Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.

Suppression of GLUT1; a new strategy to prevent diabetic complications.

High blood glucose results in high glucose levels in retina, because GLUT1, the sole glucose transporter between blood and retina, transports more glucose when blood glucose is high. This is the ultimate cause of diabetic retinopathy. Knockdown of GLUT1 by intraocular injections of a pool of siRNAs directed against SLC2A1 mRNA which codes for GLUT1 significantly reduced mean retinal glucose levels in diabetic mice. Systemic treatment of diabetic mice with forskolin or genistein, which bind GLUT1 and inhibit glucose transport, significantly reduced retinal glucose to the same levels seen in non-diabetics. 1,9-Dideoxyforskolin, which binds GLUT1 but does not stimulate adenylate cyclase had an equivalent effect to that of forskolin regarding lowering retinal glucose in diabetics indicating that cyclic AMP is noncontributory. GLUT1 inhibitors also reduced glucose and glycohemoglobin levels in red blood cells providing a peripheral biomarker for the effect. In contrast, brain glucose levels were not increased in diabetics and not reduced by forskolin. Treatment of diabetics with forskolin prevented early biomarkers of diabetic retinopathy, including elevation of superoxide radicals, increased expression of the chaperone protein ß2 crystallin, and increased expression of vascular endothelial growth factor (VEGF). These data identify GLUT1 as a promising therapeutic target for prevention of diabetic retinopathy.

Escherichia coli-induced epithelial hyporesponsiveness to secretagogues is associated with altered CFTR localization.

Both pathogenic and commensal strains of Escherichia coli colonize the human intestinal tract. Pathogenic strains differ only in the expression of virulence factors, many of which comprise a type III secretion system (TTSS). Little is known regarding the effect of E. coli on the intestinal epithelial response to the secretagogues that drive ion secretion, despite its importance in causing clinically significant diarrhoea. Using Ussing chambers to measure electrogenic ion transport of T84 intestinal epithelial cell monolayers, we found that all strains of E. coli tested (pathogenic, commensal, probiotic and lab strain) significantly reduced cAMP-dependent ion secretion after 4-8 h exposure. Enteropathogenic E. coli mutants lacking a functional TTSS caused similar hyposecretion while not causing significant apoptosis (as shown by caspase-3 cleavage) or necrosis (lactate dehydrogenase release), as did the commensal strain F18, indicating that epithelial cell death was not the cause of hyposecretion. Enteropathogenic E. coli and the TTSS mutant significantly reduced cell surface expression of the apical anion channel, cystic fibrosis transmembrane conductance regulator, which is likely the mechanism behind the pathogen-induced hyposecretion. However, F18 did not cause cystic fibrosis transmembrane conductance regulator mislocalization and the commensal-induced mechanism remains unclear.

Olprinone and colforsin daropate alleviate septic lung inflammation and apoptosis through CREB-independent activation of the Akt pathway.

Olprinone, a specific phosphodiesterase III inhibitor, and corforsin daropate, a direct adenylate cyclase activator, are now being used in critical conditions. We investigated whether their therapeutic use provides protection against septic acute lung injury (ALI) and mortality. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. Olprinone or colforsin daropate was continuously given through an osmotic pump that was implanted into the peritoneal cavity immediately following CLP. These treatments prevented the ALI development in CLP mice, as indicated by the findings that severe hypoxemia, increased pulmonary vascular permeability, and histological lung damage were strikingly remedied. Furthermore, continued administration of olprinone or colforsin daropate suppressed apoptosis induction in septic lungs and improved the survival of CLP mice. Olprinone and corforsin daropate enhanced Akt phosphorylation in septic lungs. Wortmannin, which inhibits the Akt upstream regulator phosphatidylinositol 3-kinase, abrogated the protective effects of olprinone and corforsin daropate on sepsis-associated lung inflammation and apoptosis. In vivo transfection of cyclic AMP response element binding protein (CREB) decoy oligodeoxynucleotide failed to negate the abilities of these agents to increase Akt phosphorylation and to inhibit IκBα degradation in septic lungs. These results demonstrate for the first time that CREB-independent Akt-mediated signaling is a critical mechanism contributing to the therapeutic effects of olprinone and corforsin daropate on septic ALI. Moreover, our data also suggest that these cyclic AMP-related agents, by blocking both nuclear factor-κB activation and apoptosis induction, may represent an effective therapeutic approach to the treatment of the septic syndrome.

