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Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.

Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Gαi2-fused GB2R (GB2R-Gαi2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Gαqi5 (a chimeric Gαq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl− currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Gαi2 or GB1aR-Gαqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Gαqi5 caused FRET and Ca2+-activated Cl− currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Gαqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R.

The objective of this study was to evaluate serum nitrite and nitrate (nitrite/nitrate) concentrations that affect adversely pregnancy outcome. Pregnant rats, from day 2 to day 8 of pregnancy, were daily given subcutaneously several doses (5, 10, and 30 mg/rat) of diethylenetriamine-nitric oxide (DETA/NO). Serum nitrite/nitrate concentrations were measured using an HPLC system. Serum nitrite/nitrate concentrations increased dose-dependently with DETA/NO. Effects of DETA/NO on pregnancy outcome were assessed on day 14 of pregnancy. In rats given 5 mg DETA/NO, there was a significant increase in serum nitrite/nitrate concentrations (49.2 vs 24.6 μmol/l, P

Characterization of the γ-aminobutyric acid (GABA)B receptor involved in the motility of dog small intestine was analyzed by application of the microdialysis method to the small intestine of the whole body of the dog. The reverse transcription-polymerase chain reaction (RT-PCR) was used. Intraarterial administration of muscimol induced acceleration of motility associated with acetylcholine (ACh) release, these responses being antagonized by bicuculline. Intraarterial administration of baclofen induced inhibition of motility associated with ACh release, these responses being antagonized by CGP62349. GABA induced inhibition of motility associated with decrease in ACh release. CGP62349 alone induced acceleration of motility associated with increase in ACh release. RT-PCR revealed the presence of mRNAs for both subunits of GABAB receptor, GABAB1 and GABAB2, in the dog small intestine, although GABAB1 subunits were 6 isoforms of GABAB1 (GABAB1(a) – GABAB1(g)), except GABAB1(d). Thus, the GABAB receptor located at cholinergic neurons as a heterodimer with subunits of GABAB1 and GABAB2 in the dog small intestine operates predominantly relative to the GABAA receptor in physiological motility. Keywords:: GABAB1 subunit, GABAB2 subunit, microdialysis, RT-PCR

Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

UNLABELLED Alphaxalone is a neurosteroid anesthetic, but its mechanisms of action are not completely understood. Muscarinic receptors are involved in a variety of neuronal functions in the brain and autonomic nervous system, and much attention has been paid to them as targets of anesthetics. In this study, we investigated the effects of alphaxalone on M(1) and M(3) muscarinic receptors using the Xenopus oocyte expression system. Alphaxalone inhibited acetylcholine-induced currents in oocytes expressing M(1) receptors at clinically relevant concentrations. Alphaxalone also suppressed acetylcholine-induced currents in oocytes expressing M(3) receptors. The half-maximal inhibitory concentration values for the inhibition of M(1)- and M(3)-mediated currents were 1.8 +/- 0.6 micro M and 5.3 +/- 1.0 micro M, respectively. GF109203X, a selective protein kinase C inhibitor, had little effect on the inhibition of acetylcholine-induced currents by alphaxalone in oocytes expressing these receptors. Alphaxalone inhibited the specific binding of [(3)H]quinuclidinyl benzilate to oocytes expressing M(1) or M(3) receptors. These findings suggest that alphaxalone at clinically relevant concentrations inhibits the function of M(1) and M(3) receptors through a protein kinase C-independent mechanism by interfering with the [(3)H]quinuclidinyl benzilate binding sites on the receptors. IMPLICATIONS Alphaxalone, a neurosteroid anesthetic, inhibited the function of muscarinic M(1) and M(3) receptors and the specific binding of [(3)H]quinuclidinyl benzilate ([(3)H]QNB) to oocytes expressing these receptors. These findings suggest that alphaxalone inhibits these receptors by interfering with the QNB binding sites.

Involvement of protein kinase C (PKC) in the release of gamma-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [3H]GABA showed spontaneous release of [3H]GABA, approximately 0.5% of GABA content per 1 min. Stimulation with either Ca2+ ionophore (A23187) or a high K+ concentration increased the release of [3H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12-O-Tetradecanoylphorbol 13-acetate (10-300 nM) but not 4alpha-phorbol 12,13-didecanoate (300 nM) potentiated the A23187-stimulated release of [3H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.

Tramadol is a widely used analgesic, but its mechanism of action is not completely understood. Muscarinic receptors are involved in neuronal function in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets of analgesic drugs in the central nervous system. In this study, we investigated the effects of tramadol on type-3 muscarinic (M(3)) receptors using the Xenopus oocyte expression system. Tramadol (10 nM-100 micro M) inhibited acetylcholine-induced currents in oocytes expressing M(3) receptor. Although GF109203X, a protein kinase C inhibitor, increased the basal current, it had little effect on the inhibition of acetylcholine-induced currents by tramadol. Moreover, tramadol inhibited the specific binding sites of [(3)H]quinuclidinyl benzilate. These findings suggest that tramadol at clinically relevant concentrations inhibits M(3) function via quinuclidinyl benzilate-binding sites. This may explain the modulation of neuronal function and the anticholinergic effects of tramadol.Muscarinic receptors are involved in neuronal function and are targets of analgesic drugs. We here report that tramadol inhibits type-3 muscarinic receptors function via quinuclidinyl benzilate-binding sites at clinically relevant concentrations. These findings may explain the modulation of neuronal function and the anticholinergic effects of tramadol.

