Larry Hryshko - Academia.edu (original) (raw)

Papers by Larry Hryshko

Experimental Parasitology, 2013

Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis mus... more Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis muscle larvae, have collagenolytic and elastolytic activities, and may be related to the larval invasion of intestinal epithelial cells. In this study, the serine protease gene (TspSP-1.2, GenBank accession No. EU302800) encoding a 35.5 kDa protein from T. spiralis was cloned, and recombinant TspSP-1.2 protein was produced in an Escherichia coli expression system. An anti-TspSP-1.2 serum recognized the native protein migrating at 35.5 kDa by the Western blotting of the crude or ES antigens from muscle larvae at 42 days post infection. An immunolocalization analysis identified TspSP-1.2 in the cuticle and internal organs of the parasite. Transcription and expression of the TspSP-1.2 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). An in vitro invasion assay showed that, when anti-TspSP-1.2 serum, serum of infected mice and normal mouse serum were added to the medium, the invasion rate of the infective larvae in an HCT-8 cell monolayer was 33.0%, 89.4%, and 96.2%, respectively (P<0.05), indicating that the anti-TspSP-1.2 serum partially prevented the larval invasion of intestinal epithelial cells. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TspSP-1.2 protein displayed a 34.92% reduction in adult worm burden and 52.24% reduction in muscle larval burden. The results showed that the recombinant TspSP-1.2 protein induced a partial protective immunity in mice and could be considered as a potential vaccine candidate against T. spiralis infection.

Developments in cardiovascular medicine, 1989

Dihydropyridine Ca channel agonists bear close structural resemblance to several Ca channel antag... more Dihydropyridine Ca channel agonists bear close structural resemblance to several Ca channel antagonists. The most extensively investigated agent in this group is BAY k 8644, first described by Schramm and coworkers. 1−4 Other Ca channel agonists are YC170, 5 202–791, 6 H 160–51. 7 R202–791 6 and CGP28–392. 8,9 The characteristic property of these agents is to increase the ‘open time’ of the ‘L’ type of calcium channels resulting either in an increase in calcium current during an action potential or during depolarisation by other means. 10 The cardiovascular consequence of this is an increase in cardiac contractility and vasoconstriction. 2 It is this latter property which has precluded the clinical use of calcium channel agonists in heart failure. However, the calcium channel agonists hold an important place as pharmacological tools. This review will consider some of the anomalous actions of BAY k 8644 which result in “negative” inotropy under certain circumstances. A possible effect of these drugs on sarcoplasmic reticular function, in addition to their better known effect of enhancing trans-sarcolemmal calcium current, will be discussed.

British Journal of Pharmacology, Jun 1, 1988

Positive inotropic concentration of the Ca-channel agonist, Bay K 8644, depressed contraction of ... more Positive inotropic concentration of the Ca-channel agonist, Bay K 8644, depressed contraction of canine right ventricular trabecula immediately after a rest period of 8min, without decreasing action potential plateau amplitude. In contrast, high external Ca and ouabagenin caused only a slight decrease in post-rest contraction. Bay K 8644-induced post-rest depression was inversely related to the extracellular Ca concentration. Hence it could not be due to cellular Ca overload. Since post-rest potentiation is due to increased contribution of Ca from the sarcoplasmic reticulum, these results suggest that Bay K 8644 decreases the amount of releasable Ca from this structure during rest.

Canadian Journal of Physiology and Pharmacology, Sep 1, 1988

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle d... more Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sarcoplasmic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 μM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 μM) decreased SLIF at all levels of inotropic response. The β-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.

