Modulation of cardiac connexin-43 by omega-3 fatty acid ethyl-ester supplementation demonstrated in spontaneously diabetic rats.

Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.

Diabetes and thyroid hormones affect connexin-43 and PKC-epsilon expression in rat heart atria.

We have examined the changes of intercellular electrical coupling protein connexin-43 (Cx43) and of PKC-epsilon in heart atria of diabetic rats and/or after the treatment with triiodothyronine (T(3)). Diabetes was induced in Wistar-Kyoto rats by streptozotocin (50 mg/kg, i.v.) and atria were examined after 5 (acute stage) and 10 (chronic stage) weeks. T(3) (10 microg/100 g/day) was applied via a gastric tube for the last 10 days prior to the end of the experiments to non-diabetic and to the half of diabetic rats. Expression and phosphorylated status of Cx43, as well as expression of PKC-epsilon, were analyzed by Western blots using mouse monoclonal anti-Cx43 and rabbit polyclonal anti-PKC-epsilon antibodies. We found that the Cx43 expression was significantly increased after the treatment with T(3) and in the acute diabetes. Both in diabetes and after T(3) treatment the phosphorylation of Cx43 isoforms was markedly suppressed compared to the non-diabetic and T(3)-untreated controls. Such a down-regulation was less pronounced in diabetic rats after the T(3)-treatment. The expression of atrial PKC-epsilon was increased in diabetic rats. This increase was suppressed after T(3) administration and the expression was decreased in T(3)-treated non-diabetic rats. We suggest that the reduced Cx43 phosphorylation in diabetic and hyperthyroid rats can deteriorate a cell-to-cell coupling and consequently facilitate a development of atrial tachyarrhythmia in diabetic or hyperthyroid animals.

Thyroid hormones suppress epsilon-PKC signalling, down-regulate connexin-43 and increase lethal arrhythmia susceptibility in non-diabetic and diabetic rat hearts.

We examined whether thyroid hormones affect myocardial epsilon-PKC signalling, downstream target substrate, connexin-43 (Cx43) and arrhythmogenesis in non-diabetic and diabetic rats. Diabetes was induced by a single streptozotocin injection (50mg/kg, i.v.). Triiodothyronine (T(3)) was applied by gavage (1microg/kg of body weight for 10 days) to 4 weeks and 9 weeks diabetic and age-matched non-diabetic rats. Western blot analysis of Cx43 and epsilon-PKC, immunofluorescence of Cx43, ultrastructure of cardiomyocytes and myocardial conduction velocity were performed. Isolated perfused heart preparation was used to test ventricular fibrillation susceptibility. T(3) significantly decreased epsilon-PKC expression in non-diabetic and suppressed in diabetic rat heart ventricles. Decline of epsilon-PKC signalling was associated with decrease of Cx43 phosphorylation in diabetic and to a greater extent in non-diabetic rat hearts. However, conduction velocity was significantly decreased in diabetic while enhanced due to T(3) and increased in non-diabetic T(3)-treated rat heart ventricles compared to non-treated. T(3)-induced down-regulation of Cx43 was associated with increased cardiac propensity to ventricular fibrillation. Findings indicate that activation of epsilon-PKC signalling linked with phosphorylation of Cx43 is one of the mechanisms involved in the adaptation of the heart to hyperglycemia. Suppression of epsilon-PKC and Cx43 phosphorylation by T(3) abolish benefit of adaptation rendering the heart prone to lethal arrhythmias.

Factors involved in the susceptibility of spontaneously hypertensive rats to low K+-induced arrhythmias.

