α1-And β-adrenergic and muscarinic-cholinergic regulation in the spontaneous beating and Ca2+ oscillations in cultured neonatal rat cardiac myocytes

α₁-and β-adrenergic and muscarinic-cholinergic regulation in spontaneous beating and Ca²⁺ oscillations in neonatal rat cardiac myocytes at day 6 of culture was investigated. The spontaneous beating in myocytes decreased in the presence of 10 μM norepinephrine (NE). This negative chronotropic action was antagonized by prazosin. Carbachol (CCh) also showed negative chronotropic action which was inhibited by atropine. On the other hand, isoproterenol (ISP) increased the beating rate which was antagonized by propranolol. NE increased inositol phosphate formation whereas CCh and ISP did not. NE and CCh suppressed the frequency of the spontaneous Ca²⁺ oscillations but ISP increased. The present results suggest that α₁-adrenergic and muscarinic receptors regulate chronotropism to be negative whereas β-adrenoceptor regulates chronotropism to be positive in cultured neonatal rat cardiac myocytes.     

Atrial natriuretic factor (ANF) effects on L-, N-, and P/Q-type voltage-operated calcium channels

1. We have previously reported that atrial natriuretic factor (ANF) decreases neuronal norepinephrine (NE) release. The mechanism that mediates NE release from presynaptic membrane to synaptic cleft is a strongly calcium-dependent process. The modulator effect of ANF may be related to modifications in calcium influx at the presynaptic nerve ending by interaction with voltage-operated calcium channels (VOCCs).2. On this basis we investigated the effects of ANF on K⁺-induced ⁴⁵Ca²⁺ uptake and evoked neuronal NE release in the presence of specific L-, N-, and P/Q-type calcium channel blockers in the rat hypothalamus.3. Results showed that ANF inhibited K⁺-induced ⁴⁵Ca²⁺ uptake in a concentration-dependent fashion. Concentration–response curves to VOCC blockers nifedipine (NFD, L-type channel blocker), -conotoxin GVIA (CTX, N-type channel blocker), and -agatoxin IVA (AGA, P/Q-type channel blocker) showed that all the blockers decreased NE release. Incubation of ANF plus NFD showed an additive effect as compared to NFD or ANF alone. However, when the hypothalamic tissue was incubated in the presence of ANF plus CTX or AGA there were no differences in neuronal NE release as compared to calcium channel blockers or ANF alone.4. These results suggest that ANF decreases NE release by an L-type calcium channel independent mechanism by inhibiting N- and/or P/Q-type calcium channels at the neuronal presynaptic level. Thus, ANF modulates neuronal NE release through different mechanisms involving presynaptic calcium channel inhibition.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Swimming training promotes cardiac remodeling and alters the expression of mRNA and protein levels involved in calcium handling in hypertensive rats

Aim

The aim of this study was to identify the effects of swimming training on the mRNA expression and protein levels of the calcium handling proteins in the hearts of renovascular hypertensive rats submitted to swimming protocol during 6 weeks.

Main methods

Fischer rats with renovascular hypertension 2-kidney 1-clip (2K1C) and SHAM groups were divided among sedentary and exercised groups. The exercise protocol lasted for 6 weeks (1 h/day, 5×/week), and the mean arterial pressure, cardiomyocytes hypertrophy parameters, mRNA expression and protein levels of some calcium handling proteins in the left ventricle were evaluated.

Key findings

Swimming training was able to reduce the levels of mean arterial pressure in the hypertensive group compared to 2K1C SED, and to promote cardiac hypertrophy in SHAM EX and 2K1C EX groups in comparison to the respective control groups. The mRNA levels of B-type natriuretic peptide were reduced in the 2K1C EX when compared to 2K1C SED. The mRNA and protein levels of the sarcoplasmic reticulum Ca^2 +-ATPase increased after the swimming training in SHAM and 2K1C groups. The mRNA and protein levels of phospholamban, displayed an increase in their levels in the exercised SHAM and in hypertensive rats in comparison to their respective controls; while mRNA levels of Na⁺/Ca^2 + exchanger was reduced in the left ventricle comparing to the sedentary hypertensive rats.

