Neonatal Rat Ventricular Cells Research Articles

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells.

To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5'-triphosphate (ATP)-sensitive K+ (KATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal KATP currents (at 0 mV) induced by metabolic inhibition were 48.9+/-2.8 pA/pF in control (n=48), 34.3+/-3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5+/-3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na+ current and inward-rectifying K+ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of KATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional KATP channels. Furthermore, in four out of seven control cells tested, outward K+ currents were stimulated by diazoxide, which is a potent K+ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional KATP channels. The larger population of KATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2.

Expression environment determines K+ current properties: Kv1 and Kv4 alpha-subunit-induced K+ currents in mammalian cell lines and cardiac myocytes.

Voltage-gated K+ channel (Kv) pore-forming (alpha) subunits of the Kv1 and Kv4 subfamilies have been cloned from heart cDNA libraries, and are thought to play roles in the generation of the transient outward K+ current, Ito. Heterologous expression of these subunits in Xenopus oocytes, however, reveals K+ currents that are quite distinct from Ito. In the experiments here, the detailed time- and voltage-dependent properties of the currents expressed in mammalian cell lines and in cardiac myocytes by Kv1.4 and Kv4.2 were examined and compared to previous findings in studies of oocytes, as well as to Ito characterized in various myocardial cells. As in oocytes, expression of Kv1.4 in HEK-293, Ltk- or neonatal rat ventricular cells reveals rapidly activating K+ currents. In contrast to the currents in oocytes, however, there are two components of inactivation of the Kv1.4-induced currents in mammalian cells, and both components are significantly slower in myocytes than in either HEK-293 or Ltk- cells. In addition, in all three cell types, recovery of Kv1.4 from steady-state inactivation is very slow, proceeding with mean time constants in the range of 6-8 s. The properties of Kv4.2-induced currents also vary with cell type and, importantly, the rates of activation, inactivation and recovery from inactivation are significantly faster in mammalian cells than in Xenopus oocytes. In HEK-293, Chinese hamster ovary (CHO) and neonatal rat ventricular cells, for example, the currents recover from steady-state inactivation with mean (+/-SD) time constants of 153+/-32 (n=12), 245+/-112 (n=10) and 86+/-38 (n=11) ms, respectively; therefore, recovery proceeds 5-10 times faster than observed for Kv4.2 in oocytes. These results emphasize the importance of the cellular expression environment in efforts to correlate endogenous K+ currents with heterologously expressed K+ channel subunits. In addition, the finding that Kv alpha subunits produce distinct K+ currents in different cells suggests that cell-type-specific associations with endogenous Kv alpha or accessory beta subunits and/or post-translational processing play roles in determining the properties of functional K+ channels.

スルホニルウレア受容体のアンチセンスオリゴによる新生児ラット心筋ＡＴＰ感受性Ｋ｀＋´チャネルの抑制効果

スルポニルウレア剤やK+チャネル開口薬のATP感受性K+ (KATP) チャネルに対する作用は, チャネルのサブユニットであるスルポニルウレア受容体 (SUR) によって制御されていると考えられている.本研究では, 心筋KATPチャネルの機能的なSURを同定するため, 新生児ラット心室筋細胞を培養し, SURのmRNAに対するアンチセンスオリゴ (AS-SUR) を6日間処置し, KATPチャネルへの影響をパッチクランプ法にて検討した.SUR1, SUR2に対するアンチセンス (AS-SUR1, AS-SUR2) ならびに各々に対するスクランブル配列のオリゴを用いた.代謝阻害により誘発された (0mVにおける) KATPチャネル電流は, 無処置群 (Ctrl) では48.9±2.8pA/pF (n=48) であったが, AS-SUR1ならびにAS-SUR2処置により各々34.3±3.5pA/pF (n=21) , 23.5±3.4pA/pF (n=17) に減少した (: P<0.05, : P<0.01vsCtrl) .対照として用いたスクランブル配列のオリゴはKATPチャネル電流量に影響しなかった.したがって, ラット新生児心室筋のKATPチャネルでは, 心筋タイプとされるSUR2のみならず, SUR1もチャネルの構成成分として機能していることが示唆された.