Disorders of intracellular Ca2+ homeostasis and intercellular coupling are thought to be crucial in the initiation and maintenance of malignant arrhythmias. The aim of this study was to investigate possible arrhythmogenic factors in spontaneously hypertensive rats (SHR) as well as their susceptibility to low K+-related arrhythmias. The experiments were performed on isolated hearts of 13 weeks-old SHR and age-matched Wistar Kyoto rats (WKY). Equilibration of the heart by Langendorff perfusion with oxygenated, 37 degrees C warm, standard Krebs solution at a constant pressure was followed by perfusion with low K+ solution for 60 min, unless sustained ventricular fibrillation occurred earlier. Electrocardiogram and epicardial monophasic action potentials (MAPs) were continuously monitored for incidence of arrhythmias and action potential changes. Myocardial tissue was taken for ultrastructural analysis and immunodetection of the main gap junction protein, connexin-43. The results showed that hypertrophic hearts of SHR exhibited prolongation of MAPs and a decrease in phosphorylation of connexin-43. Moreover, they were more prone to low K+-induced early after-depolarisations and ventricular premature beats as well as to connexin-43 and ultrastructural alterations than WKY rats. Consequently, the incidence of ventricular tachycardia (70% vs. 50%) and both transient (50% vs. 25%) and sustained (60% vs. 25%) ventricular fibrillation was higher in SHR than WKY rats. The results suggest that both prolongation of MAP and connexin-43 alterations are important arrhythmogenic factors facilitating arrhythmias in the setting of Ca2+ disorders due to hypokalaemia.

Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells.

Store-operated Ca2+ entry (SOCE) is the Ca2+ influx that is activated on depletion of intracellular Ca2+ stores. Although SOCE is found in a variety of cell types, its activation mechanism and molecular identity remain to be clarified. Current experimental results suggest that SOCE channels are activated by direct coupling with Ca2+ release channels on depleted stores. Here we report SOCE in cardiac myocytes, that was prominently sensitive to Zn2+ but resistant to inhibitors for voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers. The SOCE activity may be developmentally regulated, because the SOCE was easily detected during embryonic and neonatal stages but not in mature myocytes from adult hearts. In cardiac myocytes, ryanodine receptor type 2 (RyR-2) is thought to be the sole Ca2+ release channel on the intracellular store, and junctophilin type 2 (JP-2) contributes to formation of the junctional complex between the cell surface and store membranes. Using the knockout mice, we also examined possible involvement of the Ca2+ release channel and junctional membrane complex in cardiac SOCE. Apparently normal SOCE activities were retained in mutant myocytes lacking RyR-2 or JP-2, suggesting that neither the Ca2+ release channel nor junctional membrane complex is involved in activation of cardiac SOCE.

Role of ATP decrease in secretion induced by mitochondrial dysfunction in guinea-pig adrenal chromaffin cells.

The mechanism related to mitochondrial dysfunction-induced catecholamine (CA) secretion in dispersed guinea-pig adrenal chromaffin cells was investigated using amperometry and confocal laser microscopy. Application of CCCP, which does not stimulate generation of reactive oxygen species (ROS), reversibly induced CA secretion, whereas application of either cyanide or oligomycin (OL), a stimulator for ROS, enhanced CA secretion to a smaller extent. The CCCP-induced secretion was abolished by removal of external Ca2+ ions and was markedly diminished by D600. The mitochondrial membrane potential, measured using rhodamine 123, was rapidly lost in response to CCCP, but did not change noticeably during a 3 min exposure to OL. Prior exposure to OL markedly facilitated depolarization of the mitochondrial membrane potential in response to cyanide. The mitochondrial inhibitors rapidly produced an increase in Magnesium Green (MgG) fluorescence in the absence of external Ca2+ and Mg2+ ions, an increase that was larger in the cytoplasm than in the nucleus. The rank order of potency in increasing MgG fluorescence among the inhibitors was similar to that in increasing secretion. Thus, mitochondrial inhibition rapidly decreases [ATP] and the mitochondrial dysfunction-induced secretion is not due to ROS generation or to mitochondrial depolarization, but is possibly mediated by a decrease in ATP.

Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Nna(+)/Ca(2+) exchanger.

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na(+)/Ca(2+) exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha-2 repeat of NCX1 identified one residue (Gly833) that caused a large (> or = 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 microM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 microM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na(+)/Ca(2+) exchange in such a way that it becomes accessible to external drug.

Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea-pig adrenal medulla.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in catecholamine secretion from dissociated adrenal chromaffin cells of the guinea-pig was investigated using amperometry, the patch clamp technique and immunochemistry. Pretreatment of adrenal chromaffin cells with 0.3-10 nM PACAP for 2 min resulted in enhancement of nicotine- and muscarine-induced secretions in either the presence of external Ca2+ ions or nominally Ca2+-free solution, with no change in basal secretion or the holding current at -60 mV in most of the cells tested. Pretreatment with PACAP augmented the muscarine-induced non-selective cation current, but did not affect the muscarine-induced outward current or nicotine-induced current. PACAP-induced enhancement of nicotine- and muscarine-induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors 100 microM HA1004 or 2 microM H89. Application of forskolin enhanced both muscarine- and nicotine-induced secretions, whereas application of a phorbol ester augmented the nicotine-induced secretion, but suppressed the muscarine-induced secretion in a reversible manner. Immunohistochemical analysis of adrenal medullae revealed that PACAP-like immunoreactivity was present in nerve fibres surrounding putative chromaffin cells. PAC1R-like immunoreactivity was distributed diffusely in the plasma membrane, whereas nicotinic ACh receptor-like immunoreactivity was concentrated at the plasma membrane near the nucleus, where the synapses were mainly localized. These observations suggest that PACAP in the guinea-pig adrenal medulla functions as a neuromodulator to facilitate ACh-induced secretion through a cAMP-protein kinase A-dependent pathway.

The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.

We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane. We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the alpha repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca(2+) by up to 6-fold. In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the alpha repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.

Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1.

Physiological functions of the intracellular regulatory domains of the Na(+)/Ca(2+) exchanger NCX1 were studied by examining Ca(2+) handling in CCL39 cells expressing a low-affinity Ca(2+) regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na(+) inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Delta246-672). We found that D447V/D498I was unable to efficiently extrude Ca(2+) from the cytoplasm, particularly during a small rise in intracellular Ca(2+) concentration induced by the physiological agonist alpha-thrombin or thapsigargin. The same mutant took up Ca(2+) much less efficiently than the wild-type NCX1 in Na(+)-free medium when transfectants were not loaded with Na(+), although it appeared to take up Ca(2+) normally in transfectants preloaded with Na(+). XIP-4YW and, to a lesser extent, Delta246-672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na(+)-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca(2+) handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.

Retardation of cation channel deactivation by mitochondrial dysfunction in adrenal medullary cells.

The mechanism for cyanide (CN) activation of a nonselective cation (NS) channel coupled with a muscarinic receptor in a guinea pig chromaffin cell was studied with the perforated-patch method. Bath application of a protein kinase inhibitor resulted in a dose-dependent inhibition of muscarine-induced current (I(M)) but had no apparent effect on the CN-induced current (I(CN)). On the other hand, production of I(CN) occluded muscarine activation of NS channels in an amplitude-dependent manner. Deactivation of I(M) after washout was retarded while I(CN) was also active, and the extent of the retardation increased with an increase in the relative production of I(CN) on muscarinic stimulation. Restoration of Na(+) pump activity from CN suppression was conspicuously retarded below 19-20 degrees C, and the apparent diminution of I(M) and I(CN) after washout was retarded in parallel with a decrease in temperature. The results suggest that CN activation of NS channels is due to suppression of deactivation of the channel.

Effect of sphingosylphosphorylcholine on the single channel gating properties of the cardiac ryanodine receptor.

The effects of the lysosphingolipid, sphingosylphosphorylcholine (SPC), on the cardiac ryanodine receptor (RyR) were examined. The open probability of cardiac RyR incorporated in lipid bilayers was decreased by cytoplasmic, but not lumenal side application of micromolar concentrations of SPC. Modification of channel function was characterized by the appearance of a long-lived closed state in addition to the brief channel closings observed in the presence and absence of SPC. Open channel kinetics and ion conduction properties, however, were not altered by this compound. These results suggest that SPC, a putative second messenger derived from sphingomyelin, may regulate Ca(2+) release from the sarcoplasmic reticulum by modifying the gating kinetics of the RyR.