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.

The mechanism underlying orexin-induced contraction was examined in isolated preparations of guinea pig ileum, in relation to cholinergic transmission. Orexin-A caused contraction of ileal strips in a concentration-dependent manner. 1-(2-Methylbenzoxazol-6-yl)-3-[1,5]napthyridin-4-yl-urea hydrochloride (SB-334867-A) antagonized the orexin-A-induced contraction, with no effects on the acetylcholine-induced contraction and twitch contractions. The orexin-A-induced contraction was inhibited by tetrodotoxin and atropine, but not by hexamethonium, an antagonist of vasoactive intestinal peptide and a mixture of 5-hydroxytryptamine receptor antagonists. Orexin-A evoked an outflow of [3H]acetylcholine from the ileal strips preincubated with [3H]choline, in a concentration-dependent manner, and the orexin-A-evoked outflow was inhibited by tetrodotoxin, indicating that the outflow of [3H]acetylcholine originates from the nerve terminals. The orexin-A-evoked outflow of [3H]acetylcholine was antagonized by SB-334867-A. Thus, orexin-A evokes the release of acetylcholine from the enteric cholinergic neurons due to stimulation of the orexin-1 receptors and then causes contractions of guinea pig ileum.

1. The role of synaptophysin in the exocytotic release of dopamine (DA) was examined in Xenopus laevis oocytes injected with rat brain mRNA.

IN Xenopus oocytes coinjected with poly(A) + RNA derived from the rat cerebellum and cRNAs for the cloned G protein-gated inwardly rectifying K + channel (GIRK), GIRK1 and GIRK2, the GABA-B agonist baclofen elicited inwardly rectifying K + currents. The inward K + currents elicited by baclofen were inhibited by the selective GABA-B antagonists 2-OH saclofen and CGP 35348, and by the GIRK inhibitor Ba 2+ . In contrast, baclofen caused no currents in oocytes injected with the cerebellar poly(A) + RNA alone, the poly(A) + RNA and cRNA for GIRK1 or GIRK2, or only cRNAs for GIRKI and GIRK2. These findings indicate that GABA-B receptors in the rat cerebellum were functionally expressed in Xenopus oocytes and activated the cloned GIRKs composed of GIRK1 and GIRK2 as heteromultimers.

Characterization of the GABAB receptor in the human colon was performed by the reverse transcription-polymerase chain reaction (RT-PCR). mRNAs for both subunits of the GABAB receptor, GABAB1 and GABAB2, were detected in the human colon. The GABAB1(e) isoform was detected in the human colon, but not in the brain, and the other isoforms, except GABAB1(d), were detected in both tissues. Thus, the GABAB receptor may be present as a heterodimer with subunits of GABAB1 and GABAB2 in the human colon. Keywords:: GABAB1 subunit, GABAB2 subunit, RT-PCR

To investigate the clinical effects of MS-551, a Class III antiarrhythmic agent, 11 patients underwent electrophysiological study. MS-551 was given intravenously as an initial dose of 0.2 or 0.3 mg/kg for 5 minutes followed by the continuous infusion at 0.2 or 0.3 mg/kg for 30 minutes, respectively, in all patients. The rate corrected QT interval increased significantly from 3 minutes after the beginning of MS-551 infusion. The sinus heart rate decreased significantly by 8% at 10 minutes after the drug administration (P < 0.025). Mean PR and QRS intervals, and blood pressure were not significantly affected by the drug. Mean PA, AH, and HV intervals during sinus rhythm were also not affected. The effective refractory periods (ERPs) of the atrium and ventricle were significantly prolonged by 13% from 202 +/- 24 ms to 231 +/- 26 ms (P < 0.0005), and by 7% from 238 +/- 11 ms to 257 +/- 13 ms (P < 0.002), respectively, by MS-551. The ERP of the atrioventricular node and sinoatrial nodal recovery time were not changed significantly by the drug. This is a report of the effects of MS-551 in humans. This agent could be useful for treatment of tachyarrhythmias by prolongation of ERPs of the atrium and ventricle without significant variations of blood pressure and intracardiac conduction times. It is noteworthy that MS-551 slightly but significantly decreased heart rate.

