Role of soluble guanylyl cyclase in renal afferent and efferent arterioles.

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It was hypothesized that the NO-sGC-cGMP system functions differently in renal afferent arterioles (AA) compared with efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles. Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to ANG II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in ANG II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) treatment and preconstriction with ANG II. Cinaciguat-induced dilatation of l-NAME-pretreated and ANG II-preconstricted arterioles was similar to controls without l-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Furthermore, it dilated EA, but not AA, after hypoxia/reoxygenation. The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Furthermore, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

Semiautomated quantitative image analysis of glomerular immunohistochemistry markers desmin, vimentin, podocin, synaptopodin and WT-1 in acute and chronic rat kidney disease models.

Five different glomerular immunohistochemistry markers were evaluated and compared in four different acute and chronic rat kidney disease models. Progression of glomerular or podocyte damage was shown in the puromycin aminonucleoside nephrosis (PAN) and Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model. Progression and prevention of glomerular damage was demonstrated in the Zucker diabetic fatty (ZDF) and Dahl salt-sensitive (Dahl SS) rat. Immunohistochemistry was performed for desmin, vimentin, podocin, synaptopodin and Wilms tumor protein-1 (WT-1), and evaluation of glomerular immunohistochemistry markers was done by semiautomated quantitative image analysis. We found desmin and WT-1 as the most sensitive markers for podocyte damage in both acute and chronic glomerular damage followed by vimentin, podocin and synaptopodin. We were able to demonstrate that early podocyte damage as shown by increased desmin and vimentin staining together with either a phenotypic podocyte change or podocyte loss (reduced numbers of WT-1-stained podocytes) drives the progression of glomerular damage. This is followed by a reduction in podocyte-specific proteins such as podocin and synaptopodin. Our report describes the different sensitivity of glomerular or podocyte markers and gives future guidance for the selection of the most sensitive markers for efficacy testing of new drugs as well as for the selection of tissue-based toxicity markers for glomerular or podocyte injury. In addition to functional clinical chemistry markers, desmin and WT-1 immunohistochemistry offers reliable and valuable data on the morphologic state of podocytes.

Effects of the dual PPAR-α/γ agonist aleglitazar on glycaemic control and organ protection in the Zucker diabetic fatty rat.

AIMS: To evaluate the effects of aleglitazar, a dual peroxisome proliferator-activated receptor-α/γ agonist, on the development of diabetes-related organ dysfunction, in relation to glycaemic and lipid changes, in Zucker diabetic fatty (ZDF) rats. METHODS: Six-week-old, male ZDF rats received aleglitazar 0.3 mg/kg/day or vehicle as food admix for 13 weeks (n = 10 per group). Age-matched male Zucker lean rats served as non-diabetic controls. Plasma and renal markers were measured at several time points. Histopathology and quantitative immunohistochemistry were performed at 13 weeks. RESULTS: Glycated haemoglobin (5.4 vs. 9.2%) and blood glucose (8.3 ± 0.3 vs. 26.1 ± 1.0 mmol/l) were significantly reduced at 12 weeks with aleglitazar versus vehicle-treated ZDF rats (both p < 0.01), while aleglitazar preserved near-normal plasma insulin levels. Aleglitazar prevented the development of hypertriglyceridaemia (1.4 ± 0.1 vs. 8.5 ± 0.9 mmol/l) and reduced plasma non-esterified fatty acids (0.09 ± 0.02 vs. 0.26 ± 0.04 mmol/l) relative to vehicle-treated animals (both p < 0.01). Urinary glucose and protein concentrations were significantly reduced at 13 weeks with aleglitazar versus vehicle-treated rats (both p < 0.01). Consistent with its effect on glycaemic control, aleglitazar protected ß-cell morphology, as evidenced by preservation of islet integrity, and reduction of ß-cell apoptosis and islet fibrosis. Aleglitazar prevented renal glomerular hypertrophy, podocyte degeneration, glomerulosclerosis, tubulo-interstitial lesions and development of cataracts. CONCLUSIONS: Aleglitazar strongly improved glycaemic and lipid parameters while protecting key tissues, including the pancreas, kidneys and eyes, against diabetes-associated structural and functional changes in the ZDF rat.

Taspoglutide, a novel human once-weekly GLP-1 analogue, protects pancreatic ß-cells in vitro and preserves islet structure and function in the Zucker diabetic fatty rat in vivo.