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.

Na+ pump inhibition and non-selective cation channel activation by cyanide and anoxia in guinea-pig chromaffin cells.

1. Hypoxia and metabolic inhibition with cyanide (CN) evoke catecholamine secretion in adrenal chromaffin cells through depolarization. We elucidated mechanisms for a CN- or anoxia-induced inward (depolarization) current, using the perforated patch method. 2. Bath application of Ba2+ induced a dose-dependent inhibition of a muscarine-induced current (IMUS) and part of the CN-induced current (ICN) with an IC50 (concentration responsible for 50 % inhibition) of 1.3 mM. The Ba2+-sensitive component was estimated to comprise 58 % of the total ICN. 3. The Ba2+-resistant component of ICN tended to increase with shifts of membrane potential from -40 to 40 mV and was markedly suppressed by exposure to a K+-free solution or 200 microM ouabain, indicating that the majority of the Ba2+-resistant component of ICN is due to suppression of the Na+ pump current (Ipump). 4. The non-Ipump component of ICN diminished progressively in K+-free solution. Substitution of glucose for sucrose in a K+-free CN solution further diminished the CN potency to produce the non-Ipump component. 5. The I-V relationship for the non-Ipump component of ICN had a reversal potential of -3 and -47 mV at 147 and 5.5 mM Na+, respectively, and showed an outward rectification, indicating that the non-Ipump component of ICN is due to activation of non-selective cation channels. 6. Exposure to anoxia induced a current with an amplitude comparable to that of ICN, and the anoxia-induced current apparently occluded development of ICN. The anoxia-induced current diminished by ca 60 % in the absence of K+ and reversed polarity at 5 mV under K+-free conditions. 7. It is concluded that exposure to CN and to anoxia induces suppression of the Na+ pump and activation of non-selective cation channels, probably due to an ATP decrease resulting mainly from consumption by the Na+ pump.

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Are the antiarrhythmic-defibrillating effects of D-sotalol due to or despite the prolongation of the action potential duration?

These results support our hypothesis that class III compounds, with a positive inotropic effect, increase intercellular coupling and synchronization, mainly by preventing intracellular Ca overload. They act as defibrillating compound, similar to cAMP and adrenaline, most probably due to their so called sympathomimetic effect. In our opinion, their cardioprotective effects, resembling cardioversion, are not related to their ability to prolong APD and ERP. Moreover, we suggest that any compound that possesses these sympathomimetic effects, but without inducing the arrhythmogenic prolongation of APD, may exhibit a potent, safety and more efficient antiarrhythmic - defibrillating ability.

Hypoxia and cyanide induce depolarization and catecholamine release in dispersed guinea-pig chromaffin cells.

1. The perforated patch method and amperometry were used to determine whether the adrenal medullary cell itself is capable of sensing hypoxia and, if so, how such sensation is transduced to secretion of catecholamines (CA). 2. Exposure to hypoxia, cyanide (CN), or muscarine facilitated CA secretion from dissociated chromaffin cells. The CN-induced secretion was not affected by removal of glucose, indicating that the CN release is due to chemical hypoxia. 3. The secretions induced by CN and muscarine were markedly diminished by removal of Ca2+ ions or by application of Cd2+ or methoxyverapamil (D-600). 4. Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca2+ concentrations determined using the acetoxymethyl (AM) ester form of fluo-3 in the presence of external Ca2+ ions, but not in their absence. 5. Hypoxia and CN produced inward currents at an equilibrium potential for Cl- ions, irrespective of whether or not Na+ ions were present in the cells, and substitution of N-methyl-D-glucamine for 134 mM Na+ ions in the perfusate inhibited the CN current by 71 %. The reversal potential for the CN current was -24 mV in the standard perfusate. 6. The hypoxia-, CN- and muscarine-induced currents decreased in parallel with hyperpolarizations, and exposure to CN prevented muscarine, but not nicotine, from inducing a further inward current. 7. We conclude that hypoxia and CN induce CA secretion through depolarization and the subsequent activation of voltage-dependent Ca2+ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.