Carbachol inhibition of Ca2+ currents in ventricular cells obtained from neonatal and adult rats

We investigated the postnatal developmental changes produced by the muscarinic receptor agonist, carbachol, on the L-type Ca2+ current (ICa(L)) in neonatal (aged 5 to 7 days) and adult (aged 2 to 5 months) rat ventricular cells by using the whole-cell voltage clamp technique. Carbachol inhibited the isoproterenol-stimulated ICa(L). The maximal inhibition was 89.3±4.8% (n=5) in neonatal cells and 17.7±7.7% (n=9) in adult cells. Carbachol inhibited the forskolin-stimulated ICa(L) to almost same extent as the isoproterenol-stimulated ICa(L). In the cells pretreated with pertussis toxin, carbachol failed to inhibit the isoproterenol-stimulated ICa(L), indicating that carbachol produced its effect via a pertussis toxin-sensitive G-protein pathway. The effects of carbachol in adult cells became more pronounced, increasing from 17.7% to 54.8% (n=11), with the addition of the synthetic inhibitory G-protein α subunit (Giα) (1 μM) to the reaction. Conversely, the α subunit of another pertussis toxin-sensitive synthetic G-protein (Goα, 1 μM) failed to mimic the effect of Giα. These results suggest that, in rat ventricular cells, (1) the action of carbachol on ICa(L) showed a marked decrease during development; (2) the decrease in the effect of carbachol in adult cells is in part due to a decrease in the activity of pertussis toxin-sensitive G protein, especially Giα.

Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes.

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system. The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123. These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

Roles of the voltage-gated K + channel subunits, Kv 1.5 and Kv 1.4, in the developmental changes of K + currents in cultured neonatal rat ventricular cells

To investigate the roles of voltage-gated K+ channel subunits, Kv 1.5 and Kv 1.4, in the developmental regulation of K+ currents, we determined the K+ channel activities and the distributions of K+ channel subunits in the same single cultured neonatal rat ventricular cells, using a whole-cell patch-clamp technique and an immunocytochemical analysis of K+ channel proteins. In 5-day cultured cells, two types of 4-aminopyridine (4-AP)-sensitive and rapidly activating K+ currents, the transient outward current (Ito) and the ultrarapid delayed rectifier (IKur), could be distinguished. A small proportion of 5-day cells expressing sole IKur demonstrated an intense anti-Kv 1.5 antibody labeling with punctate distribution outlining the cells, while a weak staining was observed in the majority of 5-day cells expressing sole Ito. At day 15 of cell culture, only Ito was present with a lower level of the immunocytochemical expression of Kv 1.5 channel protein. Staining of the Kv 1.4 channel protein was qualitatively similar in the 5-day cells expressing either Ito or IKur. However, anti-Kv 1.4 antibody did not label the 15-day cultured cells showing remarkably increased Ito density. Our results strongly indicate that the Kv 1.5 channel expression may underlie the developmental regulation of IKur, while Kv 1.4 channel does not contribute to the postnatal increase in Ito.

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells.

1. Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin-receptor antagonists and the AT1-receptor antagonist losartan on AII- and endothelin-induced inositol phosphate (IP)-formation (assessed as accumulation of total [3H]-IPs in myo-[3H]-inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [3H]-phenylalanine incorporation). 2. Endothelin (10 pM-1 microM) concentration-dependently increased IP-formation (max. increase at 100 nM ET-1: 130 +/- 14% above basal, n = 25) and [3H]-phenylalanine incorporation (max. increase at 1 microM: 52 +/- 4% above basal, n = 16) with an order of potency: ET-1 > > ET-3. Both effects were antagonized by the ETA/ETB-receptor antagonist bosentan and the ETA-receptor antagonist BQ-123, but not affected by the ETB-receptor antagonist IRL 1038 and the AT1-receptor antagonist losartan. 3. Pretreatment of the cells with 500 ng ml-1 pertussis toxin (PTX) overnight that completely inactivated PTX-sensitive G-proteins did not attenuate but rather enhance ET-1-induced IP-formation. On the other hand, in PTX-pretreated cardiomyocytes ET-1-induced [3H]-phenylalanine incorporation was decreased by 39 +/- 5% (n = 5). 4. All (1 nM-1 microM) concentration-dependently increased IP-formation (max. increase at 1 microM: 42 +/- 7% above basal, n = 16) and [3H]-phenylalanine incorporation (max. increase at 1 microM: 29 +/- 2%, n = 9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123. 5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) All failed to increase [3H]-phenylalanine incorporation: addition of non-myocyte cells to the cardiomyocytes restored All-induced increase in [3H]-phenylalanine incorporation. 6. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET-1-induced increase in rate of protein synthesis (through ETA-receptor stimulation) involves at least two signalling pathways: one via a PTX-insensitive G-protein coupled to IP-formation, and the other one via a PTX-sensitive G-protein, and (b) the trophic effects of All are brought about via local ET-1 secretion upon AT1-receptor stimulation in neonatal rat ventricular non-myocyte cells.

