Metabolic syndrome-related SNPs in HLA and TNF7L2 may be risk factors for generalized pustular psoriasis in Chinese Han population.

Background: Generalized pustular psoriasis (GPP) is a rare and severe type of psoriasis. Previous studies have reported that metabolic syndrome and its components have been associated with psoriasis. Objective: To investigate the association of metabolic syndrome-related single-nucleotide polymorphisms (SNPs) and GPP in Chinese Han population. Materials and Methods: One hundred and thirty-six (136) GPP patients and 965 healthy controls were recruited in the study. Approximately, 4 ml peripheral venous blood was collected from each participant. After collection, second-generation sequencing was used to detect genetic polymorphism of 15 SNPs. The plink 1.07 software package was used for statistical analysis. Results: Rs805303 (p = 0.01, OR = 0.70) and rs3177928 (p = 3.18E-07, OR = 2.66) in HLA were significantly different between the two groups. Moreover, rs4506565 (p = 1.41E-03, OR = 2.72) and rs7901695 (p = 9.39E-04, OR = 2.82) in TCF7L2 were significantly associated with GPP in patients without a previous history of PsV. Genotype analysis of rs4506565 and rs7901695 showed that under the recessive model, genotype frequencies of rs4506565 (p = 0.00, OR = 18.52) and rs7901695 (p = 0.00, OR = 18.44) were significantly different between GPP patients and healthy controls. Conclusion: Rs805303 and rs3177928 in HLA may increase the risk of GPP in the Chinese Han population. TCF7L2 may be a risk factor for GPP in patients without a previous history of PsV.

Serine-385 phosphorylation of inwardly rectifying K+ channel subunit (Kir6.2) by AMP-dependent protein kinase plays a key role in rosiglitazone-induced closure of the K(ATP) channel and insulin secretion in rats.

AIMS/HYPOTHESIS: Rosiglitazone, an insulin sensitiser, not only improves insulin sensitivity but also enhances insulin secretory capacity by ameliorating gluco- and lipotoxicity in beta cells. Rosiglitazone can stimulate insulin secretion at basal and high glucose levels via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. We hypothesised that regulation of phosphorylation of the ATP-sensitive potassium (K(ATP)) channel might serve as a key step in the regulation of insulin secretion. METHODS: Insulin secretory responses were studied in an isolated pancreas perfusion system, cultured rat islets and MIN6 and RINm5F beta cells. Signal transduction pathways downstream of PI3K were explored to link rosiglitazone to K(ATP) channel conductance with patch clamp techniques and insulin secretion measured by ELISA. RESULTS: Rosiglitazone stimulated AMP-activated protein kinase (AMPK) activity and induced inhibition of the K(ATP) channel conductance in islet beta cells; both effects were blocked by the PI3K inhibitor LY294002. Following stimulation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator, both AICAR-stimulated insulin secretion and inhibition of K(ATP) channel conductance were unaffected by LY294002, indicating that AMPK activation occurs at a site downstream of PI3K activity. The serine residue at amino acid position 385 of Kir6.2 was found to be the substrate phosphorylation site of AMPK when activated by rosiglitazone or AICAR. CONCLUSIONS/INTERPRETATION: Our data indicate that PI3K-dependent activation of AMPK is required for rosiglitazone-stimulated insulin secretion in pancreatic beta cells. Phosphorylation of the Ser(385) residue of the Kir6.2 subunit of the K(ATP) channel by AMPK may play a role in insulin secretion.

Electromechanical characterization of cinnamophilin, a natural thromboxane A2 receptor antagonist with anti-arrhythmic activity, in guinea-pig heart.