Pharmacological stimulation of type 5 adenylyl cyclase stabilizes heart rate under both microgravity and hypergravity induced by parabolic flight.

We previously demonstrated that type 5 adenylyl cyclase (AC5) functions in autonomic regulation in the heart. Based on that work, we hypothesized that pharmacological modulation of AC5 activity could regulate the autonomic control of the heart rate under micro- and hypergravity. To test this hypothesis, we selected the approach of activating AC5 activity in mice with a selective AC5 activator (NKH477) or inhibitor (vidarabine) and examining heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater under micro- and hypergravity in the vidarabine group, while there were no significant changes in the NKH477 group, suggesting that autonomic regulation was unstable in the vidarabine group. The ratio of low frequency and high frequency (HF) in heart rate variability analysis, a marker of sympathetic activity, became significantly decreased under micro- and hypergravity in the NKH477 group, while there was no such decrease in the vidarabine group. Normalized HF, a marker of parasympathetic activity, became significantly greater under micro- and hypergravity in the NKH477 group. In contrast, there was no such increase in the vidarabine group. This study is the first to indicate that pharmacological modulation of AC5 activity under micro- and hypergravity could be useful to regulate the autonomic control of the heart rate.

Forskolin and derivatives as tools for studying the role of cAMP.

Forskolin (7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one) is the first main labdane diterpenoid isolated from the roots of the Indian Plectranthus barbatus ANDREWS and one of the most extensively studied constituents of this plant. The unique character of forskolin as a general direct, rapid and reversible activator of adenylyl cyclase not only underlies its wide range of pharmacological effects but also renders it as a valuable tool in the study of the role of cAMP. The purpose of this review is to provide data presenting the utility of forskolin--as a cAMP activator--for studying the function of cAMP from different biological viewpoints as follows: 1) Investigation on the role of cAMP in various cellular processes in different organs such as gastrointestinal tract, respiratory tract, reproductive organs, endocrine system, urinary system, olfactory system, nervous system, platelet aggregating system, skin, bones, eyes, and smooth muscles. 2) Studies on the role of cAMP activation and inhibition to understand the pathogenesis (e.g. thyroid autoimmune disorders, leukocyte signal transduction defect in depression, acute malaria infection, secretory dysfunction in inflammatory diseases) as well as its possibly beneficial role for curing diseases such as the regulation of coronary microvascular NO production after heart failure, the attenuation of the development or progression of fibrosis in the heart and lungs, the augmentation of myo-protective effects of ischemic preconditioning especially in the failing hearts after myocardial infarction, the stimulation of the regeneration of injured retinal ganglion cells, the curing of glaucoma and inflammatory diseases, the reducing of cyst formation early in the polycystic kidney disease, and the management of autoimmune disorders by enhancing Fas-mediated apoptosis. 3) Studies on the role of cAMP in the mechanism of actions of a number of drugs and substances such as the effect of the protoberberine alkaloid palmatine on the active ion transport across rat colonic epithelium, the inhibitory effect of retinoic acid on HIV-1-induced podocyte proliferation, the whitening activity of luteolin, the effect of cilostazol on nitric oxide production, an effect that is involved in capillary-like tube formation in human aortic endothelial cells, the apoptotic effect of bullatacin, the effects of paraoxon and chlorpyrifos oxon on nervous system. Moreover, cAMP was found to play a role in acute and chronic exposure to ethanol, in morphine dependence and withdrawal and in behavioral sensitization to cocaine as well as in the protection against cisplatin-induced oxidative injuries.

Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells.

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Intra-arterial colforsin daropate for the treatment of cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

INTRODUCTION: Cerebral vasospasm (CV) remains a major cause of mortality and morbidity in patients with subarachnoid hemorrhage (SAH). Here, we examined the effectiveness and safety of intra-arterial injection of colforsin daropate hydrochloride (CDH). METHODS: A consecutive series of 29 patients with angiographically confirmed CV received intra-arterial CDH (IAC) therapy. Angiographic changes in spastic vessels and the cerebral circulation time (CCT) were assessed before and after IAC treatment, together with the change in clinical status. RESULTS: IAC treatment was performed in 53 procedures in 29 patients. Angiographic improvement was observed following all procedures (100%), and clinical improvement was observed following 36 of 42 procedures (86%) in symptomatic cases. CCT improved significantly. At the 3-month follow-up, 19 patients (66%) showed good recovery or moderate disability on the Glasgow Outcome Scale. Major adverse effects were headache and increased heart rate. CONCLUSIONS: IAC treatment was effective and safe for the treatment of CV after SAH.