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats.

We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca2+]i) of cultured neonatal rat ventricular cells using microfluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 11.5% of control length), moderately so (to 115%-125% of control length), or extensively (to over 125% of the control length). "Quick stretches" (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca2+]i in the early phase of stretch (30 s-2 min), followed by a small but sustained increase during the late phase of stretch (5-10 min). The initial transient increase in [Ca2+]i after the "quick stretch" was preserved in the presence of gallopamil (10(-7) M) or ryanodine (10(-5) M), but was absent in Ca(2+)-free medium or in the presence of gadolinium (10(-7) M). The late or steady state [Ca2+]i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca(2+)-free medium. A steady-state increase in [Ca2+]i was also evoked by "slow stretch" in which cells were slowly pulled to the final length within 1-2 min. As the presence of external Ca2+ was indispensable, increased trans-sarcolemmal Ca2+ influx appears to be involved in both initial and steady-state increases in [Ca2+]i. The initial increase in [Ca2+]i after the "quick stretch" can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.

Modulated expression of transient outward current in cultured neonatal rat ventricular myocytes: comparison with development in situ

Developmental changes of cardiac ion channels have been characterized in freshly isolated mammalian heart cells. To investigate the regulatory factors of postnatal development in transient outward current (Ito) in cardiomyocytes, the modulated expression of Ito was studied in cultured neonatal rat ventricular cells. These changes in vitro were compared with those in situ in acutely isolated ventricular myocytes. Ventricular cells were enzymatically isolated from day-old Wistar rats and cultured under various growth conditions from day 6 to 15. Whole-cell patch-clamp recording was used to study the functional expression of Ito. During development in situ from 5- to 15-day-old stages, Ito density was doubled at day 15 with a significant increase in membrane capacitance (Cm) of the myocytes. Some cells were incubated in serum-rich medium from day 6 to 15 during primary culture, revealing marked increases in both Cm and Ito density at day 15. However, no developmental changes in the Cm and Ito density were observed in serum-free medium. Under the serum-free condition, neither the addition of acidic fibroblast growth factor (aFGF) nor basic FGF (bFGF) to culture medium influenced the Cm. aFGF (10-60 ng/ml) failed to stimulate Ito expression. 72-h treatment with bFGF significantly promoted the Ito density in a concentration-dependent manner; nevertheless, prolonged administration from day 6 to 15 did not induce a further increase, resulting in lower Ito density than in age-matched freshly isolated and serum-treated preparations. The increase in Ito in cultured cells induced by serum and bFGF may be attributable to paralleling changes in the ionic selectivity of the channel, but was not caused by changes in the voltage-dependence of steady-state Ito activation and inactivation. bFGF and some other unknown serum factors may play important roles in the postnatal expression of Ito in the neonatal cardiomyocytes. The developmental increase in Ito and postnatal cell hypertrophy of neonatal cardiomyocytes can be regulated independently.

Changes in action potentials and ion currents in long-term cultured neonatal rat ventricular cells.

A primary culture of neonatal ventricular myocytes isolated from day-old rats was established for investigating the changes in action potentials and ion currents over long periods. Cells at days 5 and 15 in culture were studied. These changes in vitro were compared with those in situ derived from the age-matched freshly isolated cells. During primary culture, quiescent cells demonstrated shortening of action potential durations (APD) resembling the developmental changes observed in situ. The beating cultured cells were not associated with APD shortening. Despite constant current amplitudes, the densities of Ca2+ currents (ICa) decreased in the quiescent cultures at later ages as a result of cell enlargement. ICa densities were maintained in the beating cultured and freshly isolated cells. Acceleration in the inactivation of ICa was observed during developments both in vitro and in situ. In addition, the densities of transient outward currents (Ito) tripled and doubled in the quiescent and beating cells during 15-day cultures. However, Ito in beating cultured cells made less contribution to APD in contrast to the quiescent cultured and freshly isolated myocytes. These findings demonstrate that electrophysiological properties differ between two types of long-term cultured cells. ICa densities remained constant in the beating cultures, suggesting that cell beating may be required for the maintenance of ICa density in developing cardiomyocytes.