Significance

Taken altogether, we provide evidence that the aerobic training may lead to cardiac remodeling, and modulate the calcium handling proteins expression in the heart of hypertensive rats.     

Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes

We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or -myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca²⁺-ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations. cardiac myocytes; cell-specific expression; adenovirus vectors; calcium transport     

Expression of protein phosphatases during postnatal development of rabbit heart

Protein phosphatases play a major role in the regulation of L-type calcium current (I_Ca) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of I_Ca, with greater stimulatory effects on I_Ca observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of and subunits of PP 1 and the subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using ³²P labeled cDNA probes specific for PP 1, PP 1 and PP 2A showed that the expression of steady state mRNA levels for PP 1, PP 1 and PP 2A were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of -agonist isoproterenol on I_Ca.     

Increased calcium release from sarcoplasmic reticulum stimulated by inositol trisphosphate in spontaneously hypertensive rat heart cells

It is known that inositol (1, 4, 5)-trisphosphate (IP₃) stimulates Ca²⁺ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP₃ on Ca²⁺ release from SR in cardiac myocytes. The effect of IP₃ on Ca²⁺ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP₃ on Ca²⁺ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP₃ on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with⁴⁵Ca²+, and then incubated with the indicated concentrations of IP³ for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca²⁺ release stimulated by 10 M (1, 4, 5)-IP₃ was detected by fura-2. In⁴⁵Ca²⁺ prelabeled SR, the maximal Ca²⁺ release was achieved at 10 M IP₃ incubated for 1 min. The release of Ca²⁺ was higher in Sr of SHR than in the SR of WKY. IP₃ stimulates Ca²⁺ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.     

Hearts of surviving MLP-KO mice show transient changes of intracellular calcium handling

The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca _i ²⁺ ) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca _i ²⁺ cycling, myocardial Ca _i ²⁺ -transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with β-agonist isoproterenol and the sarcoplasmic reticular Ca²⁺-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca²⁺ handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. β-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca _i ²⁺ -level and a decrease in Ca²⁺-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca _i ²⁺ handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.     

Mitochondrial Ca2+ Transients in Cardiac Myocytes During the Excitation–Contraction Cycle: Effects of Pacing and Hormonal Stimulation

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca²⁺ in adult rabbit cardiac myocytes loaded with Ca²⁺-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37°C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4°C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 M) increased the magnitude of Ca²⁺ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca²⁺ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca²⁺ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca²⁺ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca²⁺ transients occur during excitation–contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

Modulation of the ATP induced [Ca2+]c increase in as-30D hepatoma cells

• 1.1. The regulation of the increase in the cytosolic calcium concentration ([Ca²⁺]c) induced by extracellular ATP in AS-30D hepatoma cells was studied.
• 2.2. Homologous desensitization involving the refilling of intracellular calcium pools and the participation of protein kinase C was found.
• 3.3. Isoproterenol, forskolin and dibutyril-cyclic AMP also induced an increase in [Ca²⁺]c.
• 4.4. Interestingly, synergism was found for isoproterenol or forskolin and ATP.
• 5.5. The results suggest that there are two pathways for mobilizing [Ca²⁺] in AS-30D hepatoma cells; one is activated by ATP receptors and the other by cyclic AMP.

     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.     