BACKGROUND AND PURPOSE: Cinnamophilin, a thromboxane A(2) receptor antagonist, has been identified as a prominent anti-arrhythmic agent in rat heart. This study aimed to determine its electromechanical and anti-arrhythmic effects in guinea-pig hearts. EXPERIMENTAL APPROACH: Microelectrodes were used to study action potentials in ventricular papillary muscles. Fluo-3 fluorimetric ratio and whole-cell voltage-clamp techniques were used to record calcium transients and membrane currents in single ventricular myocytes, respectively. Intracardiac electrocardiograms were obtained and the anti-arrhythmic efficacy was determined from isolated perfused hearts. KEY RESULTS: In papillary muscles, cinnamophilin decreased the maximal rate of upstroke (V(max)) and duration of action potential, and reduced the contractile force. In single ventricular myocytes, cinnamophilin reduced Ca(2+) transient amplitude. Cinnamophilin decreased the L-type Ca(2+) current (I(Ca,L))(IC(50)=7.5 microM) with use-dependency, induced a negative shift of the voltage-dependent inactivation and retarded recovery from inactivation. Cinnamophilin also decreased the Na(+) current (I(Na)) (IC(50)=2.7 microM) and to a lesser extent, the delayed outward (I(K)), inward rectifier (I(K1)), and ATP-sensitive (I(K,ATP)) K(+) currents. In isolated perfused hearts, cinnamophilin prolonged the AV nodal conduction interval and Wenckebach cycle length and the refractory periods of the AV node, His-Purkinje system and ventricle, while shortening the ventricular repolarization time. Additionally, cinnamophilin reduced the occurrence of reperfusion-induced ventricular fibrillation. CONCLUSIONS AND IMPLICATIONS: These results suggest that the promising anti-arrhythmic effect and the changes in the electromechanical function induced by cinnamophilin in guinea-pig heart can be chiefly accounted for by inhibition of I(Ca,L) and I(Na).

Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death.

Overproduction of reactive oxygen species is one of the major causes of cell death in ischemic-reperfusion (I/R) injury. In I/R animal models, electron microscopy (EM) has shown mixed apoptotic and necrotic characteristics in the same cardiomyocyte. The present study shows that H(2)O(2) activates both apoptotic and necrotic machineries in the same myocyte and that the ultrastructure seen using EM is very similar to that in I/R animal studies. The apoptotic component is caused by the activation of clotrimazole-sensitive, NAD(+)/ADP ribose/poly(ADP-ribose) polymerase (PARP)-dependent transient receptor potential M2 (TRPM2) channels, which induces mitochondrial [Na(+)](m) (and [Ca(2+)](m)) overload, resulting in mitochondrial membrane disruption, cytochrome c release, and caspase 3-dependent chromatin condensation/fragmentation. The necrotic component is caspase 3-independent and is caused by PARP-induced [ATP](i)/NAD(+) depletion, resulting in membrane permeabilization. Inhibition of either TRPM2 or PARP activity only partially inhibits cell death, while inhibition of both completely prevents the ultrastructural changes and myocyte death.

1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces the differentiation of normal human keratinocytes, in part by increasing their basal intracellular calcium levels (Cai) over a period of hours. Agonists such as ATP acting through membrane receptors cause an immediate but transient increase in Cai accompanied by an increase in inositol trisphosphate (IP3). Treatment of keratinocytes for 24 h with 1 nM 1,25(OH)2D3 resulted in a two- to four-fold potentiation of the Cai response of these cells to ATP. This potentiation was inhibitable with cycloheximide, unaccompanied by a change in total intracellular calcium pools, but associated with an increase in basal IP3 levels and ATP-stimulated IP3 production. Treatment with 1,25(OH)2D3 raised the protein and mRNA levels of phospholipase C isoenzymes, particularly phospholipase C-beta 1 in a dose-dependent manner. These studies indicate that 1,25(OH)2D3 modulates the keratinocyte signal transduction pathway by induction of phospholipase isoenzymes, a previously undescribed action for this hormone.

Possible mechanism(s) of arachidonic acid-induced intracellular acidosis in rat cardiac myocytes.