Endocardial catheter mapping of the right atrium during sinus rhythm and programmed atrial stimulation were performed in 50 patients with sick sinus syndrome to investigate the relationship between abnormal atrial electrograms recorded during sinus rhythm and some determinants of the atrial vulnerability such as repetitive atrial firing and fragmented atrial activity elicited by single extrastimulus. The patients were divided into 2 groups on the basis of the presence (Group I) or absence (Group II) of abnormal atrial electrograms recorded during sinus rhythm. In Group I (N = 32), repetitive atrial firing was induced in 23 (72%) patients, and in Group II (N = 18) in 6 (33%) patients; p less than 0.01. The repetitive atrial firing zone was 41 +/- 37 ms in Group I and 12 +/- 18 ms in Group II; p less than 0.001. Fragmented atrial activity was induced in 30 (94%) patients from Group I, and in 8 (44%) patients from Group II; p less than 0.0001. The fragmented atrial activity zone was 47 +/- 42 ms in Group I and 14 +/- 19 ms in Group II; p less than 0.0001. The atrial electrogram width at the premature beat (A2; p < 0.02) and the maximum A2/A1 ratio (p < 0.002) were 178 +/- 53 ms and 196% +/- 40%, respectively in Group I, and 141 +/- 36 ms and 159% +/- 30%, respectively in Group II. Atrial fibrillation was induced in 13 (41%) patients from Group I, and in 1 (6%) patient from Group II (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate whether isoproterenol (Iso) could suppress the initiation of repetitive atrial firing (RAF), we investigated its effect on RAF in comparison with that of disopyramide (Diso). Extrastimuli at a basic cycle length of 500 ms were delivered from the high right atrium in 49 patients who received an intravenous infusion of Iso (0.01 microgram/kg per min) and in 39 patients given intravenous Diso (2 mg/kg per 10 min). Induction of RAF, the atrial effective refractory period (A-ERP), and the maximum conduction delay (MCD) were measured. Iso abolished the induction of RAF in 13/19 (68%) patients, while Diso did so in 13/22 (59%) patients. Thirty-four of the 41 patients with RAF in the baseline study had an A-ERP250 ms and an MCD40 ms. Iso significantly decreased the A-ERP from 205 +/- 26 to 194 +/- 23 ms (P0.01) and significantly decreased the MCD from 67 +/- 24 to 39 +/- 16 ms (P0.0001) in 19 patients with RAF. On the other hand, Diso significantly increased the A-ERP from 203 +/- 31 to 235 +/- 36 ms (P0.0001), and significantly diminished the MCD from 68 +/- 31 to 55 +/- 30 ms (P0.01) in 22 patients with RAF. In patients with new RAF (n = 7) or re-induced RAF (n = 14) during Iso or after Diso, the MCD was more than 40 ms. Our results suggest that there are two different modes of RAF suppression, i.e. shortening or lengthening of the A-ERP.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between abnormal atrial electrograms (AAE) recorded during sinus rhythm by endocardial catheter mapping of the right atrium and the atrial conduction defects of sinus impulses or single atrial extrastimuli was investigated in 44 patients with sick sinus syndrome. The patients were divided into two groups on the basis of the presence (n = 29) or absence (n = 15) of AAE recorded during sinus rhythm. The P wave duration in the AAE (+) Group patients was 137 +/- 14 msec, and 125 +/- 15 msec in the AAE (-) Group; P0.02. The intraatrial conduction time of sinus impulses in the AAE (+) Group was 54 +/- 12 msec, and 39 +/- 9 msec in the AAE (-) Group; P0.001. The interatrial conduction time in the AAE (+) Group was 101 +/- 14 msec, and 78 +/- 16 msec in the AAE (-) Group; P0.001. In the AAE (+) Group, 11 (38%) patients had a sinus node recovery time4 seconds, whereas in the AAE (-) Group there was only one (6%) patient; P0.03. AAE showed a specificity of 93% and a positive predictive accuracy of 91% in predicting inducibility of atrial fibrillation. The sensitivity was 35% and the negative predictive accuracy was 42%. Sustained atrial fibrillation was induced in ten (35%) patients of the AAE (+) Group, and in one (7%) patient of the AAE (-) Group; P0.05. These data suggest that in patients with sick sinus syndrome who possess abnormal endocardial electrograms in sinus rhythm within the right atrium have: (1) a significantly longer P wave duration; (2) a significantly longer intraatrial and interatrial conduction time of sinus impulses; and (3) a significantly greater sinus node dysfunction and higher incidence of induction of sustained atrial fibrillation. It is concluded that there are significantly greater atrial conduction defects in patients with sick sinus syndrome who possess AAE within the right atrium during sinus rhythm.

E-4031 is classified as a class III antiarrhythmic agent of the Vaughan Williams classification. Most class III agents have other antiarrhythmic effects in addition to the potassium channel-blocking action.1–3 In contrast, E-4031 could be considered as the selective blocker of the potassium channel responsible for prolonging the action potential duration without affecting the conduction time.4–6 From several experimental studies, it is believed that E-4031 suppresses reentrant tachyarrhythmias due to prolongation of the effective refractory period.7–12 However, the effects of E-4031 on electrophysiologic properties of the heart in humans are unknown. The present report investigates the electrophysiologic effects and toxicity of E-4031 in humans by means of electrophysiologic studies.

We report a patient with slow-fast atrioventricular (AV) nodal reentrant tachycardia, in which double ventricular response was demonstrated during rapid pacing at cycle length of 300 msec or less from the high right atrium. The determinants of double ventricular response during transient entrainment in the present case were: (1) a crucial conduction delay in the slow pathway; (2) the collision between the activation via the antegrade fast pathway (antidromically) of the last paced beat and the activation via the antegrade slow pathway (orthodromically) of the previous paced beat, instead of the unidirectional block in the slow pathway; and (3) the enhanced AV nodal conduction over the antegrade fast pathway.