Colforsin-induced vasodilation in chronic hypoxic pulmonary hypertension in rats.

PURPOSE: Colforsin, a water-soluble forskolin derivative, directly activates adenylate cyclase and thereby increases the 3',5'-cyclic adenosine monophosphate (cAMP) level in vascular smooth muscle cells. In this study, we investigated the vasodilatory action of colforsin on structurally remodeled pulmonary arteries from rats with pulmonary hypertension (PH). METHODS: A total of 32 rats were subjected to hypobaric hypoxia (380 mmHg, 10% oxygen) for 10 days to induce chronic hypoxic PH, while 39 rats were kept in room air. Changes in isometric force were recorded in endothelium-intact (+E) and -denuded (-E) pulmonary arteries from the PH and control (non-PH) rats. RESULTS: Colforsin-induced vasodilation was impaired in both +E and -E arteries from PH rats compared with their respective controls. Endothelial removal did not influence colforsin-induced vasodilation in the arteries from control rats, but attenuated it in arteries from PH rats. The inhibition of nitric oxide (NO) synthase did not influence colforsin-induced vasodilation in +E arteries from controls, but attenuated it in +E arteries from PH rats, shifting its concentration-response curve closer to that of -E arteries from PH rats. Vasodilation induced by 8-bromo-cAMP (a cell-permeable cAMP analog) was also impaired in -E arteries from PH rats, but not in +E arteries from PH rats, compared with their respective controls. CONCLUSIONS: cAMP-mediated vasodilatory responses without beta-adrenergic receptor activation are impaired in structurally remodeled pulmonary arteries from PH rats. In these arteries, endothelial cells presumably play a compensatory role against the impaired cAMP-mediated vasodilatory response by releasing NO (and thereby attenuating the impairment). The results suggest that colforsin could be effective in the treatment of PH.

Isoforskolin and Cucurbitacin IIa promote the expression of anti-inflammatory regulatory factor SIGIRR in human macrophages stimulated with Borrelia burgdorferi basic membrane protein A.

Certain natural products, derived from medicinal plants, exhibit anti-inflammatory properties, but the mechanism of action of many remains unclear. Borrelia burgdorferi spirochetes are responsible for causing Lyme arthritis through activation of the Toll-like receptor (TLR) signaling pathway. In this study, we investigated the mechanisms by which Isoforskolin (ISOF) and Cucurbitacin IIa (CuIIa), compounds derived from Chinese herbs, can exert anti-inflammatory effects by modulating single immunoglobulin interleukin-1 receptor-related receptor (SIGIRR; also known as Toll/interleukin-1 receptor 8, TIR8) and thereby inhibiting B. burgdorferi basic membrane protein A (BmpA)-induced TLR signaling in human macrophages, specifically the THP-1 human monocytic cell line. After THP-1 cells were exposed in vitro to: i) recombinant (r)BmpA, ii) rBmpA and ISOF or iii) rBmpA and CuIIa, Cytotoxicity assay (Cell Counting Kit-8, CCK-8) are used to measure the effects of ISOF and CuIIa on cell viability. Meanwhile, real-time polymerase chain reaction and Western blotting were used to quantify SIGIRR mRNA and protein levels, respectively, at 6, 12, 24 and 48 h time points post-stimulation. In addition, proinflammatory cytokine tumor necrosis factor-α (TNF-α) was determined by ELISA analysis. Our study showed that rBmpA stimulation of THP-1 cells resulted in a drop in SIGIRR levels in THP-1 cells. More importantly, SIGIRR levels increased significantly in rBmpA-stimulated THP-1 cells following ISOF or CuIIa administration, and the results of ELISA analysis suggested that ISOF or CuIIa reduced the secretion of the proinflammatory cytokine TNF-α. In conclusion, These results reveal new possibilities for the treatment of Lyme arthritis.

Characterization of mouse heart adenylyl cyclase.

Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces beta-adrenoceptor-stimulated Ca(2+) inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg(2+) were similar. Moreover, the inhibitory profile of eight 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-([gamma-thio])triphosphates on mouse heart in the presence of Mg(2+) was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K(i) values in the 15 to 25 nM range in the presence of Mg(2+) and in the 1 to 5 nM range in the presence of Mn(2+). However, in the presence of Mn(2+), we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg(2+), AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn(2+) masking AC5 effects were problems.

Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation.

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.

[Studies on the chemical constituents of Coleus forskohlii].

OBJECTIVE: To study the chemical constituents in the aerial parts of Coleus forskohlii. METHODS: The compounds were isolated by various column chromatographic methods, and their structures were identified by spectroscopic methods. RESULTS: Twelve compounds were isolated and identified as chamaecydin (1), 6 alpha-hydroxydemethylcryptojaponol (2), alpha-cedrene (3), oleanolic acid (4), forskolin G (5), forskolin J (6), 1,6-diacetyl-9-deoxyforskolin (7), forskolin A (8), forskolin H (9), 6-acetyl-1-deoxyforskolin (10), betulinic acid (11), beta-sitosterol (12). CONCLUSION: Compounds 1 - 3 are isolated from Coleus genus for the first time, and compound 4 is isolated from C. forskohlii for the first time.

Cardiovascular effects of intravenous colforsin in normal and acute respiratory acidosis canine models: A dose-response study.

In acidosis, catecholamines are attenuated, and higher doses are often required to improve cardiovascular function. Colforsin activates adenylate cyclase in cardiomyocytes without beta-adrenoceptor. Here, six beagles were administered colforsin or dobutamine four times during eucapnia (partial pressure of arterial carbon dioxide 35-40 mm Hg; normal) and hypercapnia (ibid 90-110 mm Hg; acidosis) conditions. The latter was induced by CO2 inhalation. Anesthesia was induced with propofol and maintained with isoflurane. Cardiovascular function was measured by thermodilution and a Swan-Ganz catheter at baseline and 60 min after 0.3 µg/kg/min (low), 0.6 µg/kg/min (middle), and 1.2 µg/kg/min (high) colforsin administration. The median pH was 7.38 [range 7.33-7.42] and 7.01 [range 6.96-7.08] at baseline in the Normal and Acidosis conditions, respectively. Endogenous adrenaline and noradrenaline levels at baseline were significantly (P < 0.05) higher in the Acidosis than in the Normal condition. Colforsin induced cardiovascular effects similar to those caused by dobutamine. Colforsin increased cardiac output in the Normal condition (baseline: 3.9 ± 0.2 L/kg/m2 [mean ± standard error], low: 5.2 ± 0.4 L/kg/min2, middle: 7.0 ± 0.4 L/kg/m2, high: 9.4 ± 0.2 L/kg/m2; P < 0.001) and Acidosis condition (baseline: 6.1 ± 0.3 L/kg/m2, low: 6.2 ± 0.2 L/kg/m2, middle: 7.2 ± 0.2 L/kg/m2, high: 8.3 ± 0.2 L/kg/m2; P < 0.001). Colforsin significantly increased heart rate and decreased systemic vascular resistance compared to values at baseline. Both drugs increased pulmonary artery pressure, but colforsin (high: 13.3 ± 0.6 mmHg in Normal and 20.1 ± 0.2 mmHg in Acidosis) may have lower clinical impact on the pulmonary artery than dobutamine (high: 19.7 ± 0.6 in Normal and 26.7 ± 0.5 in Acidosis). Interaction between both drugs and experimental conditions was observed in terms of cardiovascular function, which were similarly attenuated with colforsin and dobutamine under acute respiratory acidosis.

Modulation of calcium currents by a D1 dopaminergic protein kinase/phosphatase cascade in rat neostriatal neurons.

In rat neostriatal neurons, D1 dopamine receptors regulate the activity of cyclic AMP-dependent protein kinase (PKA) and protein phosphatase 1 (PP1). The influence of these signaling elements on high voltage-activated (HVA) calcium currents was studied using whole-cell voltage-clamp techniques. The application of D1 agonists or cyclic AMP analogs reversibly reduced N- and P-type Ca2+ currents. Inhibition of PKA antagonized this modulation, as did inhibition of PP1, suggesting that the D1 effect was mediated by a PKA enhancement of PP1 activity directed toward Ca2+ channels. In a subset of neurons, D1 receptor-mediated activation of PKA enhanced L-type currents. The differential regulation of HVA currents by the D1 pathway helps to explain the diversity of effects this pathway has on synaptic integration and plasticity in medium spiny neurons.