Arachidonic acid (AA) and other nonesterified fatty acids (FAs) have been shown to exert harmful effects during cardiac ischemia. By continuously measuring intracellular pH (pH(i)) changes in neonatal and adult cardiac myocytes, we have found, for the first time, that 10 micromol/L AA induces a substantial intracellular acidosis (0.3 to 0.4 pH units). We have ruled out the possibilities that the AA-induced acidosis is caused by (1) inhibition or stimulation of the pH(i) regulators, (2) protein kinase C activation or the generation of AA metabolites or free radicals, or (3) activation of NADPH oxidase or an inward H(+) current. The AA-induced acidosis fits to a simple diffusion mechanism, as proposed by Kamp and Hamilton (flip-flop model) for artificial phospholipid bilayers. The important properties found in the cardiac myocyte are that (1) the initial rate of acid flux (J(H)) increases with the AA concentration (2 to 50 micromol/L), (2) FAs with a (-)COOH group (eg, AA, oleic acid, and linoleic acid) induce intracellular acidification, but FAs with a (-)COOCH(3) group (eg, AA methyl ester) have little effect on the pH(i), (3) tetradecylamine (FA amine) induces intracellular alkalosis, and, most importantly, (4) both the AA- and tetradecylamine-induced pH(i) changes can be reversed by 0.3% BSA. Because a low concentration of AA (10 micromol/L) can induce a substantial acidosis, the possible involvement of the FA-evoked acidosis in the negative inotropic effect during cardiac ischemia is discussed. The full text of this article is available at http://www. circresaha.org.

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling.

OBJECTIVES: We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca(2+)-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation. BACKGROUND: Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear. METHODS: A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription-polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. RESULTS: The mRNA of L-type calcium channel and of Ca(2+)-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36+/-0.26 vs. 0.90+/-0.88 for L-type calcium channel; 0.69+/-0.42 vs. 1.21+/-0.68 for Ca(2+)-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation. CONCLUSIONS: L-type calcium channel and the sarcoplasmic reticular Ca(2+)-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes.

BACKGROUND: The major objective of the present study was to examine the cardioprotective effect of resveratrol, an antioxidant presents in red wines, in the rat after ischemia and ischemia-reperfusion (I-R). METHODS: The left main coronary artery was occluded for 30 or 5 min followed by a 30-min reperfusion in anesthetized rats. Animals were preinfused with and without resveratrol before occlusion and the severity of ischemia- and I-R-induced arrhythmias and mortality were compared. RESULTS: Resveratrol pretreatment had no effect on ischemia-induced arrhythmias nor on mortality. In contrast, a dramatic protective effects were observed against I-R-induced arrhythmias and mortality. Resveratrol pretreatment both reduced the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF). During the same period, resveratrol pretreatment also increased nitric oxide (NO) and decreased lactate dehydrogenase levels in the carotid blood. CONCLUSIONS: Resveratrol is a potent antiarrhythmic agent with cardioprotective properties in I-R rats. The cardioprotective effects of resveratrol in the I-R rats may be correlated with its antioxidant activity and upregulation of NO production.

Squamous carcinoma cell lines fail to respond to 1,25-Dihydroxyvitamin D despite normal levels of the vitamin D receptor.

Squamous carcinoma cells (SCC) fail to differentiate under conditions that are favorable for the growth and differentiation of normal human keratinocytes. Human keratinocytes differentiate from a highly proliferative basal cell to a terminally differentiated cornified cell in culture in the presence of physiological levels of extracellular calcium. 1,25-Dihydroxyvitamin D (1,25[OH]2D3) potentiates this process. Previous studies have shown that the differentiation process in keratinocytes is associated with increased expression of the genes for involucrin and transglutaminase, the products of which participate in cornified envelope formation. The mRNA for involucrin and transglutaminase was not detected in the SCC lines studied (viz. SCC4, 12B2, 12F2, A431, and HACAT) when they were grown in serum free medium. Addition of at least 2% fetal bovine serum for 48 h triggered the expression of these genes, which could then be maintained in the absence of serum. Serum was not required for induction of these genes in keratinocytes. In these cells, 1,25(OH)2D3 stimulated the expression of involucrin and transglutaminase in a concentration-dependent manner, while the SCC lines failed to respond to 1,25(OH)2D3 regardless of whether these cells had been pre-exposed to serum. An important factor that mediates 1,25(OH)2D3-stimulated gene expression is the vitamin D receptor, but vitamin D receptor mRNA levels in all the SCC lines examined were comparable to those in keratinocytes. Furthermore, the vitamin D receptor protein levels in SCC lines as assessed by ligand-binding analysis were comparable to those of keratinocytes. Thus, the mediators of 1,25(OH)2D3 action on gene expression other than the vitamin D receptor may be missing or defective in SCC lines, whereas the mediators of as yet undefined agents in serum may be better expressed in SCC lines than in keratinocytes. Our results indicate that, although SCC lines are capable of expressing the genes for the proteins involved in differentiation, the control of the expression of these genes by 1,25(OH)2D3 is abnormal in SCC despite the presence of a functional vitamin D receptor in concentrations equivalent to those in keratinocytes.