Background —Prolonged and fractionated right atrial endocardial electrograms are characteristic of paroxysmal atrial fibrillation (idiopathic or associated with sick sinus syndrome). The distribution of these abnormal atrial electrograms within the right atrium and the way it is related to the likelihood that patients with sick sinus syndrome will develop paroxysmal atrial fibrillation was studied. Methods —Endocardial catheter mapping of the right atrium during sinus rhythm was performed in 41 control patients with normal sinus node function and without paroxysmal atrial fibrillation, in 33 patients with sick sinus syndrome but without tachycardia, and in 27 patients with sick sinus syndrome and paroxysmal atrial fibrillation (group 3). The bipolar electrograms were recorded at 12 sites in the right atrium and an abnormal atrial electrogram was defined as lasting ≥100 ms and/or showing eight or more fragmented deflections. Results —1195 atrial endocardial electrograms were assessed and quantitatively measured. In patients with sick sinus syndrome and paroxysmal atrial fibrillation 54% of the abnormal atrial electrograms were recorded from the high right atrium, 28% from the mid right atrium, and 18% from the low right atrium. However, in patients with sick sinus syndrome without tachycardia 78% of the abnormal atrial electrograms were recorded from the high right atrium and 22% from the mid right atrium. No abnormal electrograms were recorded from the low right atrium. Conclusions —In patients with sick sinus syndrome without tachycardia abnormal atrial electrograms generally came from the high right atrium but in patients with sick sinus syndrome and paroxysmal atrial fibrillation abnormal atrial electrograms were more widely distributed in the right atrium. In patients with sick sinus syndrome the greater the extent of the compromised atrial muscle, the greater the likelihood that paroxysmal atrial fibrillation will develop.

An improved method for isolating cardiac myocytes is described. This method consists of the initial dispersion of the myocytes with a new type of collagenase and the following treatment of the cells with a non-specific protease. This method gives more than 80% rod-shaped, viable, and quiescent cells, which are suitable for biochemical as well as electrophysiological experiments. Formation of a G omega seal and subsequent whole-cell recording is easily performed using these cells.

The Third Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, JapanSummaryBackground: The prevalence of atrial fibrillation (AF) hasbeen reported to increase with advancing age. Histologic stud-ies in AF have demonstrated that the percentage of fibrosis anddegenerative changes in the atrial muscle increase significant-ly with age.Hypothesis: This study was undertaken to assess the influ-ence of advancing age on atrial endocardial electrograms re-corded during sinus rhythm in patients with paroxysmal atrialfibrillation (PAF), which had not been assessed previously.Methods: Right atrial endocardial catheter mapping duringsinus rhythm was performed in 111 patients with PAF to eval-uate the influence of advancing age on atrial endocardial elec-trograms. The bipolar electrograms were recorded at 12 sitesin the right atrium, and an abnormal atrial electrogram was de-fined as lasting ≥100 ms, and/or showing eight or more frag-mented deflections.Results: In all, 1,332 right atrial endocardial electrogramswere assessed and measured quantitatively. The number of ab-normal atrial electrograms in patients with PAF showed a sig-nificantly positive correlation with age (r = 0.34; p 60 years had a significantly greater mean num-ber of abnormal electrograms (2.58 ± 2.05) than those aged

Acetazolamide, a carbonic anhydrase inhibitor, is used empirically in neuromuscular diseases with episodic ataxia, weakness, and myotonia, although not all of the mechanisms responsible for its therapeutic effects are understood. To elucidate whether acetazolamide acts directly on the human skeletal muscle voltage-gated chloride channel (ClC-1), which is associated with myotonia, we evaluated the effects of acetazolamide on ClC-1 expressed in cultured mammalian cells, using whole-cell recording. Acetazolamide significantly shifted the voltage dependency of the open probability (P(o)) toward negative potentials in a dose-dependent manner, resulting in an increase of chloride conductance at voltages near the resting membrane potential. This effect was attenuated when using a pipette solution containing 30 mmol/L Hepes. These results suggest that acetazolamide can influence the voltage-dependent opening gate of ClC-1 through a mechanism related to intracellular acidification by inhibiting carbonic anhydrase, and that the therapeutic effects of acetazolamide in neuromuscular diseases may be mediated by activation of ClC-1.

The influence of advancing age on atrial endocardial electrograms recorded during sinus rhythm in humans who do not have atrial fibrillation has not been assessed thus far. The prevalence of atrial arrhythmias has been reported to increase with advancing age. Right atrial endocardial catheter mapping during sinus rhythm was performed in 106 patients who had normal sinus node function and no paroxysmal atrial fibrillation to evaluate the influence of advancing age on atrial endocardial electrograms. The bipolar electrograms were recorded at 12 sites in the right atrium, and an abnormal atrial electrographic result was defined as lastingor =100 ms and/or showingor =8 fragmented deflections. A total of 1,272 right atrial endocardial electrograms was assessed and quantitatively measured. The mean number of abnormal atrial electrograms per patient (0.61 vs 0.14, p0.02) and the incidence of abnormal atrial electrograms (11% vs 30%, p0.02) were significantly greater in patients who were60 years of age than in younger patients. The longest duration (r = 0.24, p0.02) and the maximal number of fragmented deflections (r = 0.28, p0.005) of atrial electrograms among the 12 right atrial sites showed a slight positive correlation with age. The process of aging modifies the electrophysiologic properties of the atrial muscle. There is a progressive increment in the extension of altered atrial muscle with advancing age in humans who do not have atrial fibrillation. Patients who are60 years of age have significantly greater abnormalities on atrial endocardial electrograms than do younger patients.