Canadian Journal of Physiology and Pharmacology, Jul 1, 1989

Contraction of canine ventricular trabeculae were recorded at a stimulation frequency of 0.5 Hz a... more Contraction of canine ventricular trabeculae were recorded at a stimulation frequency of 0.5 Hz and after rest periods of 2 and 8 min to analyze the effect of the Ca channel agonist BAY k 8644, on sarcoplasmic reticular function. Short periods of rest interposed between steady trains of stimuli caused a potentiation of the postrest beat. This is believed to be due to the mobilization of activator Ca from the sarcoplasmic reticulum (SR). Racemic BAY k 8644 and its Ca channel agonist enantiomer, (−) BAY k 8644, both produced an increase in contraction in response to a steady train of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole. Addition of Ca channel antagonists, (+) BAY k 8644, nitrendipine, or nifedipine, to reverse the agonistic effect of (−) and racemic BAY k 8644 on the Ca channel did not convert the rest depression into rest potentiation. In the presence of any of the Ca channel antagonist alone, rest potentiation was not reversed but enhanced. The rank order of rest potentiation after these drugs was nifedipine > nitrendipine > (+) BAY k 8644. It is concluded that (−) BAY k 8644 enhances the ongoing loss of Ca from the SR during diastole in a manner similar to that of racemic BAY k 8644. However, its depressant effect on the postrest tension cannot be attributed to an increase in Ca current because (+) BAY k 8644 and other Ca channel antagonists did not reverse rest depression due to (−) or racemic BAY k 8644 even though they normalized steady-state tension. The difference between the effects of the three Ca channel antagonists on the postrest contraction suggests that they may have variable effects on the release of Ca from the SR in addition to causing inhibition of transsarcolemmal Ca influx. This difference may be related to different degrees of intracellular penetration of these compounds.Key words: cardiac, contractility, excitation – contraction coupling, heart.

Springer eBooks, 1987

Ca channel agonists are novel compounds which increase cardiac contractile force by increasing sl... more Ca channel agonists are novel compounds which increase cardiac contractile force by increasing slow inward Ca current through the sarcolemma (1,2,3). The presently available compounds are dihydropyridine analogs with structural similarities with agents such as nifedipine, which are Ca channel antagonists. The present study resulted from some chance observation which were made with BAY K 8644, a prototype Ca channel agonist first discovered by Schramm et al (4). This compound was being used by us to evaluate an experimental model designed to test the effects of cardioactive agents on the contribution of transsarcolemmal and transsarcoplasmic reticular Ca movement to inotropy. We observed that BAY K 8644, impaired Ca release from the sarcoplasmic reticulum in addition to its well known effect of increasing transsarcolemmal Ca influx.

American Journal of Physiology-heart and Circulatory Physiology, Aug 1, 1989

Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium,... more Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium, was converted to rest depression by BAY K 8644 (1 microM). Plateau height and duration of the postrest beat were enhanced by BAY K 8644, suggesting an enhancement of extracellular calcium entry. Caffeine (3 mM) also produced depression at all rest intervals, although to a lesser extent than BAY K 8644. Compared with BAY K 8644, treatment with caffeine resulted in an elevation of plateau amplitude and a shortening of action potential duration. Action potential configuration changes induced by rest were unaltered by caffeine despite reduction in rest potentiation. Caffeine-induced rest depression was associated with an increase in the time to peak tension. This was not observed with BAY K 8644. Treatment with both caffeine (3 mM) and BAY K 8644 (1 microM) greatly prolonged time to peak tension. Action potential duration and plateau height were either maintained or increased. Less rest depression was observed with the combination than with either agent alone. These results suggest that 1) BAY K 8644 and caffeine inhibit rest potentiation by different mechanisms, and 2) caffeine-induced inhibition of calcium uptake by the sarcoplasmic reticulum may enhance the effect of BAY K 8644-induced increase in calcium influx on the contractile apparatus.

American Journal of Physiology-heart and Circulatory Physiology, Aug 1, 1989

The calcium-channel agonist-antagonist racemic mixture of BAY K 8644 as well as the pure calcium-... more The calcium-channel agonist-antagonist racemic mixture of BAY K 8644 as well as the pure calcium-channel agonist enantiomer, converted rest potentiation in canine ventricular muscle to rest depression. The depressed postrest beats had increased action potential plateau heights and durations, suggesting that extracellular calcium entry was enhanced. BAY K 8644 did not alter the time to peak tension of either steady-state or postrest beats. Although BAY K 8644 slightly increased the amplitude of rapid-cooling contractures (RCC) immediately after an electrically evoked steady-state contraction (RCCss), the RCC amplitude rapidly decreased with increasing durations of rest. These results suggest that increased calcium influx because of BAY K 8644 increases the pool of releasable calcium during steady-state stimulation. However, they also indicate that BAY K 8644 causes acceleration of diastolic loss of calcium from the sarcoplasmic reticulum (SR). Scattered light intensity fluctuation measurements showed that the negative inotropic effect of BAY K 8644 on postrest contraction was not caused by calcium overloading of the SR, since BAY K 8644 reduced asynchronous myofilament motion. A negative relationship found between extracellular calcium and BAY K 8644-induced postrest depression also rules out the possibility that the latter effect is caused by intracellular calcium overload. BAY K 8644 seems to accelerate the loss of calcium from the SR during diastole by a pathway that does not appear to pass through the myofilaments.