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst.

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I(e)) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I(e). In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 microM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

Beta-adrenoceptor-mediated vasodilation of retinal blood vessels is reduced in streptozotocin-induced diabetic rats.

We investigated the effects of epinephrine and dopamine on retinal blood vessels in streptozotocin (STZ, 80 mg/kg, i.p.)-treated rats and age-matched control rats to determine whether diabetes mellitus alters the retinal vascular responses to circulating catecholamines. Experiments were performed 6-8 weeks after treatment with STZ or the vehicle. The fundus images were captured with the digital fundus camera system for small animals we developed and diameters of retinal blood vessels contained in the digital images were measured. Epinephrine increased the diameters of retinal blood vessels, but the vasodilator responses were reduced in diabetic rats. Dopamine produced a biphasic retinal vascular response with an initial vasoconstriction followed by a vasodilation. The vasoconstrictor effects of dopamine on retinal arterioles were enhanced in diabetic rats, whereas the difference between the two groups was abolished by treatment with propranolol. The vasodilator effect of isoproterenol, but not of the activator of adenylyl cyclase colforsin, on retinal blood vessels was reduced in diabetic rats. No difference in vasoconstriction of retinal blood vessels to phenylephrine between non-diabetic and diabetic rats was observed. The vasodilator responses of retinal blood vessels to 1,1-dimethyl-4-phenylpiperazinium, a ganglionic nicotinic receptor agonist, were also attenuated in diabetic rats. These results suggest that diabetes mellitus alters the retinal vascular responses to circulating catecholamines and the impairment of vasodilator responses mediated by beta-adrenoceptors contributes to the alteration.

[The effect of PDEIII inhibitors and colforsin daropate on hypoxic pulmonary vasoconstriction in rabbit lungs].

BACKGROUND: PDEIII inhibitors and colforsin daropate hydrochloride (CDH) exert positive inotropic and vasodilatory effects by increasing intracellular cAMP The effect of olprinone (OLP), milrinone (MIL) and CDH on hypoxic pulmonary vasoconstriction (HPV) was evaluated using isolated blood-perfused lung preparations from the rabbit in situ. METHODS: We prepared a rabbit constant-flow lung-perfusion model in which HPV was induced by decreasing the FI(O2) from 21% to 3%. We conducted 2 sets of experiments by administering different doses of OLP MIL or CDH into the reservoir. In experiment 1, we administered OLP: 0.02, 0.2, 2, 20, 200 (microg), MIL: 0.1, 1, 10, 100, 1000, or CDH: 0.01, 0.1, 1, 10, 100 (microg). In experiment 2, we administered OLP 2microg or CDH 1 microg after premedication with thapsigargin (TH) 0.1 microM. Following drug administration, changes in pulmonary artery perfusion pressure were measured. RESULTS: Experiment 1:HPV was inhibited by the administration of 200 microg OLP 100 and 1000 microg MIL, and 10 and 100 microg CDH. Experiment 2:HPV was inhibited by pretreatment with TH, although supplementation with 2 microg OLP or 1 microg CDH did not change the level of inhibition of HPV. CONCLUSIONS: These results suggest that PDEIII inhibitors and CDH inhibit HPV only at high concentrations, the mechanism of inhibition being a decrease in the sensitivity of vascular smooth muscle to Ca2+.

Retinal waves trigger spindle bursts in the neonatal rat visual cortex.

During visual system development, the light-insensitive retina spontaneously generates waves of activity, which are transmitted to the lateral geniculate nucleus. The crucial question is whether retinal waves are further transmitted to the cortex and influence the early cortical patterns of activity. Using simultaneous recordings from the rat retina and visual cortex during the first postnatal week in vivo, we found that spontaneous retinal bursts are correlated with spindle bursts (intermittent network bursts associated with spindle-shape field oscillations) in the contralateral visual cortex (V1). V1 spindle bursts could be evoked by electrical stimulation of the optic nerve. Intraocular injection of forskolin, which augments retinal waves, increased the occurrence of V1 spindle bursts. Blocking propagation of retinal activity, or removal of the retina reduced the frequency, but did not completely eliminate the cortical spindle bursts. These results indicate that spontaneous retinal waves are transmitted to the visual cortex and trigger endogenous spindle bursts. We propose that the interaction between retinal waves and spindle bursts contributes to the development of visual pathways to the cortex.