This study characterized the functional effects of a novel gastroprokinetic agent, N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1, 3-thiazole-4-carboxyamide monohydrochloride trihydrate (Z338), on the muscarinic M1, M2, and M3 receptors expressed in Xenopus oocytes using the two-electrode voltage clamp method. Z-338 did not produce by itself any currents in oocytes expressing muscarinic M1, M3 receptors or muscarinic M2 receptors/G protein-gated inward rectifying K+ channels (Kir3.1 channels). In oocytes expressing muscarinic M1 receptors, Z-338 inhibited the acetylcholine-induced Ca2+ -activated Cl- current with an IC50 of 1.8 microM. In oocytes expressing muscarinic M2 receptors/Kir3.1 channels, Z-338 inhibited the acetylcholine-induced K+ currents with an IC50 of 10.1 microM, whereas in oocytes expressing muscarinic M3 receptors, Z-338 did not inhibit the acetylcholine-induced Ca2+ -activated Cl- current in a concentration-dependent manner. These results indicate that Z-338 is a potent antagonist not for muscarinic M3 receptor but for both muscarinic M1 and M2 receptors. Thus, Z-338 is a gastrokinetic agent with a unique profile.

We studied the functions of βγ-subunits of Gi/oprotein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl−currents in oocytes expressing β2-adrenoceptor and the protein kinase A-dependent Cl−channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [d-Ala2, d-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing β2-adrenoceptor-CFTR and 5-HT1Areceptor (5-HT1AR), δ-opioid receptor, or GABABreceptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin α (Gtα). The 5-HT-induced enhancement was further augmented by coexpression of the Gβγ-activated form of adenylate cyclase (AC) type II but not AC type III. Thus βγ-subunits of Gi/oprotein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or δ-opioid receptor alone. They elicited Ca2+-activated Cl−currents in oocytes coexpressing these receptors with the Gβγ-activated form of phospholipase C (PLC)-β2 but not with PLC-β1. These currents were inhibited by pretreatment with PTX and coexpression of Gtα, suggesting that βγ-subunits of Gi/oprotein activate PLC-β2 and then cause intracellular Ca2+mobilization. Our results indicate that βγ-subunits of Gi/oprotein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+mobilization.

Interaction of function and localization of receptors, especially the 5-hydroxytryptamine4 (5-HT4) receptor was examined in relation to the motility of gastrointestinal tract. The responses mediated by stimulation of 5-HT4 receptor appear to differ with different localization of receptor. The 5-HT4 receptor-mediated acceleration of acetylcholine (ACh) release in the preparations isolated from gastric antrum and corpus, but not fundus, of guinea pig corresponded to the presence of 5-HT4 receptor in the myenteric plexus. The 5-HT4 receptor-mediated response was predominant in the 5-HT-induced acceleration of motility associated with ACh release in the intestine of the whole body of dogs, and the 5-HT4 receptor was localized on the myenteric plexus of dog intestine. Local administration of GABA into intestinal marginal artery reduced the motility associated with ACh release in the intestine of whole body of dogs, and thus response via the inhibitory GABAB receptor was more predominant than that via excitatory GABAA receptor. The GABAB receptor is the first G protein-coupled receptor discovered to form heterodimers, consisted of GABAB1 and GABAB2. There are isoforms of GABAB1, from GABAB1(a) to GABAB1(g), and therefore the function and localization of the GABAB receptor may vary with the different isoforms of GABAB1.

The function of 5-hydroxytryptamine (5-HT) receptors, especially the 5-HT 4 receptor, in the urinary bladder were examined in preparations isolated from the guinea pig by in vitro receptor autoradiography and determinations of mechanical activity and acetylcholine (ACh) release. Specific [ 1 2 5 I]SB207710 binding sites were detected evenly throughout the urinary bladder. 5-HT (3 x 10 - 8 - 10 - 4 M) caused contractions of strips of the urinary bladder, in a concentration dependent manner. Ketanserin antagonized the 5-HT-induced contractions, while granisetron and SB204070 antagonized the contractions induced by high concentrations of 5-HT. Atropine inhibited the contractions induced by high concentrations of 5-HT. Ketanserin prevented the 5-HT-induced contractions in the presence of atropine, but granisetron and SB204070 did not affect the contractions under such a condition. 5-HT enhanced the electrically-stimulated (5 Hz, 0.5 ms) outflow of [ 3 H]acetylcholine from strips preloaded with [ 3 H]choline, and the enhancement was antagonized by granisetron and SB204070. Thus, the contractile response to 5-HT was mediated by activations of 5-HT 2 , 5-HT 3 and 5-HT 4 receptors. The 5-HT 2 receptor may be a property of high affinity to 5-HT and located on the smooth muscle cells. The 5-HT 4 as well as 5-HT 3 receptor may be a property of low affinity to 5-HT and located on the cholinergic neurons.

In native Xenopus oocytes, injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (30 mM, 5 nl) did not induce Cl- current in 11 out of 22 oocytes. Injection of increased concentration of GTPgammaS (100 mM, 5 nl) into the oocytes induced Cl- currents in 16 out of 17 oocytes; however, the size of the induced currents was extremely varied. In oocytes overexpressing Gq alpha, GTPgammaS (30 mM, 5 nl) faithfully evoked Ca2+-activated Cl- currents. These results indicate that heterogeneous expression of Gq alpha in Xenopus oocytes provides a useful system for studying the functional roles of Gq alpha in regulating cellular events.