22-Oxa calcitriol is a less potent regulator of keratinocyte proliferation and differentiation due to decreased cellular uptake and enhanced catabolism.

22-oxa calcitriol (OCT) is a recently synthesized analog of calcitriol (1,25(OH)2D3) with potent biologic actions both in vivo and in vitro. Because it is considerably less hypercalcemic than 1,25(OH)2D3 when given in vivo, OCT is of potential use for the treatment of diseases, such as psoriasis, that respond to the antiproliferative, prodifferentiating actions of 1,25(OH)2D3. To determine the potential usefulness of OCT in hyperproliferative skin diseases, we compared the ability of OCT to that of 1,25(OH)2D3 with respect to regulation of keratinocyte proliferation and differentiation in vitro. These studies were performed in serum-free media to eliminate differences in potency secondary to differences in binding to the serum vitamin D-binding protein. We observed that OCT was considerably less effective than 1,25(OH)2D3 in inhibiting keratinocyte proliferation and stimulating differentiation. The decreased potency of OCT appeared to be due to decreased uptake and increased catabolism rather than decreased affinity for the vitamin D receptor. We conclude that under the conditions of our experiments OCT was less potent than 1,25(OH)2D3 because it failed to achieve comparable concentrations within the cell.

Beneficial effects of astringinin, a resveratrol analogue, on the ischemia and reperfusion damage in rat heart.

Oxidative stress plays an important role in the pathogenesis of myocardial ischemia and infarction. Antioxidants might then be beneficial in the prevention of these diseases. Astringinin (3,3',4',5-tetrahydroxystilbene), a resveratrol (3,4',5-trihydroxystilbene) analogue with considerably higher antioxidative activity and free radical scavenging capacity, was introduced to examine its cardioprotective effects in ischemia or ischemia-reperfusion (I/R) rats. In the present study, the left main coronary artery was occluded by the following procedures: (i) 30 min occlusion, (ii) 5 min occlusion followed by 30 min reperfusion, and (iii) 4 h occlusion. Animals were infused with and without astringinin before coronary artery occlusion. Mortality, and the severity of ischemia- and I/R-induced arrhythmias were compared. Pretreatment of astringinin dramatically reduced the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) during either ischemia or I/R period. Astringinin at 2.5 x 10(-5) and 2.5 x 10(-4) g/kg completely prevented the mortality of animals during ischemia or I/R. During the same period, astringinin pretreatment also increased nitric oxide (NO) and decreased lactate dehydrogenase (LDH) levels in the carotid blood. In animals subjected to 4 h coronary occlusion, the cardiac infarct size (expressed as a percentage of occluded zone) was reduced from 44.4 + or - 4.1% to 19.1 + or - 2.4% by astringinin (2.5 x 10(-4) g/kg). We conclude that, astringinin is a potent antiarrhythmic agent with cardioprotective activity in ischemic and ischemic-reperfused rat heart. The beneficial effects of astringinin in the ischemic and ischemic-reperfused hearts may be correlated with its antioxidant activity and upregulation of NO production.

Regulation of involucrin gene expression by calcium in normal human keratinocytes.

Calcium is essential for normal epidermal differentiation. Data from Northern and nuclear run-on analysis indicate that involucrin gene transcription is induced by 1.2 mM extracellular calcium. A 3.7 Kbp fragment of the involucrin gene, which contains 2.5 Kbp of upstream region, the transcription start site, and the first intron, was sub-cloned into the pGL3-basic luciferase reporter vector and transfected into pre-confluent normal human keratinocytes (NHK). The stimulated activities of this clone were above basal levels and was further enhanced eight-fold by 1.2 mM extracelluar calcium. The results from a series of truncation and internal deletion experiments revealed multiple calcium-independent enhancer elements between -2476 and -2131 bp of the transcription start site and a calcium-dependent element between -2131 and -2028 bp. This 103 bp fragment contains sequences of an AP-1 site (TGAGTCA), a SP-1 site (GGGCGG), and shares homology with two elements in the human keratin-1 promoter, within the regions identified as mediating the calcium responsiveness of that gene in keratinocytes. One or more of these putative elements may be involved in the calcium-dependent regulation of the involucrin gene transcription in NHK.