AIM: Glucagon-like peptide-1 (GLP-1) has protective effects on pancreatic ß-cells. We evaluated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, on ß-cells in vitro and in vivo. METHODS: Proliferation of murine pancreatic ß (MIN6B1) cells and rat islets in culture was assessed by imaging of 5-ethynyl-2'-deoxyuridine-positive cells after culture with taspoglutide. Apoptosis was evaluated with the transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labelling assay in rat insulinoma (INS-1E) cells and isolated human islets exposed to cytokines (recombinant interleukin-1ß, interferon-γ, tumour necrosis factor-α) or lipotoxicity (palmitate) in the presence or absence of taspoglutide. Islet morphology and survival and glucose-stimulated insulin secretion in perfused pancreata were assessed 3-4 weeks after a single application of taspoglutide to prediabetic 6-week-old male Zucker diabetic fatty (ZDF) rats. RESULTS: Proliferation was increased in a concentration-dependent manner up to fourfold by taspoglutide in MIN6B1 cells and was significantly stimulated in isolated rat islets. Taspoglutide almost completely prevented cytokine- or lipotoxicity-induced apoptosis in INS-1E cells (control 0.5%, cytokines alone 2.2%, taspoglutide + cytokines 0.6%, p < 0.001; palmitate alone 8.1%, taspoglutide + palmitate 0.5%, p < 0.001) and reduced apoptosis in isolated human islets. Treatment of ZDF rats with taspoglutide significantly prevented ß-cell apoptosis and preserved healthy islet architecture and insulin staining intensity as shown in pancreatic islet cross sections. Basal and glucose-stimulated insulin secretion of in situ perfused ZDF rat pancreata was normalized after taspoglutide treatment. CONCLUSIONS: Taspoglutide promoted ß-cell proliferation, prevented apoptosis in vitro and exerted multiple ß-cell protective effects on islet architecture and function in vivo in ZDF rats.

Taspoglutide, a novel human once-weekly analogue of glucagon-like peptide-1, improves glucose homeostasis and body weight in the Zucker diabetic fatty rat.

AIM: Glucagon-like peptide-1 (GLP-1) receptor agonists are a novel class of pharmacotherapy for type 2 diabetes. We investigated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, in the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. METHODS: Blood glucose and plasma levels of insulin, peptide YY (PYY), glucose-dependent insulinotropic polypeptide (GIP) and triglycerides were measured during oral glucose tolerance tests (oGTT) conducted in ZDF rats treated acutely or chronically with a single long-acting dose of taspoglutide. Pioglitazone was used as a positive control in the chronic study. Postprandial glucose, body weight, glycaemic control and insulin sensitivity were assessed over 21 days in chronically treated animals. RESULTS: Acute treatment with taspoglutide reduced glucose excursion and increased insulin response during oGTT. In chronically treated rats, glucose excursion and levels of GIP, PYY and triglycerides during oGTT on day 21 were significantly reduced. Postprandial glucose levels were significantly lower than vehicle controls by day 15. A significant reduction in body weight gain was noticed by day 8, and continued until the end of the study when body weight was approximately 7% lower in rats treated with taspoglutide compared to vehicle. Glycaemic control (increased levels of 1,5-anhydroglucitol) and insulin sensitivity (Matsuda index) were improved by taspoglutide treatment. CONCLUSIONS: Taspoglutide showed typical effects of native GLP-1, with improvement in glucose tolerance, postprandial glucose, body weight, glycaemic control and insulin sensitivity.

Dalcetrapib: no off-target toxicity on blood pressure or on genes related to the renin-angiotensin-aldosterone system in rats.

BACKGROUND AND PURPOSE: The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model. EXPERIMENTAL APPROACH: Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg kg(-1) day(-1); dalcetrapib 100, 300 or 500 mg(-1) kg day(-1); or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days' treatment with torcetrapib 40 mg kg(-1) day(-1), dalcetrapib 500 mg kg(-1) day(-1) or vehicle was measured by quantitative polymerase chain reaction. KEY RESULTS: Torcetrapib transiently increased mean AP in normotensive rats (+3.7 +/- 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 +/- 0.6 mmHg with 40 mg kg(-1) day(-1) at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas. CONCLUSIONS AND IMPLICATIONS: In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.