The multiple 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes are distinguished. In this article, we described mainly the 5-HT4 receptor of four subtypes of functional 5-HT receptors, 5-HT1, 5-HT2, 5-HT3, and 5-HT4, recognized in the gastrointestinal tract. In-vivo microdialysis experiments determined that activation of the 5-HT4 receptor stimulated intestinal motor activity associated with a local increase in acetylcholine (ACh) release from the intestinal cholinergic neurons in the whole body of dogs. The 5-HT4 receptor-mediated response of ACh release in the antral, corporal, and fundic strips isolated from guinea pig stomach corresponds to the presence of 5-HT4 receptor in the myenteric plexus. In-vitro receptor autoradiograms of the stomach and colon indicate that the distribution of 5-HT4 receptors in human tissues is similar to that in the guinea pig, although density of 5-HT4 receptors in the myenteric plexus of human tissues is lower than that in guinea pig tissues. The 5-HT4 receptors located in the myenteric plexus may participate in gastrointestinal motility, and thus the 5-HT4 agonists and antagonists may be available for treatment of dysfunction of gastrointestinal motility.

We investigated the inactivation process of macroscopic cardiac L-type Ca2+channel currents using the whole cell patch-clamp technique with Na+as the current carrier. The inactivation process of the inward currents carried by Na+through the channel consisted of two components >0 mV. The time constant of the faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased with depolarization, but the time constant of the slower inactivating component (489 ± 21 ms at 0 mV) was not significantly influenced by the membrane potential. The inactivation process in the presence of isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents and attenuated the effects of isoproterenol. In the presence of cAMP (500 μM), the inactivation process consisted of a single slow component. We propose that the faster inactivating component represents a kinetic of the dephosphorylated or partially phosphorylated channel, and phosphorylation converts the kinetics into one with a different voltage dependency.

The effects of propofol, 2,6-diisopropylphenol, an intravenous general anesthetic, on signal transduction mediated by the rat M1 muscarinic acetylcholine (ACh) receptor (M1 receptor) were examined in electrophysiological studies by analyzing receptor-stimulated, Ca2+-activated Cl--current responses in the Xenopus oocyte expression system. In oocytes expressing the M1 receptor, ACh induced the Ca2+-activated C1- current, in a dose-dependent manner (EC50= 114 nM). Propofol (5-50 microM) reversibly and dose-dependently inhibited induction of the Ca2+-activated Cl- current by ACh (100 nM) (IC50=5.6 microM). To determine a possible site affected by propofol in this signal transduction, we tested the effects of this anesthetic (10 microM) on the activation of current by injection of CaCl2 and aluminum fluoride (AlF4-). Propofol did not affect activation of the current by the intracellular injected Ca2+, or activation of the current by the intracellular injected AlF4-. These results indicate that propofol does not affect G protein, the inositol phosphate turnover, release of Ca2+ from Ca2+ store or the Ca2+-activated Cl- channel. Propofol apparently inhibits the M1 receptor-mediated signal transduction at the receptor site and/or the site of interaction between the receptor and associated G protein.

We have identified two novel splice variants of the metabotropic γ-aminobutyric acid receptor (GABABR1), designated GABABR1c and GABABR1d, when screening a rat cerebellum cDNA library. GABABR1c has an amino acid sequence identical to GABABR1b, a member of GABABR1 isoforms, and an additional 93-bp insertion that generates an additional 31-amino-acid sequence in the fifth transmembrane region of GABABR1b. Thus, GABABR1c may have a structural variation in the second extracellular loop and fifth transmembrane region. GABABR1d also has an amino acid sequence identical to GABABR1b and an additional insertion of 566 bp that generates a divergent amino acid sequence in the carboxyl-terminal end. Reverse-transcription polymerase chain reaction analysis showed that in various rat tissues GABABR1c mRNA was ubiquitously expressed and GABABR1d mRNA in forebrain, cerebellum, eye, kidney, and urinary bladder. GABABR1 isoforms may function not only in the central nervous system but also in various peripheral tissues.

In order to quantify underlying atrial conduction properties in patients with atrial fibrillation (AF) using clinical electrophysiology techniques, atrial conduction curves relating intra-atrial conduction times to extrastimulus prematurities during programmed atrial stimulation were drawn. Based on the presence or absence of AF episodes, 95 subjects were divided into 2 groups: control (n=42); and AF (n=53). During programmed stimulation introduced from the right atrial appendage, an atrial conduction curve was drawn for each patient. For most of the control subjects, when the extrastimulus prematurity was increased by 10-ms steps, the intra-atrial conduction times also increased gradually; the maximum stepwise prolongation in intraatrial conduction time was 11.O±3.4msec. For patients with AF, a 10-msec increase in extrastimulus prematurity often produced a sudden marked prolongation in the intra-atrial conduction time; the maximum stepwise prolongation of intra-atrial conduction time was 21.4±5.9msec. In contrast to the gradual atrial conduction curves recorded in control subjects, the sudden prolongation of intra-atrial conduction time was remarkable on the curves obtained in patients with AF. Statistical significance was clearly established (p

