Philip Palade

TNFα receptor expression in rat cardiac myocytes: TNFα inhibition of L-type Ca2+ current and Ca2+ transients

Tumor necrosis factor‐α (TNFα) is a potentially powerful anti‐neoplastic agent; however, its therapeutic usefulness is limited by its cardiotoxic and negative inotropic effects. Accordingly, studies were undertaken to gain a better understanding of the mechanisms of TNFα‐mediated cardiodepression. Single cell RT‐PCR, [125I]TNFα ligand binding and Western immunoblotting experiments demonstrated that rat cardiac cells predominantly express type I TNFα receptors (TNFRI or p60). TNFα inhibited cardiac L‐type Ca2+ channel current (I Ca) and contractile Ca2+ transients. Thus, it is possible that the negative inotropic effects of TNFα are the result of TNFRI‐mediated blockade of cardiac excitation‐contraction coupling.

Perforated patch recording with beta-escin.

Abstract Perforated patch recording with nystatin, amphotericin B and gramicidin can be more difficult in the hands of some investigators than others. In addition, it is difficult to introduce low molecular weight substances such as dyes into the cytoplasm in such experiments. We have determined that β-escin represents a convenient, easy-to-use alternative to less water-soluble ionophores.

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca2+ release are here tentatively classified as activators of a Ca2+-induced Ca2+ release channel preferentially localized in SR terminal or as likely activators of other Ca2+ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca2+ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca2+ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (“sarcoballs”) designed to make the pharmacological study of SR Ca2+ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.

A model of the L‐type Ca2+ channel in rat ventricular myocytes: ion selectivity and inactivation mechanisms

1 We have developed a mathematical model of the L‐type Ca2+ current, which is based on data from whole‐cell voltage clamp experiments on rat ventricular myocytes. Ion substitution methods were employed to investigate the ionic selectivity of the channel. Experiments were configured with Na+, Ca2+ or Ba2+ as the majority current carrier. 2 The amplitude of current through the channel is attenuated in the presence of extracellular Ca2+ or Ba2+. Our model accounts for channel selectivity by using a modified Goldman‐Hodgkin‐Katz (GHK) configuration that employs voltage‐dependent channel binding functions for external divalent ions. Stronger binding functions were used for Ca2+ than for Ba2+. 3 Decay of the ionic current during maintained depolarization was characterized by means of voltage‐ and Ca2+‐dependent inactivation pathways embedded in a five‐state dynamic channel model. Particularly, Ca2+ first binds to calmodulin and the Ca2+‐calmodulin complex is the mediator of Ca2+ inactivation. Ba2+‐dependent inactivation was characterized using the same scheme, but with a decreased binding to calmodulin. 4 A reduced amount of steady‐state inactivation, as evidenced by a U‐shaped curve at higher depolarization levels (>40 mV) in the presence of [Ca2+]o, was observed in double‐pulse protocols used to study channel inactivation. To characterize this phenomenon, a mechanism was incorporated into the model whereby Ca2+ or Ba2+ also inhibits the voltage‐dependent inactivation pathway. 5 The five‐state dynamic channel model was also used to simulate single channel activity. Calculations of the open probability of the channel model are generally consistent with experimental data. A sixth state can be used to simulate modal activity by way of introducing long silent intervals. 6 Our model has been tested extensively using experimental data from a wide variety of voltage clamp protocols and bathing solution manipulations. It provides: (a) biophysically based explanations of putative mechanisms underlying Ca2+‐ and voltage‐dependent channel inactivation, and (b) close fits to voltage clamp data. We conclude that the model can serve as a predictive tool in generating testable hypotheses for further investigation of this complex ion channel.

Calcium‐induced calcium release in crayfish skeletal muscle.

1. Cut crayfish skeletal muscle fibres were mounted in a triple Vaseline‐gap voltage clamp with the Ca(2+)‐sensing dye Rhod‐2 allowed to diffuse in via the cut ends. Ca2+ currents across the surface/T‐tubule membranes (ICa) were recorded simultaneously with changes in myoplasmic Ca2+ concentration (Ca2+ transients). 2. Excitation‐contraction coupling in crayfish skeletal muscle fibres is abolished when calcium in the extracellular solution is replaced by Mg2+. 3. The amplitude of the Ca2+ transients elicited by voltage clamp pulses closely followed the amplitude of the peak calcium currents recorded simultaneously across the surface/T‐tubule membranes. This included decreases in both parameters as the pulse potential approached ECa (reversal potential for Ca2+), as well as secondary Ca2+ transients accompanying large tail calcium currents occurring upon repolarization from very large depolarizations. 4. A large contribution of sarcoplasmic reticulum (SR) Ca2+ release to the Ca2+ transients was revealed by a large decrease in the transient caused by the calcium‐induced calcium release (CICR) blockers procaine and tetracaine. 5. Short pulses which interrupted the calcium current while SR Ca2+ release was in progress at high rates caused the Ca2+ transient to stop rising nearly immediately after the end of the pulse in most fibres. In about 15% of the fibres the Ca2+ transients continued to rise, albeit at a slower rate, for 10‐20 ms after the end of the pulse, as if released Ca2+ was able to elicit some further Ca2+ release from the SR for a while. 6. Even with fibres displaying little sign of continued release after termination of short pulses under control conditions, procaine accelerated the decay of Ca2+ transients elicited by short pulses, indicating that continued release was taking place even as the transient was declining. 7. These results suggest that CICR in crayfish fibres is more closely controlled by a small entry of Ca2+ via surface/T‐tubule membrane Ca2+ current than by a larger amount of Ca2+ released from the SR. The limited positive feedback of released Ca2+ on further Ca2+ release allows CICR to remain graded (according to ICa) rather than all‐or‐none.