Synthesis and biological evaluation of novel hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines as potent and selective 5-HT(2C) receptor agonists.

Further lead optimization efforts on previously described 1,2,3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indoles led to the new class of 5,5a,6,7,8,9-hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines culminating in the discovery of (5aR,9R)-2-[(cyclopropylmethoxy)methyl]-5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[3', 2':4,5]pyrrolo[1,2-a]pyrazine 18 as a potent, full 5-HT(2C) receptor agonist with an outstanding selectivity profile and excellent hERG and phospholipidosis properties.

Identification of 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT2C receptor agonists.

Synthesis and evaluation of the activity of new 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT(2C) receptor agonists are described. Appropriately substituted, several analogs displayed selectivity against the other 5-HT(2) receptor subtypes of 1 order of magnitude or more. Selectivity was improved for several compounds versus the lead 1, increasing the therapeutic interest in this series of 5-HT(2C) receptor agonists.

Effects of verapamil on atrial fibrillation and its electrophysiological determinants in dogs.

BACKGROUND: Atrial tachycardia-induced remodeling promotes the occurrence and maintenance of atrial fibrillation (AF) and decreases L-type Ca(2+) current. There is also a clinical suggestion that acute L-type Ca(2) channel blockade can promote AF, consistent with an AF promoting effect of Ca(2+) channel inhibition. METHODS: To evaluate the potential mechanisms of AF promotion by Ca(2+) channel blockers, we administered verapamil to morphine-chloralose anesthetized dogs. Diltiazem was used as a comparison drug and autonomic blockade with atropine and nadolol was applied in some experiments. Epicardial mapping with 240 epicardial electrodes was used to evaluate activation during AF. RESULTS: Verapamil caused AF promotion in six dogs, increasing mean duration of AF induced by burst pacing, from 8+/-4 s (mean+/-S.E.) to 95+/-39 s (P<0.01 vs. control) at a loading dose of 0.1 mg/kg and 228+/-101 s (P<0.0005 vs. control) at a dose of 0.2 mg/kg. Underlying electrophysiological mechanisms were studied in detail in five additional dogs under control conditions and in the presence of the higher dose of verapamil. In these experiments, verapamil shortened mean effective refractory period (ERP) from 122+/-5 to 114+/-4 ms (P<0.02) at a cycle length of 300 ms, decreased ERP heterogeneity (from 15+/-1 to 10+/-1%, P<0.05), heterogeneously accelerated atrial conduction and decreased the cycle length of AF (94+/-4 to 84+/-3 ms, P<0.005). Diltiazem did not affect ERP, AF cycle length or AF duration, but produced conduction acceleration similar to that caused by verapamil (n=5). In the presence of autonomic blockade, verapamil failed to promote AF and increased, rather than decreasing, refractoriness. Neither verapamil nor diltiazem affected atrial conduction in the presence of autonomic blockade. Epicardial mapping suggested that verapamil promoted AF by increasing the number of simultaneous wavefronts reflected by separate zones of reactivation in each cycle. CONCLUSIONS: Verapamil promotes AF in normal dogs by promoting multiple circuit reentry, an effect dependent on intact autonomic tone and not shared by diltiazem.

Differential efficacy of L- and T-type calcium channel blockers in preventing tachycardia-induced atrial remodeling in dogs.

BACKGROUND: Tachycardia-induced remodeling likely plays an important role in atrial fibrillation (AF) maintenance and recurrence after cardioversion, and Ca(2+) overload may be an important mediator. This study was designed to evaluate the relative efficacies of selective T-type (mibefradil) and L-type (diltiazem) Ca(2+)-channel blockers in preventing tachycardia-induced atrial remodeling. METHODS: Dogs were given daily doses of mibefradil (100 mg), diltiazem (240 mg) or placebo in a blinded fashion, beginning 4 days before and continuing through a 7-day period of atrial pacing at 400 bpm. An electrophysiological study was then performed to assess changes in refractoriness, refractoriness heterogeneity and AF duration. RESULTS: Mean duration of burst-pacing induced AF was similar in placebo (567+/-203 s) and diltiazem-treated (963+/-280 s, P=NS) animals, but was much less in mibefradil-treated dogs (3.6+/-0.9 s, P<0.002) and non-paced controls (6.6+/-2.7 s). In contrast to mibefradil, diltiazem did not alter tachycardia-induced refractoriness abbreviation or heterogeneity. To exclude inadequate dosing as an explanation for diltiazem's inefficacy, we studied an additional group of dogs treated with 720 mg/day of diltiazem, and again noted no protective effect. Acute intravenous administration of diltiazem to control dogs failed to alter atrial refractoriness or AF duration, excluding a masking of remodeling suppression by offsetting profibrillatory effects of the drug. CONCLUSIONS: Whereas the selective T-type Ca(2+)-channel blocker mibefradil protects against atrial remodeling caused by 7-day atrial tachycardia, the selective L-type blocker diltiazem is without effect. These findings are potentially important for understanding the mechanisms and prevention of clinically-relevant atrial-tachycardia-induced remodeling.