The function of the cloned rat calcitonin receptors, C1a and C1b, was studied in Xenopus oocytes using the two-electrode voltage clamp method. In oocytes expressing the C1a receptors and the cystic fibrosis transmembrane conductance regulator (CFTR), C1a/CFTR, application (30 sec) of either salmon calcitonin (sCT) or human calcitonin (hCT) activated currents through CFTR. In C1b/CFTR, sCT activated the currents, whereas hCT failed to elicit a response. The sCT induced currents in C1a/CFTR were similar in size to those in C1b/CFTR. Both the activation and the deactivation of sCT-induced currents were slower in C1a/CFTR. In oocytes expressing C1a or C1b alone, application of relatively high concentrations of sCT induced small oscillatory inward currents. Application of hCT induced small inward currents in C1a alone, but failed to activate currents in C1b alone. These results demonstrate new insights into the signal transduction of calcitonin receptors.

Possible mechanisms for run-down in the Ca2+ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca2+ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l-5 microM), phosphate (l20 mM) and leupeptin (l0 microM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5-2 microM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NPo (the product of the number of channels N and the open probability Po) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca2+ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein.

Electrophysiologic findings suggesting the coexistence of dual atrioventricular (AV) nodal pathways and accessory AV connections have been previously described. Anterograde conduction through the accessory pathway (AP) may preclude the diagnosis of AV nodal dual pathway physiology during atrial stimulation. This study reports on a patient with manifest Wolff-Parkinson-White syndrome with clinically documented paroxysmal atrial fibrillation, in whom dual AV nodal pathways were unmasked after successful radiofrequency ablation of two accessory AV connections. In spite of detailed investigation, fast and slow AV nodal pathways were not detected in the first electrophysiologic study 8 years before ablation, nor were they detected during preablation study because of exclusive anomalous anterograde conduction. The anterograde AP effective refractory period was shorter than that of the fast and slow AV nodal pathways, and was limited by atrial refractoriness at 190 ms. The present findings strongly suggest the necessity for a careful postablation electrophysiologic study before and after isoproterenol administration with specific evaluation of AV nodal conduction. This is the first documented report on the finding of AV nodal dual pathway physiology and reentry after successful radiofrequency ablation of two APs. This finding may be of great therapeutic significance in light of the feasibility of slow pathway ablation also during a single session, had AV nodal reentry been induced in a sustained manner after ablation of the AP to prevent late recurrence of tachycardia.

The effects of aprindine on atrial vulnerability were studied in 11 patients; 9 with paroxysmal atrial fibrillation (PAF), and 2 with Wolff-Parkinson-White syndrome, aged 19 to 69 (55.9 +/- 16.5; mean +/- SD). Before and 10 min after the intravenous injection of aprindine (1.5 mg/kg), programmed extrastimulation was performed from the right atrial appendage. Atrial vulnerability was assessed by evaluating the repetitive atrial firing zone (RAFZ), conduction delay zone (CDZ), maximum conduction delay (Max. CD) and fragmented atrial activity zone (FAAZ). After the injection, the duration of the P wave and QTc interval was significantly prolonged without any change in blood pressure or heart rate. RAF was observed in 8 patients under control conditions. However, after the injection of aprindine, the RAFZ completely disappeared in 2 patients, was narrowed in 4, and became wider in 1. AF was induced in the remaining patient. The zone significantly reduced (p < 0.01) without any change in CDZ or Max. CD. While FAA was observed in 5 patients under control conditions, it completely disappeared in 2 patients, was narrowed in 1, and did not change in the remaining 7 after the injection of aprindine. In patients whose RAFZ narrowed after administration of aprindine, the wavelength, as determined from the atrial effective refractory period and conduction velocity, was augmented. These results indicate that aprindine suppresses atrial vulnerability with an augmentation of the wavelength. However aprindine exaggerated atrial vulnerability in some patients, such that atrial fibrillation was induced.

Catheter ablation was attempted in 2 patients with atrioventricular node reentry tachycardia which showed fast, intermediate and slow anterograde atrioventricular node pathways. Radiofrequency currents were applied within a restricted area of the tricuspid annulus between the His bundle and the ostium of the coronary sinus where presumed slow pathway potentials were identified. Eliminaition of both the intermediate and the slow pathways, with preservation of anterograde and retrograde fast pathway conduction, was achieved in both patients.

The present study investigated the possible involvement of a Na(+)-H+ antiporter in the regulation of L-type Ca2+ channels by angiotensin II (Ang II) in isolated rabbit ventricular cardiac myocytes by using both cell-attached and whole-cell patch-clamp current recording techniques. In cell-attached patch-clamp current recordings, an increase in the open-state probability of the Ca2+ channel (144.8 +/- 9.8% [mean +/- SEM], n = 11) was seen after exposure of the cells to Ang II (100 nmol/L). This effect was inhibited by pretreatment with losartan (10 mumol/L), a synthetic antagonist of the AT1 receptor. 5(N,N-Dimethyl)amiloride (100 mumol/L), an amiloride analogue, as well as Na(+)-deficient bath solution abolished Ang II-induced stimulation of the Ca2+ channel activities. In whole-cell patch-clamp current recordings, Ang II also increased the L-type Ca2+ current when a pipette solution of pH 7.1 containing 5 mmol/L HEPES (139 +/- 5%, n = 4) was used but did not significantly increase the current when a pipette solution of pH 7.5 containing 5 mmol/L HEPES or a pipette solution of pH 7.1 containing 30 mmol/L HEPES was used. These results suggest that Ang II-induced stimulation of the Ca2+ channels is mediated by a Na(+)-H+ antiporter and therefore provide a novel insight into signal transduction of Ang II receptor stimulation in cardiac myocytes.

