and Giα were measured by Western blot analysis, B max and K d did not differ significantly between the two groups; B max; l, 14.7 ± 1.3 v C, 13.4 ± 0.9 f mol/mg protein; K d: l, 345 ± 31 v C, 340 ± 28 pM (mean ± standard error, S.E.). There were no significant differences in Gsα and Giα concentrations between the two groups as measured by Northern blot analysis (Gsα: l, 91.6 ± 4.5 v C, 96.5 ± 2.3%; Giα l, 95.4 ± 3.6 v C. 90.0 ± 3.0%) or by Western blot analysis (Gsα: l, 95.2 ± 2.0 v C. 94.5 ± 2.6%; Giα l, 91.5 ± 3.0 v C. 95.1 ± 2.9%). Activity of basal and MnCl 2-stimulated adenylate cyclase did not differ significantly in the two groups; basal: l, 7.5 ± 0.7 v C, 8.1 ± 0.5 pmol cAMP/mg protein/min; MnCl 2 l, 80.8 ± 5.8 v C, 86.4 ± 6.7 pmol cAMP/mg protein/min. Sodium fluoride and forskolin-stimulated adenylate cyclase activity were significantly lower in the hearts with ischemic failure compared with controls (sodium fluoride: l, 68.5 ± 5.6 v C. 103 ± 4.8 pmol cAMP/mg protein/min: forskolin; l, 84.6 ± 6.5 v C, 117.1 ± 5.6 pmol cAMP/mg protein/min). These data suggest the presence of myocardial Gsα dysfunction in ischemic heart failure. We conclude that such a dysfunction in Gsα may contribute to the contractile abnormalities in ischemic heart failure.

Interactions of lesopitron (E-4424) with central 5-HT 1A receptors: in vitro and in vivo studies in the rat

Previous studies have shown that the 5-HT 1A receptor ligand, lesopitron (E-4424, 2-[4-[4-(4-chloro-1-pyrazolyl)butyl]-1-piperazinyl]pyrimidine), exerts potent anxiolytic-like effects in rodents and monkeys (Costall et al., 1992, J. Pharmacol. Exp. Ther. 262, 90). In an attempt to determine whether these effects are really mediated through the interaction of lesopitron with central 5-HT 1A receptors, we investigated the agonistic and/or antagonistic nature of this interaction under in vitro and in vivo conditions in the rat. In vitro binding and autoradiographic studies with [ 3H]8-hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT) and [ 3H]lesopitron as radioligands confirmed that lesopitron binds to 5-HT 1A receptors in the rat brain with a relatively high affinity (p K i = 7.35). As expected of a full agonist at postsynaptic 5-HT 1A receptors, lesopitron (IC 50 = 125 nM) inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes to the same extent as 5-HT, and this effect was preventable by potent 5-HT 1A receptor antagonists such as (−)-tertatolol, (−)-propranolol and N-tert-butyl-3,4-(2-methoxyphenyl)piperazin-1-yl-2-phenyl-propanamide ((+)-WAY 100135). As previously shown for agonists acting at the somato-dendritic 5-HT 1A autoreceptors in the dorsal raphe nucleus, lesopitron inhibited the firing of serotoninergic neurons both in vitro (in brainstem slices, IC 50 = 120 nM) and in vivo (in chloral hydrate-anaesthetized rats, ID 50 = 35 μg/kg i.v.), and this effect was preventable by (−)-tertatolol. Interestingly, the inhibition of the discharge due to lesopitron lasted for only a few minutes both in vitro and in vivo whereas the anxiolytic-like properties of this drug lasted for hours after a single injection in mice (Costall et al., 1992). In addition, the doses required for the stimulation of pre- and postsynaptic 5-HT 1A receptors were markedly higher than those producing significant anxiolytic-like effects in rodents (Costal et al., 1992). It is therefore unlikely that the anxiolytic-like properties of lesopitron involve its stimulatory action at central 5-HT 1A receptors.

Association and agonistic action of DN-2327, a novel isoindoline derivative, on GABA B receptor in brain

The association and action of DN-2327, 2-(7-chloro-1,8-naphthyridin-2-yl)-3-[(1,4-dioxa-8-azaspiro[4,5]dec-8-yl)carbonylmethyl]isoindolin-1-one, on the γ-aminobutyric acid (GABA) B receptor in rat brain have been examined. DN-2327 inhibited the binding of [ 3H]GABA to GABA B receptor in crude synaptic membrane obtained from rat brain. The Scatchard analysis of [ 3H]GABA binding to GABA B receptor indicated that DN-2327 induced the decrease in affinity of both high and low affinity binding sites without changing the B max values. The forskolin-stimulated adenylate cyclase activity in slices from rat cerebral cortex was significantly suppressed by the addition of DN-2327. Furthermore, this inhibition by DN-2327 was eliminated by the simulataneous additions of 2-hydroxy saclofen or CGP 55845A, GABA B receptor antagonists. These results suggest that DN-2327 may have not only a high association with GABA B receptor but also an agonistic action on the receptor.

Adrenalectomy-induced alterations of calmodulin-dependent hippocampal adenylate cyclase activity: role of guanine nucleotide-binding proteins.

Ca2+/calmodulin-dependent processes are altered by manipulations of the hypothalamic-pituitary-adrenal axis. In particular, adrenalectomy (ADX) attenuates hippocampal, but not cortical, calmodulin-dependent adenylate cyclase activity measured during the active (waking) phase of rats. The involvement of calmodulin- and guanine nucleotide (G)-binding proteins in the effects of ADX on the activity of calmodulin-dependent adenylate cyclase were investigated. In hippocampal membranes, inclusion of the GTP antagonist guanosine 5'-O-(2-thiodiphosphate) (250 microM) caused pronounced inhibition of calmodulin-stimulated adenylate cyclase activity. Guanosine 5(1)-O-(2-thiodiphosphate) had much smaller effects on calmodulin-independent (basal and forskolin-stimulated) enzyme activity. Substitution of Mn2+ for Mg2+ in the assay medium increased basal and forskolin-stimulated adenylate cyclase activity, but abolished calmodulin-dependent activation of this enzyme in both hippocampal and cortical membranes. These treatments blunted ADX-induced attenuation of hippocampal adenylate cyclase. ADX, with or without corticosterone administration (40 mg/kg, sc, once daily), failed to alter either Gi alpha or Gs alpha membrane protein content in either hippocampus or cortex. The levels of major membrane calmodulin-binding proteins in hippocampus and cortex also were not significantly altered by ADX. These results confirm that hormonal and biochemical regulation of calmodulin-dependent adenylate cyclase is distinct from that of other adenylate cyclase family members. Changes in Gs alpha and Gi alpha protein content alone cannot account for the effects of ADX on this enzyme. Overall, our studies suggest that the effects of ADX on calmodulin-dependent adenylate cyclase may occur through a reduction in the absolute amount of the catalytic subunit or an alteration(s) in the efficiency of coupling between adenylate cyclase and its modulators.