British Journal of Pharmacology, 1989

R.A. BOUCHARD et al. suggest that this reversal is a result of depressed intracellular calcium bu... more R.A. BOUCHARD et al. suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644. This project was supported by grants from the Medical Research Council of Canada, Manitoba Health Research Council and Manitoba Heart Foundation. R.A.B. is the recipient of a Canadian Heart Foundation studentship. We are very grateful to Theresa Chau and Proma Tagore for technical assistance.

Journal of Biological Chemistry, 1996

Journal of Biological Chemistry, Jul 1, 1994

The Na+-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this... more The Na+-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this transporter, NCX1, has been cloned previously (Nicoll, D. A, Longoni, S., and Philipson, K. D. (1990) Science 250, 562-565). We now report the cloning of a second isoform (NCX2) of the Na+-Ca2+ exchanger which was present in a rat brain cDNA library. NCX2 is predicted to code for a protein of 921 amino acids. NCXl and NCX2 are 61 and 65% identical at the nucleotide and amino acid levels, respectively, and are the products of different genes. The genes for NCXl and NCX2 are located on human chromosomes 2 and 14, respectively. Hydropathy profiles of the two exchangers are very similar. Transcripts of NCX2 are detected in brain and skeletal muscle. NCX2 was expressed in Xenopus oocytes and Na+-Ca2+ exchange activity was analyzed electrophysiologically by the giant inside-out, excised patch technique. Outward currents were evoked by the application of Na+ with the exchanger operating in the reversed mode (extracellular Ca2+ exchanging for intracellular Na'). The affinity for Na+ (30 m~) and the current-voltage relationship of NCX2 are similar to those for NCX1. Like NCX1, NCX2 is secondarily regulated by intracellular Ca2+, but the affinity of NCX2 for regulatory Ca2+ (1.5 p~) upon initial application of Na' is lower than that of NCXl (0.3 w). The existence of multiple Na+-Ca2+ exchanger isoforms may provide flexibility for regulation and expression. Na+-Caz+ exchange across the plasma membrane is an important determinant of intracellular Ca2+ levels. The primary role of Na+-Ca2+ exchange is Ca2+ extrusion. Net efflux of Ca2+ is accomplished using the energy of the Na+ gradient set up by the ATP-dependent Na+ pump. Three Na+ ions are exchanged for one Ca2+ ion in each reaction cycle. The importance of Na+-Ca2+ exchange is most well established in cardiac tissue. Na+-Ca2+ exchange, however, has also received considerable attention in other tissues such as brain, kidney, and smooth muscle. Recent reviews on Na+-Ca2+ exchange can be found in Philipson and Nicoll (1992, 1993).

Steinkopff eBooks, 1998

Na-Ca exchange proteins are involved in Ca homeostasis in a wide variety of tissues. Unique Na

Journal of Biological Chemistry, 1996

The Journal of general physiology, 1995

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ b... more The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at...

British Journal of Pharmacology, Nov 1, 1988

The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolat... more The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolated ventricular muscle. 2 Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced the amplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibited post-extrasystolic potentiation and converted rest-potentiation into rest depression. Restpotentiation was blocked preferentially by ryanodine compared to post-extrasystolic potentiation. An increase in the contribution of extracellular Ca to the extrasystolic contraction could not entirely account for the post-extrasystolic potentiation. 3 Prolonged rest, by itself, also caused depression of the first post-rest contraction. During restpotentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx. However, the ability of the 'release pool' of Ca in the SR to be reprimed after a contraction was reduced. This was seen as a decrease in post-extrasystolic potentiation elicited immediately after rest. 4 A decrease in stimulus interval was associated with a transient decrease in contraction amplitude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryanodine blocked the initial transient changes and accelerated the delayed changes. These results suggest that the transient changes in contraction after sudden changes in drive interval are dependent on the SR. 5 Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in the SR after a contraction seem to be interval-dependent. The data also indicate that different mechanisms are involved in post-extrasystolic and rest-potentiation. 6 The results are consistent with a model which proposes 'recirculation' of activator Ca within the SR after a contraction or of the presence of an appreciable amount of inactivation of the SR Ca release process during normal stimulation. An increased pool of releasable Ca due to longer recirculation time or a time-dependent decay in the level of inactivation of Ca release from the SR may give rise to rest-potentiation.