BFGF promotes functional expressions of transient outward currents in cultured neonatal rat ventricular cells

To investigate the effect of basic fibroblast growth factor (bFGF) on ion channel expressions in developing cardiac cells, a short-term primary culture of ventricular myocytes isolated from day-old newborn rats was established. Functional expressions of the transient outward currents (Ito) were measured by whole-cell voltage-clamp method. Addition of 40 ng/ml bFGF to the serum-free culture medium for 72 hours increased the Ito density without influences on membrane capacitance of the myocytes, an estimate of cell size. bFGF also promoted expressions of the steady-state outward currents. Methyl 2,5-dihydroxycinnamate (3 microM), a tyrosine kinase inhibitor, showed preferential inhibition of the bFGF-stimulated increase in Ito density. These results suggest that bFGF may play a role in regulating ion channel expressions of developing cardiac cells via activation of bFGF receptor tyrosine kinase activity.

Retinoid-dependent pathways suppress myocardial cell hypertrophy.

Utilizing an in vitro model system of cardiac muscle cell hypertrophy, we have identified a retinoic acid (RA)-mediated pathway that suppresses the acquisition of specific features of the hypertrophic phenotype after exposure to the alpha-adrenergic receptor agonist phenylephrine. RA at physiological concentrations suppresses the increase in cell size and induction of a genetic marker for hypertrophy, the atrial natriuretic factor (ANF) gene. RA also suppresses endothelin 1 pathways for cardiac muscle cell hypertrophy, but it does not affect the increase in cell size and ANF expression induced by serum stimulation. A trans-activation analysis using a transient transfection assay reveals that neonatal rat ventricular myocardial cells express functional RA receptors of both the retinoic acid receptor and retinoid X receptor (RAR and RXR) subtypes. Using synthetic agonists of RA, which selectively bind to RXR or RAR, our data indicate that RAR/RXR heterodimers mediate suppression of alpha-adrenergic receptor-dependent hypertrophy. These results suggest the possibility that a pathway for suppression of hypertrophy may exist in vivo, which may have potential therapeutic value.

Effects of three fragments of parathyroid hormone on calcium channel currents in neonatal rat ventricular cells

The effects of different fragments of bovine parathyroid hormone, bPTH-(1–34), bPTH-(1–84) and bPTH-(3–34), on two types of calcium channel currents in neonatal rat ventricular cells were compared in the present study. bPTH-(1–34) increased the amplitude of L channel currents, but not of the T channel currents. This effect of bPTH-(1–34) was sustained after a complete washout of the peptide from the bath. The intact PTH molecule, bPTH-(1–84), also increased L channel currents but not affecting T channel currents. While bPTH-(3–34) did not affect the amplitudes of either L or T channel currents by itself, pretreatment of cells with bPTH-(3–34) abolished the effects of both bPTH-(1–34) and bPTH-(1–84) on L channel currents. Moreover, the kinetics of L channel currents in the presence of bPTH-(1–34) or bPTH-(3–34) were different. bPTH-(1–34) increased the time constant of activation, but not of inactivation, of L channel currents from 1.8 to 2.5 ms ( P<0.05). In contrast, bPTH-(3–34) decreased the time constant of inactivation, but not of activation, of L channel currents from 159 to 117 ms ( P<0.05). These results indicate that different fragments of PTH exert different effects on the amplitudes or kinetics of cardiac calcium channel currents.

Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes.

Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5' end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3',5'-cyclic monophosphate (cAMP). It was determined by column chromatography that cAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met5-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-1-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Met5-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells.

Electrical uncoupling induced by gossypol in neonatal rat ventricular cells: Hervé J.C., B. Bastide, F. Pluciennik and J. Délèze, Physiologie Cellulaire, URA CNRS n o 290, Poitiers (France)

Electrical uncoupling induced by gossypol in neonatal rat ventricular cells: Hervé J.C., B. Bastide, F. Pluciennik and J. Délèze, Physiologie Cellulaire, URA CNRS n o 290, Poitiers (France)

Tetrandrine inhibits both T and L calcium channel currents in ventricular cells.