Enhancement of L-type Ca(2+) current from neonatal mouse ventricular myocytes by constitutively active PKC-betaII

The cardiacL-type calcium current (I_Ca) can be modified byactivation of protein kinase C (PKC). However, the effect of PKC activation on I_Ca is still controversial. Somestudies have shown a decrease in current, whereas other studies havereported a biphasic effect (an increase followed by a decrease incurrent or vice versa). A possible explanation for the conflictingresults is that several isoforms of PKC with opposing effects onI_Ca were activated simultaneously. Here, weexamined the influence of a single PKC isoform (PKC-II) on L-typecalcium channels in isolation from other cardiac isoforms, using atransgenic mouse that conditionally expresses PKC-II. Ventricularcardiac myocytes were isolated from newborn mice and examined forexpression of the transgene using single cell RT-PCR afterI_Ca recording. Cells expressing PKC-II showeda twofold increase in nifedipine-sensitive I_Ca. The PKC-II antagonist LY-379196 returned I_Caamplitude to levels found in non-PKC-II-expressing myocytes. Theincrease in I_Ca was independent ofCa_v1.2-subunit mRNA levels as determined by quantitativeRT-PCR. Thus these data demonstrate that PKC- is a potent modulatorof cardiac L-type calcium channels and that this specific isoformincreases I_Ca in neonatal ventricular myocytes.

     

In vivo regulation of Na/Ca exchanger expression by adrenergic effectors

The Na/Ca exchanger encoded by the NCX1 gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca(2+). To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 microg x kg(-1) x h(-1)), isoproterenol (150 microg x kg(-1) x h(-1)), and phenylephrine (200 microg x kg(-1) x h(-1)) each stimulated a significant increase in NCX1 mRNA levels (35-85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance (P < 0.05), a 20% decrease in relaxation duration (P < 0.05), and a 25% reduction in the fluorescence decay constant (P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of alpha- and beta-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

Patch clamp analysis of chemically activated and modulated ionic channels in isolated mammalian cardiomyocytes

Techniques routinely utilized in this laboratory for recording currents through single ionic channels of isolated atrial and ventricular rat cardiomyocytes are described. Emphasis is placed in two main areas: first, on methods for obtaining a sufficient yield of Ca⁺⁺-tolerant myocytes suitable for patch clamp experiments, and secondly, on methods for analyzing the temporal characteristics of patched ionic channels. These methods were used on acetylcholine activated K⁺ channels in isolated atrial myocytes and on an inwardly-rectifying K⁺ channel in ventricular myocytes. The latter is an example of a hormonally modulated K⁺ channel, since its activity could be substantially increased by norepinephrine. Analysis of the closed and open time distributions suggested that one of the closed states of this channel is markedly abbreviated by norepinephrine, whereas the open state is nearly unaffected. Norepinephrine was effective when channel activity was recorded from on-cell patches and the hormone was added to the solution bathing the cell membrane outside of the patched area. This indicates that a second messenger substance is probably mediating the action of norepinephrine.     

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line

The effects of the -galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca²⁺]_i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca²⁺]_i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by -galactosides. The observed Ca²⁺-response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca²⁺]_i compared to wild type lectin.Abbreviation [Ca²⁺]_i concentration of intracytoplasmic free calcium - CM carboxamidomethylation - CRD earbohydrate recognition domain - C2S mutant lectin protein in which Cys2 was replaced by serine - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N,N - N-tetraacetic acid HEPES,N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPL14 human -galactoside-binding placental lectin - Rec H recombinant human 14 kDa lectin - W68Y mutant lectin protein in which Trp68 was substituted to tyrosine     

Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

TRPV4 protein forms a Ca²⁺-permeable channel that is sensitive to osmotic and mechanical stimuli and responds to warm temperatures, and expresses widely in various kinds of tissues. As for cardiac myocytes, TRPV4 has been detected only at the mRNA level and there were few reports about subcel-lular localization of the protein. The purpose of the present study was to investigate the expression profile of TRPV4 protein in cultured neonatal rat ventricular myocytes. Using Western blots, immunofluorescence, confocal microscopy and immuno-electron microscopy, we have shown that TRPV4 protein was predominantly located in the nucleus of cultured neonatal myocytes. Furthermore, cardiac myocytes responded to hypotonic stimulation by translocating TRPV4 protein out of the nucleus. The significance and mechanism concerning the unusual distribution and translocation of TRPV4 protein in cardiac myocytes remain to be clarified.Key words: TRPV4, nucleus, hypotonicity, translocation, ventricular myocytes, neonatal rat.