The Journal of Physiology, Feb 1, 2005

Chinese hamster ovary cells expressing the bovine cardiac Na+–Ca2+ exchanger (NCX1.1) accumulated... more Chinese hamster ovary cells expressing the bovine cardiac Na+–Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, Δ(241-680), that does not require allosteric activation by Ca2+ for activity. Non-transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5–2 pm markedly stimulated the subsequent uptake of Ca2+ by Na+–Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pm Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pm Cd2+ were 40% smaller than currents activated by 1 μm cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pm Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.

American Journal of Transplantation, Mar 1, 2016

American Journal of Transplantation, Mar 1, 2016

Abbreviations: C a O 2 , arterial oxygen content; CO, cardiac output; DCD, donation after circula... more Abbreviations: C a O 2 , arterial oxygen content; CO, cardiac output; DCD, donation after circulatory death; LV, left ventricle; MRI, magnetic resonance imaging; P a CO 2 , arterial partial pressure of carbon dioxide; P a O 2 , arterial partial pressure of oxygen; RV, right ventricle; UHPLC, ultra-high-performance liquid chromatography; WLST, withdrawal of life-sustaining therapy

American Journal of Physiology-heart and Circulatory Physiology, Sep 1, 2001

Inhibition of Na ϩ /Ca 2ϩ exchange by KB-R7943: transport mode selectivity and antiarrhythmic con... more Inhibition of Na ϩ /Ca 2ϩ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences. Am J Physiol Heart Circ Physiol 281: H1334-H1345, 2001.-The Na ϩ /Ca 2ϩ exchanger plays a prominent role in regulating intracellular Ca 2ϩ levels in cardiac myocytes and can serve as both a Ca 2ϩ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca 2ϩ entry) of Na ϩ /Ca 2ϩ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na ϩ /Ca 2ϩ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na ϩ /Ca 2ϩ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na ϩ /Ca 2ϩ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 M KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca 2ϩ entry via reverse Na ϩ /Ca 2ϩ exchange in this process. sodium/calcium; NCX1.1; giant excised patch clamp; electrophysiology TRANSSARCOLEMMAL CA 2ϩ EFFLUX via Na ϩ /Ca 2ϩ exchange is essential for cardiac muscle relaxation. On a beatto-beat basis, the Na ϩ /Ca 2ϩ exchanger is believed to

Transplantation, Jul 1, 2014

Organ Care System (OCS) device for 8 hrs, which allowed ex vivo beating heart assessment in worki... more Organ Care System (OCS) device for 8 hrs, which allowed ex vivo beating heart assessment in working mode in addition to the standard resting mode. Functional & metabolic parameters were evaluated. The hearts were maintained in resting and working modes for 4 hours each. Results: During resting perfusion, heart 1 had favourable lactate profiles (lactate extraction) suggestive of viable myocardium, however remained > 5mmol/l. Heart 2 had an adverse lactate profile suggestive of ischaemic myocardium (Figure 1). In a working state, a left atrial pressure challenge was conducted. Heart 1 functioned better than Heart 2 (figure 1), with superior cardiac output and generated pressure. The inferiority of Heart 2 was likely the result of dual BD + DCD insult, decreased post-conditioning activation, and an undiagnosed patent foramen ovale. Conclusion: We report the first 2 DCD human hearts recovered on the OCS. Although these were not the ideal donors for DCD heart donation (donor 1-age> 50, WIT > 30 mins; Donor 2-BD + DCD, PFO), both demonstrated viability and the potential for DCD cardiac allografts. Further studies of ideal criteria DCD donors are underway.