Tetrandrine, a putative Ca2+ channel blocker, is extracted from the Chinese medicinal herb, Radix stephania tetrandrae. In the present study, the whole-cell version of the patch clamp technique was used to investigate the effects of tetrandrine on both T and L calcium channel currents in primary cultured neonatal rat ventricular cells. We show that tetrandrine inhibits both T and L calcium channel currents in ventricular cells. This inhibition of inward Ca2+ currents is concentration dependent and reversible. Tetrandrine does not shift the I-V relationship of the calcium currents. These results clearly demonstrate that tetrandrine acts as a calcium channel antagonist in ventricular cells. Previous data show that tetrandrine may be regarded as a wide-spectrum calcium channel antagonist.

Microinjection of antibodies and expression vectors into living myocardial cells. Development of a novel approach to identify candidate genes that regulate cardiac growth and hypertrophy.

Microinjection approaches in the cardiac cell context have allowed delivery of various calcium dyes and monitoring of short-term physiological responses. However, unlike other cell types, it has proved difficult to microinject myocardial cells without the concomitant loss of long-term cell viability. An analysis of experimental variables was conducted to adapt microinjection techniques to the neonatal rat ventricular cell context. Among the variables optimized were the selection of culture dishes, plating substrate, microinjection parameters, and a variety of maneuvers to inhibit myocyte hypercontracture, injury, and consequent death after micropuncture. With the modified technique, the percentage of injected cells that maintained long-term viability (48 hours) increased from less than 1% to 30%. Similarly, an increased efficiency of gene transfer and expression (measured as the percentage of injected cells that express the delivered gene) was obtained after either cytoplasmic or nuclear injection of a beta-galactosidase expression vector into cardiac myocytes. Microinjection of marker immunoglobulin G does not interfere with the induction of the hypertrophic response or the expression of a coinjected atrial natriuretic factor promoter-luciferase reporter fusion gene construct. To the best of our knowledge, this study provides the first description of the efficient microinjection of neonatal cardiac muscle cells with maintenance of long-term cell viability. The microinjection technique is now a viable approach to examine cause-and-effect relations between specific gene products and any defined feature or response of cardiac myocytes that can be assayed at a single-cell level.

Protein kinase C activity in neonatal cultured rat cardiomyocytes supplemented with docosahexaenoic acid

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after α 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the “in vivo” ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.

GTP gamma S activates calcium channels in neonatal rat ventricular cells.

It has been reported that G proteins modulate voltage-dependent Ca2+ channels in cardiac muscle. G proteins may activate channels directly (acting on Ca2+ channel proteins) or indirectly (acting through intracellular second messengers, e.g., cAMP) (Brown, 1990). The effect of the G protein activator, GTPγS, on Ca2+ channels in neonatal rat ventricular cells has not been demonstrated. Therefore, the effect of the intracellular application of GTPγS, a non-hydrolyzable analogue of GTP, on Ca2+ channel currents in neonatal rat ventricular cells was investigated. Two types of Ca2+ channels have been described in the cardiac cells (Bean, 1985). One type of Ca2+ channel activates at a low threshold and inactivates very rapidly. This channel is called the low voltage-activated or transient channel (L channel). The other type of Ca2+ channel requires larger depolarization for activation and inactivates slowly. This channel is called the high voltage-activated or long-lasting channel (L channel). Therefore, two protocols were used in this study. In one protocol, cells were held at a membrane potential of -80 mV. Test pulses from this holding potential to more positive potentials evoked inward Ca2+ currents which consisted of two components (T and L channel components).

Parathyroid hormone: an endogenous modulator of cardiac calcium channels.

We have tested the effects of the active 1-34 amino acid sequence of rat parathyroid hormone (PTH) on Ca2+ channel activity in neonatal rat ventricular cells. Rat PTH (30 pM to 10 nM) increased depolarization-induced Ca2+ influx into these cells, an effect that was abolished by 1 microM nifedipine. The 1-34 amino acid sequence of bovine PTH also stimulated Ca2+ influx in control cells but not in cells pretreated with cholera toxin. Rat PTH also elevated adenosine 3',5'-cyclic monophosphate accumulation in these ventricular myocytes. Whole cell voltage-clamp recordings confirmed a stimulatory effect of rat PTH on cardiac L-type Ca2+ channels. Cell-attached single channel recordings revealed an increase in the probability of channel opening as the primary mechanism for the enhancement of Ca2+ current. Taken together these results suggest an important role for PTH as an endogenous modulator of cardiac L-type Ca2+ channels.