The purpose of this study was to evaluate prospectively the relationship between supernormal atrial conduction (SNC) and the atrial vulnerability to fibrillation in patients with sick sinus syndrome (SSS) and paroxysmal atrial fibrillation (PAF). Programmed atrial stimulation was performed in 32 age-matched control patients (group I), 26 with SSS but without tachyarrhythmias (group II), and 24 with both SSS and PAF (group III) to assess some determinants of atrial vulnerability, SNC, and atrial fibrillation inducibility. Supernormal atrial conduction was observed in 20 (63%) patients of group I, 12 (46%) patients of group II, and 5 (21%) patients of group III (group I vs group III; p < 0.002). The SNC zone was 46 ± 44 msec in group I, 36 ± 42 msec in group II, and 12 ± 24 msec in group III. (group I vs group III; p < 0.001). The absence of SNC showed a specificity of 89% and a positive predictive accuracy of 79% in predicting inducibility of atrial fibrillation. The sensitivity was 33% and the negative predictive accuracy was 52%. The SNC zone showed a significant inverse correlation with P wave duration (r = −0.32; p < 0.003), intraatrial conduction time (r = −0.28; p < 0.02), and maximum conduction delay (r = −0.23; p < 0.05). The maximum decrease in conduction time during supernormal conduction showed a significant inverse correlation with P wave duration (r = −0.27; p < 0.02), intraatrial conduction time (r = −0.26; p < 0.02), and with the maximum conduction delay (r = −0.27; p < 0.02). We conclude that the greater the atrial vulnerability and the greater the atrial conduction defects, the lower the occurrence of supernormal atrial conduction in the SSS. The absence of supernormal atrial conduction associated with atrial conduction defects may play an important role in the development of atrial fibrillation in patients with sick sinus syndrome and paroxysmal atrial fibrillation.

To investigate the effects of verapamil on indicators of atrial vulnerability, we examined 30 patients with paroxysmal supraventricular tachycardia who received intravenous verapamil during an electrophysiologic study. Single atrial extrastimuli were given before and after intravenous administration of verapamil to induce repetitive atrial firing (RAF) or atrial fibrillation, and to examine the maximum A2/A1, which was defined as the maximum ratio of the duration of the atrial electrogram resulting from premature stimulation (A2) to that resulting from the basic drive beat (A1). The maximum A2/A1 increased from 145 +/- 20% to 154 +/- 25% (p0.02) after verapamil administration. The maximum A2/A1 in patients in whom neither RAF nor atrial fibrillation were induced both before and after verapamil were smaller than those in patients in whom RAF was induced only after verapamil (before; 138 +/- 20% vs 165 +/- 15%, p0.02. after; 144 +/- 22% vs 172 +/- 17%, p0.05). RAF or atrial fibrillation was induced only after verapamil in 6 patients, who showed a maximum A2/A1 before verapamil of 150% or more. These data suggest that verapamil may induce repetitive atrial firing and possibly atrial fibrillation in some predisposed patients, especially in those that have a greater maximum A2/A1, which may be an indicator of local intraatrial conduction delay before drug infusion.

The electrophysiologic properties of left-sided accessory pathways (APs) were examined by cardiac stimulation in 55 patients with Wolff-Parkinson-White syndrome. Atrioventricular and ventriculoatrial conduction times were assessed at the coronary sinus level nearest to the AP and then plotted graphically as a function of coupling interval (for atrial and ventricular refractory period determinations). Of 29 patients with anterograde conduction over the AP, 10 (34%) exhibited decremental conduction. However, only two (7%) had a maximal decrement equal to or more than 30 msec. In the other eight (27%) patients the maximal decrement ranged from 10 to 20 msec. The longest coupling interval at which anterograde decremental conduction was demonstrated ranged from 260 to 440 msec (346 +/- 52 msec). The shortest coupling interval ranged from 240 to 320 msec (265 +/- 24 msec). The anterograde decremental conduction zone was 91 +/- 55 msec. Of 51 patients with retrograde conduction over the AP, 23 (45%) exhibited decremental conduction. However, only eight (15%) had a maximal decrement equal to or greater than 30 msec. In the other 15 (29%) patients the maximal decrement ranged from 10 to 25 msec. The longest coupling interval was 338 +/- 70 msec. The shortest coupling interval was 275 +/- 42 msec. The retrograde decremental conduction zone was 72 +/- 47 msec. There was a significant inverse correlation between the AP effective refractory period and the maximal decrement (r = -0.42; p < 0.05). The comparison of maximal ventriculoatrial conduction time with the maximal decrement revealed a positive correlation (r = 0.63; p < 0.01). These data reveal that minimal decremental conduction over left-sided APs is not an uncommon finding and stress that care should be taken in evaluation of conduction over these connections.

BACKGROUND: Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026; FINDINGS: We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy. CONCLUSIONS: Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening., PloS one, 4(10), e7655; 2009