Pharmacological properties of Ca(V)3.2, a low voltage-activated Ca2+ channel cloned from human heart.

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.

Contrasting efficacy of dofetilide in differing experimental models of atrial fibrillation.

BACKGROUND: Rapid atrial pacing (RAP) and congestive heart failure (CHF) produce different experimental substrates for atrial fibrillation (AF). We tested the hypothesis that AF maintained by different substrates responds differently to antiarrhythmic-drug therapy. METHODS AND RESULTS: The class III antiarrhythmic agent dofetilide was given intravenously at doses of 10 (D10) and 80 (D80) microg/kg to dogs with AF induced either (1) after 7 days of RAP at 400 bpm or (2) in the presence of CHF induced by rapid ventricular pacing. Dofetilide terminated AF in all CHF dogs, but D10 failed to terminate AF in any RAP dog, and D80 terminated AF in only 1 of 5 RAP dogs (20%) (P<0.01 for efficacy in CHF versus RAP dogs). Dofetilide was highly effective in preventing AF induction by atrial burst pacing in dogs with CHF but was totally ineffective in dogs with RAP. Dofetilide increased atrial effective refractory period and AF cycle length to a greater extent in CHF dogs. Epicardial mapping with 248 bipolar electrodes showed that CHF-related AF was often due to macroreentry, with dofetilide terminating AF by causing block in reentry circuits. RAP-related AF was due to multiple-wave front reentry, with dofetilide slowing reentry and decreasing the number of simultaneous waves, but not sufficiently to stop AF. CONCLUSIONS: The mechanism underlying AF importantly influences dofetilide efficacy. The dependence of drug efficacy in AF on the underlying mechanism has potentially significant implications for antiarrhythmic drug use and development and may explain the well-known therapeutic resistance of longer-duration AF.

Effects of the T-type Ca(2+) channel blocker mibefradil on repolarization of guinea pig, rabbit, dog, monkey, and human cardiac tissue.

At supratherapeutic doses (2- to 5-fold), the T-type Ca(2+) antagonist mibefradil modifies the T/U wave of the human ECG. In this study, we show that this effect is observed in conscious monkeys and is duplicated by verapamil or diltiazem. We then evaluate the proarrhythmic risk of such alterations of cardiac repolarization by examining the actions of mibefradil on cardiac action potentials (APs). In isolated cardiomyocytes from guinea pigs or humans, mibefradil dose dependently shortens the plateau of the AP; this effect is similar to other Ca(2+) antagonists and opposite to drugs having class III antiarrhythmic properties. The metabolites of mibefradil, singly or in combination, also shorten APs. In isolated rabbit hearts, noncardiodepressant concentrations of mibefradil have no effect on monophasic action potentials (MAPs), whereas cardiodepressant levels produce a slight nonsignificant lengthening. In hearts of open-chest bradycardic dogs, mibefradil has no effect on MAP dispersion or on QT interval and shortens MAPs slightly; although high doses produce atrioventricular block, likely through Ca(2+) antagonism, arrhythmias are never observed. In contrast, d-sotalol lengthens QT interval and MAPs, increases dispersion, and produces arrhythmias. Together, these in vitro and in vivo results suggest that mibefradil carries no proarrhythmic risk despite changes in T/U wave morphology. Although these changes resemble those observed with class III compounds, they also are seen with nonproarrhythmic compounds such as verapamil and diltiazem. In conclusion, the classical models used in the present study could not link the changes in T/U wave morphology produced by mibefradil and verapamil to any experimental marker of proarrhythmic liability.