Experimental Parasitology, 2013

Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis mus... more Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis muscle larvae, have collagenolytic and elastolytic activities, and may be related to the larval invasion of intestinal epithelial cells. In this study, the serine protease gene (TspSP-1.2, GenBank accession No. EU302800) encoding a 35.5 kDa protein from T. spiralis was cloned, and recombinant TspSP-1.2 protein was produced in an Escherichia coli expression system. An anti-TspSP-1.2 serum recognized the native protein migrating at 35.5 kDa by the Western blotting of the crude or ES antigens from muscle larvae at 42 days post infection. An immunolocalization analysis identified TspSP-1.2 in the cuticle and internal organs of the parasite. Transcription and expression of the TspSP-1.2 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). An in vitro invasion assay showed that, when anti-TspSP-1.2 serum, serum of infected mice and normal mouse serum were added to the medium, the invasion rate of the infective larvae in an HCT-8 cell monolayer was 33.0%, 89.4%, and 96.2%, respectively (P<0.05), indicating that the anti-TspSP-1.2 serum partially prevented the larval invasion of intestinal epithelial cells. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TspSP-1.2 protein displayed a 34.92% reduction in adult worm burden and 52.24% reduction in muscle larval burden. The results showed that the recombinant TspSP-1.2 protein induced a partial protective immunity in mice and could be considered as a potential vaccine candidate against T. spiralis infection.

Developments in cardiovascular medicine, 1989

Dihydropyridine Ca channel agonists bear close structural resemblance to several Ca channel antag... more Dihydropyridine Ca channel agonists bear close structural resemblance to several Ca channel antagonists. The most extensively investigated agent in this group is BAY k 8644, first described by Schramm and coworkers. 1−4 Other Ca channel agonists are YC170, 5 202–791, 6 H 160–51. 7 R202–791 6 and CGP28–392. 8,9 The characteristic property of these agents is to increase the ‘open time’ of the ‘L’ type of calcium channels resulting either in an increase in calcium current during an action potential or during depolarisation by other means. 10 The cardiovascular consequence of this is an increase in cardiac contractility and vasoconstriction. 2 It is this latter property which has precluded the clinical use of calcium channel agonists in heart failure. However, the calcium channel agonists hold an important place as pharmacological tools. This review will consider some of the anomalous actions of BAY k 8644 which result in “negative” inotropy under certain circumstances. A possible effect of these drugs on sarcoplasmic reticular function, in addition to their better known effect of enhancing trans-sarcolemmal calcium current, will be discussed.

British Journal of Pharmacology, Jun 1, 1988

Positive inotropic concentration of the Ca-channel agonist, Bay K 8644, depressed contraction of ... more Positive inotropic concentration of the Ca-channel agonist, Bay K 8644, depressed contraction of canine right ventricular trabecula immediately after a rest period of 8min, without decreasing action potential plateau amplitude. In contrast, high external Ca and ouabagenin caused only a slight decrease in post-rest contraction. Bay K 8644-induced post-rest depression was inversely related to the extracellular Ca concentration. Hence it could not be due to cellular Ca overload. Since post-rest potentiation is due to increased contribution of Ca from the sarcoplasmic reticulum, these results suggest that Bay K 8644 decreases the amount of releasable Ca from this structure during rest.

Canadian Journal of Physiology and Pharmacology, Sep 1, 1988

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle d... more Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sarcoplasmic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 μM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 μM) decreased SLIF at all levels of inotropic response. The β-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.