Acetylcholine hydrolysis during neuromuscular transmission in the synaptic cleft of skeletal muscle of mouse and chick.

Inhibition of acetylcholinesterase (AChE) by more than 80% by neostigmine or physostigmine resulted in a failure of tetanic contraction (100 Hz) in the isolated mouse nerve-diaphragm preparation. In the chick biventer cervicis muscle, however, the tetanic contraction was well maintained and even outlasted the period of nerve stimulation after inactivation of AChE. The concentration of (+)tubocurarine for 70% block of the indirect twitch response of the mouse diaphragm at 0.1 Hz was increased from 0.67 to 0.99 to 1.21-2.03 microM in the presence of neostigmine (0.15-1.5 microM) which inhibited AChE by 70% or more, while that to depress the tetanic contraction (50 Hz) was increased from 0.38 to 0.42 to 0.53-0.69 microM. In the chick muscle, physostigmine at 2.4 microM increased the concentration of (+)tubocurarine for 70% block of the twitch response from 1.68 to 4.14 microM, whereas that for block of the response to exogenous acetylcholine (ACh) was increased from 1.47 to 74.6 microM. On single stimulation, the relative peak concentrations of acetylcholine (ACh) at the postsynaptic receptor site of the mouse diaphragm and chick biventer cervicis were estimated to be increased by about 110 and 120% respectively, after complete inhibition of AChE. In the chick muscle, physostigmine increased the relative concentration of ACh by about 40-fold at the receptor site for exogenously applied ACh. It is concluded that the intrinsic ACh released from the nerve terminal is hydrolyzed by about 50% during the time of diffusion across the synaptic cleft whereas most of exogenous ACh is hydrolyzed before reaching the target.

A study on the interaction of crotapotin with crotoxin phospholipase A2, notexin and other presynaptic neurotoxins.

1 Crotapotin, the acidic subunit of crotoxin, greatly potentiated the presynaptic effect of isolated basic phospholipase A (PLA) of crotoxin in both mouse diaphragm and chick biventer cervicis muscles whereas the myotoxic effect was not affected significantly.2 In contrast to crotoxin PLA, the presynaptic effects of notexin and notechis-5, self-active single chain toxins, were antagonized by crotapotin while actions of beta-bungarotoxin were not affected.3 By assaying PLA activity, crotoxin PLA was found to be unstable in physiological salt solution, especially when in contact with muscle, due to massive non-specific binding to and destruction by the muscle.4 The decline of crotoxin PLA was greatly reduced by the presence of crotapotin but not by another acidic protein, volvatoxin A(2), or heparin.5 Notechis-5 was found to be stable even when in the presence of muscles.6 [(3)H]-acetylated crotoxin PLA, which retained about 40% of its original enzyme and presynaptic blocking activities, also bound rapidly to the mouse diaphragm on incubation and this binding was greatly hindered by the simultaneous addition of crotapotin.7 The prevention of binding of crotoxin PLA by crotapotin occurred mostly at those sites where the binding was easily dissociable on washing. No antagonism of binding occurred at the firmly binding site.8 The binding of [(3)H]-acetylated crotapotin was much less than that of crotoxin PLA, and interestingly, the binding was increased by the latter, suggesting that crotapotin may be first bound to the diaphragm together with crotoxin PLA.9 No specific binding at the endplate zone was found either for crotoxin PLA or for crotapotin.10 It is concluded that crotapotin potentiates the presynaptic effect of crotoxin PLA by curtailing its non-specific affinity with muscles, minimizing its dispersal and destruction en route to the nerve terminal, but not by acting as an affinity probe for the nerve terminal.

Comparative direct electrophysiological effects of propofol on the conduction system and ionic channels of rabbit hearts.