Activation of Ca(2+)-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells.

The effects of cyanide (CN) on whole cell current measured with the perforated-patch method were studied in adrenal medullary cells. Application of CN produced initially inward and then outward currents at -52 mV or more negative. As the membrane potential was hyperpolarized, amplitude and latency of the outward current (Io) by CN became small and long, respectively. A decrease in the external Na+ concentration did not affect the latency for CN-induced Io but enhanced the amplitude markedly. The CN Io reversed polarity at -85 mV, close to the Nernst potential for K+, and was suppressed by the K+ channel blockers curare and apamin but not by glibenclamide, suggesting that Io is due to the activation of Ca(2+)-dependent K+ channels. Consistent with this notion, the Ca(2+)-mobilizing agents, muscarine and caffeine, also produced Io. Exposure to CN in a Ca(2+)-deficient medium for 4 min abolished caffeine- or muscarine-induced Io without development of Io, and addition of Ca2+ to the CN-containing solution induced Io. We conclude that exposure to CN produces Ca(2+)-dependent K+ currents in an external Ca(2+)-dependent manner, probably via facilitation of Ca2+ influx.

Correlation between wave components of the second derivative of plethysmogram and arterial distensibility.

The ratio of two wave components (magnitude of b/a constituting the second derivative of the plethysmogram (SDPTG) was correlated with arterial distensibility. Eighty-two subjects (33-93 years old) were classified into three groups according to the thickness of the intima-media complex of the common carotid artery measured by B-mode ultrasonography. One group was non-atherosclerotic (without pathologic thickening) (nAS) and the other two groups atherosclerotic (mild and severe thickening, or plaque formation) (AS-1 and AS-2). Distensibility (D) of the common carotid artery was calculated from arterial dimensions and blood pressure: h/p = D, where h = (Ds-Dd)/Dd; Ds, Dd and p represent the inner diameter of the carotid artery at peak systole, at end diastole and brachial pulse pressure, respectively. The plethysmogram was recorded at the cuticle of the 2nd digit of the left hand, and the SDPTG was determined with a 10 msec time constant. D showed a significant negative correlation with age in all subjects and in the three separate groups. The correlation between age and magnitude of b/a was significantly negative in all subjects. This negative correlation was not observed in the nAS group, while it was significant in both AS-1 and AS-2. The correlation between magnitude of b/a and D was significantly positive in all subjects and in each group. Significant differences were found among the three groups for magnitude of b/a and D. These results suggest that a decrease in magnitude of b/a or in D was proportional to the thickness of the intima-media complex of the carotid artery, that is, the development of atherosclerosis. These results provide direct evidence that magnitude of b/a of the SDPTG is related to the distensibility of the peripheral artery, and suggest that magnitude of b/a is a useful non-invasive index of atherosclerosis and altered arterial distensibility.

Whole-cell currents from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell CCL39.

A conventional patch-clamp technique was used to record the whole-cell current from the cloned canine cardiac Na+/Ca2+ exchanger NCX1 overexpressed in a fibroblast cell. Ca2+ was extracellularly applied to the Na+-loaded cell to activate the outward current by operating the reverse mode of NCX1. No measurable outward current was ever elicited from the nontransfected cell. Na+/Ca2+ exchange blocker 5 mM Ni2+ or 3 microM KB-R7943 that was applied extracellularly abolished the outward current. With 140 mM external Li+ (replacing Na+), the outward current was transient during the Ca2+ application. In contrast, with 140 mM external Na+, the outward current was maintained without any inactivation during the Ca2+ application. I-V relations predicted from the whole-cell clamp protocols used were obtained both before and during the Ca2+ application. The exchanger whole-cell currents are thus successfully detectable from NCX1 which is overexpressed in this stable transfectant system.