Canadian Journal of Physiology and Pharmacology, Jul 1, 1989

Contraction of canine ventricular trabeculae were recorded at a stimulation frequency of 0.5 Hz a... more Contraction of canine ventricular trabeculae were recorded at a stimulation frequency of 0.5 Hz and after rest periods of 2 and 8 min to analyze the effect of the Ca channel agonist BAY k 8644, on sarcoplasmic reticular function. Short periods of rest interposed between steady trains of stimuli caused a potentiation of the postrest beat. This is believed to be due to the mobilization of activator Ca from the sarcoplasmic reticulum (SR). Racemic BAY k 8644 and its Ca channel agonist enantiomer, (−) BAY k 8644, both produced an increase in contraction in response to a steady train of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole. Addition of Ca channel antagonists, (+) BAY k 8644, nitrendipine, or nifedipine, to reverse the agonistic effect of (−) and racemic BAY k 8644 on the Ca channel did not convert the rest depression into rest potentiation. In the presence of any of the Ca channel antagonist alone, rest potentiation was not reversed but enhanced. The rank order of rest potentiation after these drugs was nifedipine > nitrendipine > (+) BAY k 8644. It is concluded that (−) BAY k 8644 enhances the ongoing loss of Ca from the SR during diastole in a manner similar to that of racemic BAY k 8644. However, its depressant effect on the postrest tension cannot be attributed to an increase in Ca current because (+) BAY k 8644 and other Ca channel antagonists did not reverse rest depression due to (−) or racemic BAY k 8644 even though they normalized steady-state tension. The difference between the effects of the three Ca channel antagonists on the postrest contraction suggests that they may have variable effects on the release of Ca from the SR in addition to causing inhibition of transsarcolemmal Ca influx. This difference may be related to different degrees of intracellular penetration of these compounds.Key words: cardiac, contractility, excitation – contraction coupling, heart.

Springer eBooks, 1987

Ca channel agonists are novel compounds which increase cardiac contractile force by increasing sl... more Ca channel agonists are novel compounds which increase cardiac contractile force by increasing slow inward Ca current through the sarcolemma (1,2,3). The presently available compounds are dihydropyridine analogs with structural similarities with agents such as nifedipine, which are Ca channel antagonists. The present study resulted from some chance observation which were made with BAY K 8644, a prototype Ca channel agonist first discovered by Schramm et al (4). This compound was being used by us to evaluate an experimental model designed to test the effects of cardioactive agents on the contribution of transsarcolemmal and transsarcoplasmic reticular Ca movement to inotropy. We observed that BAY K 8644, impaired Ca release from the sarcoplasmic reticulum in addition to its well known effect of increasing transsarcolemmal Ca influx.

American Journal of Physiology-heart and Circulatory Physiology, Aug 1, 1989

Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium,... more Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium, was converted to rest depression by BAY K 8644 (1 microM). Plateau height and duration of the postrest beat were enhanced by BAY K 8644, suggesting an enhancement of extracellular calcium entry. Caffeine (3 mM) also produced depression at all rest intervals, although to a lesser extent than BAY K 8644. Compared with BAY K 8644, treatment with caffeine resulted in an elevation of plateau amplitude and a shortening of action potential duration. Action potential configuration changes induced by rest were unaltered by caffeine despite reduction in rest potentiation. Caffeine-induced rest depression was associated with an increase in the time to peak tension. This was not observed with BAY K 8644. Treatment with both caffeine (3 mM) and BAY K 8644 (1 microM) greatly prolonged time to peak tension. Action potential duration and plateau height were either maintained or increased. Less rest depression was observed with the combination than with either agent alone. These results suggest that 1) BAY K 8644 and caffeine inhibit rest potentiation by different mechanisms, and 2) caffeine-induced inhibition of calcium uptake by the sarcoplasmic reticulum may enhance the effect of BAY K 8644-induced increase in calcium influx on the contractile apparatus.

American Journal of Physiology-heart and Circulatory Physiology, Aug 1, 1989

The calcium-channel agonist-antagonist racemic mixture of BAY K 8644 as well as the pure calcium-... more The calcium-channel agonist-antagonist racemic mixture of BAY K 8644 as well as the pure calcium-channel agonist enantiomer, converted rest potentiation in canine ventricular muscle to rest depression. The depressed postrest beats had increased action potential plateau heights and durations, suggesting that extracellular calcium entry was enhanced. BAY K 8644 did not alter the time to peak tension of either steady-state or postrest beats. Although BAY K 8644 slightly increased the amplitude of rapid-cooling contractures (RCC) immediately after an electrically evoked steady-state contraction (RCCss), the RCC amplitude rapidly decreased with increasing durations of rest. These results suggest that increased calcium influx because of BAY K 8644 increases the pool of releasable calcium during steady-state stimulation. However, they also indicate that BAY K 8644 causes acceleration of diastolic loss of calcium from the sarcoplasmic reticulum (SR). Scattered light intensity fluctuation measurements showed that the negative inotropic effect of BAY K 8644 on postrest contraction was not caused by calcium overloading of the SR, since BAY K 8644 reduced asynchronous myofilament motion. A negative relationship found between extracellular calcium and BAY K 8644-induced postrest depression also rules out the possibility that the latter effect is caused by intracellular calcium overload. BAY K 8644 seems to accelerate the loss of calcium from the SR during diastole by a pathway that does not appear to pass through the myofilaments.