1. Propofol, an intravenous anaesthetic agent, can affect cardiac conduction but the ionic mechanisms have not been well defined. 2. This study measured the direct effects of propofol on the cardiac conduction system by using intracardiac recording/stimulation in Langendorff-perfused rabbit hearts. The underlying ionic mechanism was elucidated by using the whole-cell voltage clamp on rabbit isolated atrial and ventricular myocytes. 3. Propofol prolonged significantly the AV conduction (AH) interval at a clinically relevant concentration (3 microM). This AH interval prolongation was dose-dependent (3 to 100 microM). At higher concentrations, the AV nodal Wenckebach cycle length and its refractory period were also prolonged (10 to 100 microM). In addition, the conduction through the His-Purkinje system (HV interval) and the atrial tissue (SA interval), as well as the spontaneous cycle length, were lengthened dose-dependently (30 to 100 microM). 4. In isolated ventricular myocytes, Na current was decreased dose-dependently by propofol. In part this was due to a negative-shift of the steady-state voltage-dependent inactivation and a slowed rate of recovery from inactivation. The INa suppression by propofol was frequency-dependent. Propofol also blocked the ICa. The ED50 for peak current inhibition was 6.9 +/- 0.9 (n = 6) and 8.3 +/- 1.5 microM (n = 7) for INa and ICa, respectively. 5. The transient outward potassium current (Ito) of atrial myocytes was suppressed with an ED50 of 5.7 +/- 0.8 microM (n = 11), which was only partly caused by a left-shift of the steady-state inactivation. The inward rectifier K current (IK1) of the ventricular cells was reduced somewhat by propofol. 6. In summary, propofol can cause direct dromotropic and chronotropic effects on the cardiac conduction system, especially the atrioventricular node. These changes can be attributed, at least in part, to its direct dose-dependent suppression of the cardiac ICa, INa and Ito. Special concerns in the use of propofol anaesthesia for cardiac patients and the therapeutic antiarrythmic potential of propofol-like compounds are addressed.

A study on the membrane depolarization of skeletal muscles caused by a scorpion toxin, sea anemone toxin II and crotamine and the interaction between toxins.

Quinquestriatus toxin (QTX) isolated from the venom of a scorpion (Leiurus quinquestriatus) and sea anemone (Anemonia sulcata) toxin II enhanced the twitch response of the rat and mouse diaphragms and like crotamine (isolated from the venom of Crotalus durissus terrificus) caused spontaneous fasciculation of the muscle. Trains of action potentials in muscles at 70-250 Hz, which could not be antagonized by (+)-tubocurarine, were triggered by single stimulation or occurred spontaneously after treatment with these toxins. QTX and toxin II prolonged the rat muscle action potential 3 to 4 fold whereas crotamine prolonged the action potential by only 30%. The membrane potential was depolarized from about -82 mV to -55 mV by crotamine 2 micrograms ml-1, -41 mV by toxin II 5 micrograms ml-1 and to -50 mV by QTX 1 microgram ml-1. The concentrations to induce 50% maximal depolarization (K0.5) were 0.07, 0.15 and greater than 0.4 microgram ml-1, respectively, for QTX, crotamine and toxin II, whereas the rates of depolarization were in the order toxin II greater than or equal to crotamine greater than QTX. The depolarizing effects of crotamine and QTX, but not of toxin II, were saturable. The depolarizing effects of all three toxins were irreversible whereas the membrane potential could be restored by tetrodotoxin non-competitively. Simultaneous treatment with crotamine and QTX or crotamine and toxin II at concentrations below K0.5 caused only additive effects on depolarization. When the muscle was depolarized by pretreating with a saturating concentration of crotamine, the onset of depolarization by QTX was greatly retarded whereas that by toxin II was unaffected. Action potentials were further prolonged in both cases. 8 It is inferred that all three peptide toxins act at sites on the sodium channel and the binding sites for QTX and crotamine overlap to a considerable extent. On the other hand, the site for toxin II appears not to overlap with that of crotamine but may overlap with that of QTX.

Mechanical and electrophysiological studies on the positive inotropic effect of 2-phenyl-4-oxo-hydroquinoline in rat cardiac tissues.