The T-type Ca(2+) channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by tachycardia-induced atrial remodeling in dogs.

BACKGROUND: Ca(2+) overload is believed to play a role in tachycardia-induced atrial electrophysiological remodeling. L-type Ca(2+) channel blockers attenuate effective refractory period (ERP) changes caused by 24 hours of atrial tachycardia but may not substantially alter atrial fibrillation (AF) inducibility. This study assessed the effects of the T-type Ca(2+) channel blocker mibefradil on tachycardia-induced atrial remodeling. METHODS AND RESULTS: Dogs subjected to rapid atrial pacing (400 bpm) for 7 days were treated with mibefradil (100 mg/d, n=8) or matching placebo (n=10) in blinded fashion. Radiofrequency ablation of atrioventricular conduction and ventricular pacing were used to control ventricular rate. Placebo dogs showed significant decreases in atrial ERP (76+/-5 ms at a cycle length of 300 ms) and increases in ERP heterogeneity (27.7+/-2.4%), AF duration (414+/-232 seconds), and AF inducibility by single extrastimuli (41+/-10% of sites) compared with 10 unpaced control dogs (ERP 114+/-3 ms, ERP heterogeneity 13.8+/-0.9%, AF duration 7+/-3 seconds, AF inducibility 1.9+/-1.0% of sites). The changes caused by atrial tachycardia were strongly attenuated in mibefradil dogs, with ERPs averaging 102+/-7 ms, ERP heterogeneity 18.8+/-1.4%, AF duration 3+/-1 seconds, and AF inducibility 9.6+/-4.0% of sites. Among mibefradil-treated dogs, ERP, AF duration, and inducibility correlated with plasma drug concentration. Acute mibefradil administration did not alter ERP or AF. CONCLUSIONS: Mibefradil, a drug with strong T-type Ca(2+) channel blocking properties, prevents AF-promoting electrophysiological remodeling by atrial tachycardia. These findings have important potential implications for the mechanisms of tachycardia-induced atrial remodeling and demonstrate the feasibility of preventing electrical remodeling caused by several days of atrial tachycardia.

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.

Primary culture of human atrial myocytes is associated with the appearance of structural and functional characteristics of immature myocardium.

We examined changes in the structural and physiological characteristics of human atrial myocytes during primary culture in the presence of serum. Action potentials and ionic currents were recorded in freshly dissociated (FM) and cultured (CM) whole-cell patch-clamped myocytes, alpha-smooth muscle actin, sarcomeric alpha-actinin and beta-myosin heavy chains (beta-MHC) were stained with monoclonal antibodies. From day 5 to day 21, myocytes lost their rod shape, spread and exhibited reorganized sarcomeres. These morphological changes were associated with a marked increase in membrane capacitance (+266%). Both beta-MHC and alpha-smooth muscle actin were expressed in CM but not in FM, indicating a dedifferentiation process. CM were characterized by a lower resting potential (-30 +/- 2 v -60 +/- 4 mV, P < 0.05) and, when repolarized, by a shorter action potential duration (APD) than FM (APD-60: 126.9 v 159.6 ms, P < 0.05). The inward rectifier K+ current was absent in CM, thus explaining the low resting potential. The density of the transient component of the voltage-activated K+ current Ito1 was not modified during culture, while that of the sustained component Isus was increased fourfold. The amplitude of ICa was increased, but its density was unchanged, indicating that CM maintained a normal density of functional calcium channels. Neither the voltage dependence nor the inactivation of ICa was modified in CM. The time constants of inactivation of ICa were unchanged, although the amplitude of the rapidly inactivating component of ICa was increased in CM compared to FM. Moreover, ICa was increased by the beta-adrenergic agonist isoproterenol (1 microM) throughout the culture period. Our results demonstrate that in long-term serum-supplemented culture, adaptation of human atrial myocytes to their new environment is associated with differential alterations of the main ionic currents and phenotypic changes characteristic of immature myocardium.

Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.