British Journal of Pharmacology, 1989

R.A. BOUCHARD et al. suggest that this reversal is a result of depressed intracellular calcium bu... more R.A. BOUCHARD et al. suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644. This project was supported by grants from the Medical Research Council of Canada, Manitoba Health Research Council and Manitoba Heart Foundation. R.A.B. is the recipient of a Canadian Heart Foundation studentship. We are very grateful to Theresa Chau and Proma Tagore for technical assistance.

Journal of Biological Chemistry, 1996

Journal of Biological Chemistry, Jul 1, 1994

The Na+-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this... more The Na+-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this transporter, NCX1, has been cloned previously (Nicoll, D. A, Longoni, S., and Philipson, K. D. (1990) Science 250, 562-565). We now report the cloning of a second isoform (NCX2) of the Na+-Ca2+ exchanger which was present in a rat brain cDNA library. NCX2 is predicted to code for a protein of 921 amino acids. NCXl and NCX2 are 61 and 65% identical at the nucleotide and amino acid levels, respectively, and are the products of different genes. The genes for NCXl and NCX2 are located on human chromosomes 2 and 14, respectively. Hydropathy profiles of the two exchangers are very similar. Transcripts of NCX2 are detected in brain and skeletal muscle. NCX2 was expressed in Xenopus oocytes and Na+-Ca2+ exchange activity was analyzed electrophysiologically by the giant inside-out, excised patch technique. Outward currents were evoked by the application of Na+ with the exchanger operating in the reversed mode (extracellular Ca2+ exchanging for intracellular Na'). The affinity for Na+ (30 m~) and the current-voltage relationship of NCX2 are similar to those for NCX1. Like NCX1, NCX2 is secondarily regulated by intracellular Ca2+, but the affinity of NCX2 for regulatory Ca2+ (1.5 p~) upon initial application of Na' is lower than that of NCXl (0.3 w). The existence of multiple Na+-Ca2+ exchanger isoforms may provide flexibility for regulation and expression. Na+-Caz+ exchange across the plasma membrane is an important determinant of intracellular Ca2+ levels. The primary role of Na+-Ca2+ exchange is Ca2+ extrusion. Net efflux of Ca2+ is accomplished using the energy of the Na+ gradient set up by the ATP-dependent Na+ pump. Three Na+ ions are exchanged for one Ca2+ ion in each reaction cycle. The importance of Na+-Ca2+ exchange is most well established in cardiac tissue. Na+-Ca2+ exchange, however, has also received considerable attention in other tissues such as brain, kidney, and smooth muscle. Recent reviews on Na+-Ca2+ exchange can be found in Philipson and Nicoll (1992, 1993).

Steinkopff eBooks, 1998

Na-Ca exchange proteins are involved in Ca homeostasis in a wide variety of tissues. Unique Na

Journal of Biological Chemistry, 1996

The Journal of general physiology, 1995

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ b... more The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at...

British Journal of Pharmacology, Nov 1, 1988

The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolat... more The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolated ventricular muscle. 2 Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced the amplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibited post-extrasystolic potentiation and converted rest-potentiation into rest depression. Restpotentiation was blocked preferentially by ryanodine compared to post-extrasystolic potentiation. An increase in the contribution of extracellular Ca to the extrasystolic contraction could not entirely account for the post-extrasystolic potentiation. 3 Prolonged rest, by itself, also caused depression of the first post-rest contraction. During restpotentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx. However, the ability of the 'release pool' of Ca in the SR to be reprimed after a contraction was reduced. This was seen as a decrease in post-extrasystolic potentiation elicited immediately after rest. 4 A decrease in stimulus interval was associated with a transient decrease in contraction amplitude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryanodine blocked the initial transient changes and accelerated the delayed changes. These results suggest that the transient changes in contraction after sudden changes in drive interval are dependent on the SR. 5 Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in the SR after a contraction seem to be interval-dependent. The data also indicate that different mechanisms are involved in post-extrasystolic and rest-potentiation. 6 The results are consistent with a model which proposes 'recirculation' of activator Ca within the SR after a contraction or of the presence of an appreciable amount of inactivation of the SR Ca release process during normal stimulation. An increased pool of releasable Ca due to longer recirculation time or a time-dependent decay in the level of inactivation of Ca release from the SR may give rise to rest-potentiation.