1. The pharmacological and electrophysiological effect of 2-phenyl-4-oxo-hydroquinoline (YT-1), a new synthetic agent, were determined in rat isolated cardiac tissues and ventricular myocytes. 2. YT-1 was found to have a positive inotropic effect in both atria and ventricular muscles but did not cause significant increases in the spontaneously beating rate of right atria. 3. The positive inotropic effect of YT-1 was antagonized neither by beta-nor by alpha-adrenoceptor antagonists but was partially antagonized by a Ca2+ channel blocker (verapamil) and a K+ channel blocker (4-AP). 4. The action potential duration and amplitude of ventricular cells were progressively increased as the concentration of YT-1 was increased from 3 to 30 microM. 5. A voltage clamp study revealed that the prolongation of action potential duration by YT-1 was associated with a prominent inhibition of 4-AP-sensitive transient outward current (I(to)). At potentials negative to the reversal potential of K1-channels, the inward current through these channels was partially reduced by YT-1. At potentials positive to the reversal potential, the outward current through these channels was affected very little. 6. Although YT-1 blocked the amplitude of I(to), the voltage-dependence of the steady-state inactivation of I(to), was unaffected. 7. Apart from the inhibition of K+ currents, YT-1 also inhibited the sodium inward current. 8. The evidence suggests that YT-1 increases the slow inward Ca2+ current (ICa) significantly. 9. It is concluded that the positive inotropic effect of YT-1 is due predominantly to the increase of ICa and inhibition of I(to).

Electrophysiological basis for the antiarrhythmic action and positive inotropy of HA-7, a furoquinoline alkaloid derivative, in rat heart.

1. HA-7, a new synthetic derivative of furoquinoline alkaloid, increased the contractile force of right ventricular strips and effectively suppressed the ischaemia-reperfusion induced polymorphic ventricular tachyrhythmias in adult rat heart (EC50 = 2.8 microM). 2. In rat ventricular myocytes, HA-7 concentration-dependently prolonged the action potential duration (APD) and decreased the maximal rate of rise of the action potential upstroke (Vmax). The action potential amplitude and resting membrane potential were also reduced, but to a smaller extent. The prolongation of APD by HA-7 was prevented by pretreating the cells with 1 mM 4-AP. 3. Voltage clamp experiments revealed that HA-7 decreased the maximal current amplitude of I(Na) (IC50 = 4.1 microM) and caused a negative shift of its steady-state inactivation curve and slowed its rate of recovery from inactivation. The use-dependent inhibition of I(Na) by HA-7 was enhanced at a higher stimulation rate. The L-type Ca2+ current (I(Ca)) was also reduced, but to a lesser degree (IC50 = 5.3 microM, maximal inhibition = 31.8%). 4. This agent also influenced the time- and voltage-dependent K currents. The prolongation of APD was associated with an inhibition of a 4-AP sensitive transient outward K current (I(to)) (IC50 = 2.9 microM) and a slowly inactivating, steady-state outward current (I(SS)) (IC50 = 2.5 microM). The inhibition of I(to) by HA-7 was associated with an acceleration of its time constant of inactivation. HA-7 suppressed I(to) in a time-dependent manner and caused a significant negative shift of the voltage-dependent steady-state inactivation curve but did not affect its rate of recovery from inactivation. 5. At higher concentrations, the inward rectifier K+ current (I(KI)) was also inhibited but to a less extent. Its slope conductance after 3, 10 and 30 microM HA-7 was decreased by 24+/-4%, 41+/-5% and 54+/-8%. respectively. 6. We conclude that HA-7 predominantly blocks I(to) and Na+ channels and that it also weakly blocks Ca2+ and I(KI) channels. These changes alter the electrophysiological properties of the heart and terminate the ischaemia reperfusion induced ventricular arrhythmia. The significant I(to) inhibition and minimal I(Ca) suppression may afford an opportunity to develop an effective antiarrhythmic agent linked with positive inotropy.

Pharmacological activity of (-)-discretamine, a novel vascular alpha-adrenoceptor and 5-hydroxytryptamine receptor antagonist, isolated from Fissistigma glaucescens.