Smooth muscle uses another promoter to express primarily a form of human Cav1.2 L-type calcium channel different from the principal heart form.

Several different first exons and amino termini have been reported for the cardiac Ca channel known as alpha(1C) or Ca(V)1.2. The aim of this study was to investigate whether the expression of this channel is regulated by different promoters in smooth muscle cells and in heart in humans. Ribonuclease protection assay (RPA) indicates that the longer first exon 1a is found in certain human smooth muscle-containing tissues, notably bladder and fetal aorta, but that it is not expressed to any significant degree in lung or intestine. On the other hand, all four smooth muscle-containing tissues examined strongly express transcripts containing exon 1b, first reported cloned from human fibroblast cells. In addition, primary cultures of human colonic myocytes and coronary artery smooth muscle cells express predominantly transcripts containing exon 1b. The promoter immediately upstream of exon 1b was cloned, and it displays functional promoter activity when luciferase-expressing constructs were transfected into three different cultured smooth muscle cells: primary human coronary artery smooth muscles cells, primary human colonocytes, and the fetal rat aorta-derived A7r5 cell line. These results indicate that expression in smooth muscle is primarily driven by a promoter different from that which drives expression in cardiac myocytes.

Negative control mechanism with features of adaptation controls Ca2+ release in cardiac myocytes

The central paradox of cardiac excitation-contraction coupling is that Ca(2+)-induced Ca2+ release (CICR), an inherently self-regenerating process, is finely graded by surface membrane Ca2+ current (ICa). By using FPL64176, a novel Ca2+ channel agonist that reduces inactivation of ICa, a rapid negative control mechanism was unmasked at the Ca2+ release level in isolated rat ventricular myocytes. This mechanism terminates CICR independently of the duration of trigger ICa and before the sarcoplasmic reticulum becomes depleted of Ca2+. In its ability to be reactivated by incremental increases in trigger ICa, this mechanism differs from conventional inactivation/desensitization and is similar to the mechanism of increment detection or adaptation described for intracellular Ca2+ release channels. These results indicate that ryanodine receptor adaptation regulates Ca2+ release in cardiac muscle, accounting for or contributing to the graded nature of CICR and, additionally, permitting stores to reload at later times during Ca2+ entry.

Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca2+ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (a fast Ca2+ chelator), (2) a stimulation of peak ICa that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak ICa that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of ICa inactivation is due to caffeines ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak ICa is probably due to phosphodiesterase inhibition, while caffeines inhibitory effect on ICa is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.

A new promoter for α1C subunit of human L-type cardiac calcium channel CaV1.2

The cardiac Ca channel known as a1C or CaV1:2 is shown to express a new longer first exon equivalent to that formerlyreported in rabbit heart or rat aorta. Ribonuclease protection assayindicates that this exon is found in the majorityof Ca V1:2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven bya promoter immediately upstream of this exon and its 5 0 untranslated region. The putative promoter exhibits 69% homologyto its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs. 2002 Elsevier

Arachidonic acid-induced Ca2+ release from isolated sarcoplasmic reticulum

Arachidonic acid has been shown to release Ca2+ from isolated skeletal and cardiac sarcoplasmic reticulum (SR) vesicles. The release took place nearly equally well from all fractions of the SR and was only partially inhibited by ruthenium red, suggesting that some other pathway is involved in addition to the SR Ca2+ release channel. Arachidonic acid increased SR Ca2+ efflux even in the presence of several different SR Ca2+ pump inhibitors. It also had considerably less effect on uptake measured in the presence of oxalate and did not appear to inhibit Ca(2+)-dependent ATPase activity. Thus, the SR Ca2+ pump also appears to be minimally perturbed by arachidonic acid. Arachidonyl CoA was more effective at releasing Ca2+ than the parent compound. Arachidonic acid effects were not inhibited by lipoxygenase or cyclooxygenase inhibitors, suggesting that no eicosanoids are involved in the effects under study here. Flunarizine, cinnarizine and propyl-methylenedioxyindene inhibited the Ca2+ release induced by arachidonic acid. The effects of arachidonic acid appear to depend on the ratio of arachidonic acid to membrane vesicles.