The Journal of Physiology, Feb 1, 2005

Chinese hamster ovary cells expressing the bovine cardiac Na+–Ca2+ exchanger (NCX1.1) accumulated... more Chinese hamster ovary cells expressing the bovine cardiac Na+–Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, Δ(241-680), that does not require allosteric activation by Ca2+ for activity. Non-transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5–2 pm markedly stimulated the subsequent uptake of Ca2+ by Na+–Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pm Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pm Cd2+ were 40% smaller than currents activated by 1 μm cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pm Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.

American Journal of Transplantation, Mar 1, 2016

American Journal of Transplantation, Mar 1, 2016

Abbreviations: C a O 2 , arterial oxygen content; CO, cardiac output; DCD, donation after circula... more Abbreviations: C a O 2 , arterial oxygen content; CO, cardiac output; DCD, donation after circulatory death; LV, left ventricle; MRI, magnetic resonance imaging; P a CO 2 , arterial partial pressure of carbon dioxide; P a O 2 , arterial partial pressure of oxygen; RV, right ventricle; UHPLC, ultra-high-performance liquid chromatography; WLST, withdrawal of life-sustaining therapy

American Journal of Physiology-heart and Circulatory Physiology, Sep 1, 2001

Inhibition of Na ϩ /Ca 2ϩ exchange by KB-R7943: transport mode selectivity and antiarrhythmic con... more Inhibition of Na ϩ /Ca 2ϩ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences. Am J Physiol Heart Circ Physiol 281: H1334-H1345, 2001.-The Na ϩ /Ca 2ϩ exchanger plays a prominent role in regulating intracellular Ca 2ϩ levels in cardiac myocytes and can serve as both a Ca 2ϩ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca 2ϩ entry) of Na ϩ /Ca 2ϩ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na ϩ /Ca 2ϩ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na ϩ /Ca 2ϩ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na ϩ /Ca 2ϩ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 M KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca 2ϩ entry via reverse Na ϩ /Ca 2ϩ exchange in this process. sodium/calcium; NCX1.1; giant excised patch clamp; electrophysiology TRANSSARCOLEMMAL CA 2ϩ EFFLUX via Na ϩ /Ca 2ϩ exchange is essential for cardiac muscle relaxation. On a beatto-beat basis, the Na ϩ /Ca 2ϩ exchanger is believed to

Transplantation, Jul 1, 2014

Organ Care System (OCS) device for 8 hrs, which allowed ex vivo beating heart assessment in worki... more Organ Care System (OCS) device for 8 hrs, which allowed ex vivo beating heart assessment in working mode in addition to the standard resting mode. Functional & metabolic parameters were evaluated. The hearts were maintained in resting and working modes for 4 hours each. Results: During resting perfusion, heart 1 had favourable lactate profiles (lactate extraction) suggestive of viable myocardium, however remained > 5mmol/l. Heart 2 had an adverse lactate profile suggestive of ischaemic myocardium (Figure 1). In a working state, a left atrial pressure challenge was conducted. Heart 1 functioned better than Heart 2 (figure 1), with superior cardiac output and generated pressure. The inferiority of Heart 2 was likely the result of dual BD + DCD insult, decreased post-conditioning activation, and an undiagnosed patent foramen ovale. Conclusion: We report the first 2 DCD human hearts recovered on the OCS. Although these were not the ideal donors for DCD heart donation (donor 1-age> 50, WIT > 30 mins; Donor 2-BD + DCD, PFO), both demonstrated viability and the potential for DCD cardiac allografts. Further studies of ideal criteria DCD donors are underway.