The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca2+ transients (Cai transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric alpha-actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca2+ current (Ica) elicited a prolonged Cai transient, with an initial rapidly activating phase (slope 1, 23.6 +/- 1.2 s-1) followed by a slowly activating phase (slope 2, 5.8 +/- 0.4 s-1; P < .001 versus slope 1), resulting in a dome-shaped Cai transient. Ryanodine (100 mumol/L) inhibited 79 +/- 6% of the Cai transient, indicating that it was due essentially to sarcoplasmic reticulum Ca2+ release. During step depolarizations, maximal activation of the Cai transient or tail current (Itail) (in cells dialyzed with Ca2+ buffer-free internal solution) preceded that of Ica and did not follow its voltage dependence (n = 12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Cai transient and Itail (n = 5). In a given cell, the two components of the Cai transient could be dissociated by altering the intracellular Ca2+ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of Ica. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, Ica only partially controls the activation of RyRs, with the prolonged and dome-shaped Cai transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca2+ channels.

Differential regulation of voltage-activated potassium currents in cultured human atrial myocytes.

To examine whether the two components of the voltage-activated outward K+ current, an initially rapidly inactivating component (Ito,1) and a slowly inactivating sustained component (Isus), in human atrial myocytes are distinct currents differentially regulated, we studied their behavior during serum-induced growth of cultured myocytes. Currents were recorded in whole cell patch clamped myocytes. After 1 wk of culture (day 8), membrane capacitance was twice the value in freshly dissociated myocytes (178.7 +/- 23 vs. 83.1 +/- 5.5 pF; P < 0.001). Ito,1 density did not differ from that in freshly dissociated myocytes (at +40 mV: 4.38 +/- 0.8 vs. 3.71 +/- 0.6 pA/pF), whereas that of Isus was markedly increased (at +40 mV: 9.76 +/- 2 vs. 2.21 +/- 0.29 pA/pF; P < 0.001). After inactivation of Ito,1 by a prepulse, sustained depolarization elicited in cultured myocytes an Isus with a density of 10.22 +/- 1.18 pA/pF and an apparent tail current reversal potential of -73.5 +/- 3.2 mV, indicating high K+ selectivity. Isus was highly sensitive to 4-aminopyridine (55.4 +/- 4.4% inhibition in 50 microM) and to D-600 (with a concentration inhibiting 50% of maximal response of 34.2 x 10(-6) M). Addition of 5-10 nM staurosporine at day 3 prevented cell growth and reduced Ito,1 density but not the increase in Isus density, which was inhibited by 10 microM staurosporine. Our results indicate that Ito,1 and Isus are regulated independently during in vitro myocyte growth in human atrial myocytes and that the increase in Isus density is not mediated by a protein kinase C-dependent pathway.

Contribution of Na+/Ca2+ exchange to action potential of human atrial myocytes.

The Ca2+ dye indo 1 was used to record internal Ca2+ (Cai) transients in order to investigate the role of the Na+/Ca2+ exchange current (INa/Ca) in whole cell patch-clamped human atrial myocytes After the activation of the L-type Ca2+ current by test pulses (20 ms) at +20 mV, a tail current (I(tail)) was activated at a holding potential of -80 mV with a density of -1.29 +/- 0.06 pA/pF. The time course of I(tail) followed that of Cai transients I(tail) was suppressed by dialyzing cells with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, applying 5 mM caffeine, or substituting external Na+ with Li+, indicating that this current was mainly generated by INa/Ca. Two types of action potential were recorded: type A, which is characterized by a narrow early plateau followed by a late low plateau phase, and type B, which is characterized by a small initial peak followed by a prolonged high plateau phase. Type B action potentials were found in larger cells than type A action potentials (membrane capacitance 81.8 +/- 4.5 and 122.4 +/- 7.0 pF in types A and B, respectively, P < 0.001). Substitution of external Na+ with Li+ shortened the late plateau of the type A action potential and the prolonged plateau of the type B action potential. Suppression of Cai transients by caffeine shortens the late part of both types of action potentials, whereas its lengthening effect on the initial phase of action potentials can result from several different mechanisms. The beat-to-beat dependent relationship between Cai transients and action potentials could be mediated by Ina/Ca- Delayed afterdepolarizations were present in a significant proportion of atrial myocytes in our experimental conditions. They were reversibly suppressed by Li+ substitution for Na+, suggesting that they are generated by INa/Ca. We conclude that INa/Ca plays a major role in the development of action potentials and delayed afterdepolarizations in isolated human atrial myocytes.

Nitric oxide regulates the calcium current in isolated human atrial myocytes.

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.