1. The pharmacological activity of (-)-discretamine, isolated from Fissistigma glaucescens, was determined in rat isolated thoracic aorta, cardiac tissues and ventricular myocytes and guinea-pig isolated trachea. 2. (-)-Discretamine was found to be an alpha 1-adrenoceptor blocking agent in rat thoracic aorta as revealed by its competitive antagonism of noradrenaline (pA2 = 7.20 +/- 0.10)- or phenylephrine (pA2 = 7.60 +/- 0.09)-induced vasoconstriction. It was as potent as phentolamine (pA2 = 7.51 +/- 0.10), but was more potent than yohimbine (pA2 = 6.18 +/- 0.06). Removal of endothelium significantly increased the antagonistic potency of (-)-discretamine on noradrenaline (pA2 = 7.52 +/- 0.09)- or phenylephrine (pA2 = 7.90 +/- 0.09)-induced vasoconstriction. 3. (-)-Discretamine was also an alpha 2-adrenoceptor blocking agent (pA2 = 6.30 +/- 0.15) and a 5-hydroxytryptamine antagonist (pA2 = 6.87 +/- 0.06), both in rat aorta denuded of endothelium. 4. (-)-Discretamine protected alpha-adrenoceptors from alkylation by the irreversible blocking agent, phenoxybenzamine. 5. [3H]-inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta was suppressed by (-)-discretamine (10 and 30 microM) and prazosin (3 microM). 6. A high concentration of (-)-discretamine (30 microM) did not affect the contraction induced by the thromboxane receptor agonist U-46619, prostaglandin F2 alpha (PGF2 alpha), angiotensin II, high K+ or endothelin in rat aorta denuded of endothelium. Neither cyclic AMP nor cyclic GMP content of rat thoracic aorta was changed by (-)-discretamine. 7. Contraction of guinea-pig trachea caused by histamine, leukotriene C4 or carbachol was not affected by (-)-discretamine (30 microM). (-)-Discretamine also did not block beta l- or beta2-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea.8. A voltage clamp study in rat ventricular single myocytes revealed that sodium inward current, slow inward Ca2+ current or transient (ItO) and steady state (I800) outward current was not affected by(-)-discretamine (30 microM).9. It is concluded that (-)-discretamine is a selective x-adrenoceptor and 5-HT receptor antagonist in vascular smooth muscle.

Mechanical and electrophysiological effects of 8-oxoberberine (JKL1073A) on atrial tissue.

The effects of 8-oxoberberine (JKL1073A) on contractions and electrophysiological characteristics of atrial tissues were examined. In driven left atria of the rat JKL1073A (10-100 microM) increased twitch tension dose-dependently. In spontaneously beating right atria, JKL1073A increased twitch tension but decreased beating rate slightly. The positive inotropic and the negative chronotropic effect of 30 microM JKL1073A was not affected by prazosin (1 microM), propranolol (1 microM) and 3-isobutyl-1-methyl-xanthine (10 microM) but significantly suppressed by 4-aminopyridine (2 mM 4-AP). Current-clamp study revealed that JKL1073A prolonged rat atrial action potential duration (APD). This prolongation of APD by JKL1073A was decreased by pretreating the cells with 2 mM 4-AP. Voltage-clamp study showed that JKL1073A inhibited the integral of the transient outward current (I(to)) dose-dependently with a KD value of 3.66 +/- 0.93 microM in rat atrial myocytes. The equilibrium dissociation constant (Kd) for JKL1073A bindings to open state I(to) was 0.50 +/- 0.08 microM. The suppression of I(to) by 3 microM JKL1073A was accompanied by shortening of its inactivation time constant from 52.5 +/- 0.9 ms to 16.8 +/- 0.7 ms. V(0.5) for the steady-state inactivation curve of I(to) was shifted from -25.7 +/- 3.3 mV to -34.8 +/- 3.2 mV. In human atrial cells, similar inhibition of I(to) and prolongation of APD by JKL1073A was found. The KD value of JKL1073A for inhibition of the integral of I(to) in human atrial cells is 4.03 +/- 0.02 microM. The Kd for bindings to open state I(to) is 0.5 microM. Currents through K1 channels of rat and human atrial myocytes were not inhibited by JKL1073A at concentrations up to 10 microM. These results indicate that JKL1073A exerts a positive inotropic effect by inhibition of I(to). JKL1073A inhibit I(to) by binding to open state channels or shifting of the steady-state inactivation curve of I(to).