Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

Cell-to-cell coupling assayed by means of electrical measurements

The importance of electrical measurements in the evaluation of cell-to-cell coupling in heart muscle was discussed. The presence of gap junctions in heart and smooth muscle, and the implications of this for electrical synchronization and healing-over, was emphasized. Moreover, the modulation of junctional resistance by Ca, protons and cAMP was reviewed.     

Biochemical events associated with rapid cellular damage during the oxygen- and calcium-paradoxes of the mammalian heart

The O2- and Ca2(+)-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e- and also oxygen radicals or redox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised conditions, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2(+)-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2(+)-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Sodium channels in cardiac Purkinje cells

Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3 omega cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.     

Platelet abnormalities and the pathophysiology of essential hypertension

The mechanisms whereby intracellular calcium concentration is controlled are briefly reviewed. With the current knowledge of both calcium homeostasis and the function and properties of cellular Ca2+-target proteins/signal transduction systems, a dysfunction of cellular calcium metabolism is considered in relation to the pathogenesis of hypertension. Although the enhanced peripheral vascular resistance characteristic of hypertension is ultimately a function of Ca2+ availability for smooth muscle cell contraction, the platelet possesses many parallel biochemical and physiological properties. Therefore, we have utilized the platelet as the cell-model for investigating the role of Ca2+ in hypertension disorders. An overview of Ca2+-linked platelet processes altered in essential hypertension is presented, and an attempt is made to integrate these multiple aberrations in a fundamental membrane lesion.     

Cell-to-cell coupling assayed by means of electrical measurements

Summary The importance of electrical measurements in the evaluation of cell-to-cell coupling in heart muscle was discussed. The presence of gap junctions in heart and smooth muscle, and the implications of this for electrical synchronization and healing-over, was emphasized. Moreover, the modulation of junctional resistance by Ca, protons and cAMP was reviewed.Nur allein der Mensch Vermag das Unmögliche, Er unterscheidet, Wählet und richtet; Er kann dem Augenblick Dauer verleihen. Goethe     

An analysis of the effects of urethane on cardiovascular responsiveness to catecholamines in terms of its interference with Ca++ mobilization from both intra and extracellular pools

Urethane (1 X 10(-2) - 1 X 10(-1) M) reduced, in a concentration-dependent manner, both intra and extracellular Ca++ dependent noradrenaline-induced contractions of perfused rabbit ear artery as well as the tonic contractions produced by perfusion with high K+ solution. However, a quantitative analysis of the data indicated that for urethane concentrations similar to those found in plasma during anesthesia urethane antagonism is confined to noradrenaline-induced contractions which depend upon the mobilization of Ca++ from intracellular storage sites. In KCl-contracted arteries, urethane enhanced the relaxant effects of isoprenaline. - Urethane reduced the amplitude of contractions of spontaneously beating guinea-pig right atrium at concentrations which have only a limited effect on frequency. In addition, it decreased in a concentration-dependent manner the amplitude of isoprenaline-activated electrically driven, and K+ depolarized guinea-pig right ventricular strips. Urethane had no effect on the chrono and inotropic actions of isoprenaline on cardiac preparations. In in vivo experiments the chronotropic response to low doses of isoprenaline was significantly higher in urethane-treated as compared to unanesthetized rats. The higher dose of isoprenaline tested produced a significant fall in systolic blood pressure in urethane-anesthetized rats. A significant correlation exists between the chronotropic response to isoprenaline and resting heart rate values in urethane-anesthetized rats. These results indicate that urethane, at concentrations similar to those found in plasma during anesthesia selectively interferes with mobilization of Ca++ from intracellular storage sites. In addition, the interference of urethane anesthesia with the isoprenaline chronotropic effect 'in vivo' cannot be explained by a direct interference of urethane with beta-adrenoceptors at cardiac level.     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Calcium signalling: a historical account, recent developments and future perspectives

Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca(2+)-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria 'buy time' by precipitating inside Ca2+ and phosphate. If injury persists, Ca2(+)-death eventually ensues.     

Cell-to-cell coupling studied in isolated ventricular cell pairs

Cell pairs isolated from adult rat and guinea pig ventricles were used to study the electrical properties of the nexal membrane. Each cell of a pair was connected to a voltage-clamp system so as to enable whole-cell, tight-seal recording. The current-voltage relationship of the nexal membrane was found to be linear, revealing a resistance rn of 2-4 M omega. rn was insensitive to the sarcolemmal membrane potential (range: -90 to +30 mV), and exerted no time-dependent gating behavior (range: 0.1 to 10 s). Lowering pHi yielded a small increase in rn. Vigorous elevations in [Ca2+]i gave rise to an increase in rn which was associated with a cell shortening. Uncoupling caused by aliphatic alcohols or halothane did not produce cell shortening. Cell pairs were also used to study action potential transfer.     

A note on the mechanism of action of UV-irradiation of amphibian embryos

The actions on amphibian embryos of UV-irradiation, exposure to Li+ or exposure to ouabain show interesting parallels with their effects on spontaneous release at the presynaptic terminals of the neuromuscular junction. It is suggested that these treatments serve to raise intracellular Ca2+ ([Ca2+]i) in these examples, and that UV-promoted abnormalities in embryogenesis are a consequence of changes in [Ca2+]i at critical stages in development.     

Potassium currents in cardiac cells

The kinetic properties of the inwardly rectifying K current and the transient outward current in cardiac cells were investigated. In sheep Purkinje fibers superfused with Na-free K-free solution, time-dependent changes in the conductance of the inward rectifier are described. In patch clamp experiments the inward rectifier inactivates during hyperpolarization, as can be seen by a decrease in the open state probability. Using whole cell clamp on ventricular myocytes it is demonstrated that the inactivation during hyperpolarization is due to blocking of the channel by external Na, Mg and Ca. The channels responsible for the transient outward current in cow, sheep and rabbit Purkinje fibers are identified using single channel recording. It is demonstrated that in all three preparations the channels are K-selective. The channel in cow Purkinje cells has a large conductance and is regulated by voltage and internal Ca concentration. The channels identified in the sheep and rabbit cells have a much smaller conductance.     

Biochemical events associated with rapid cellular damage during the oxygen-and calcium-paradoxes of the mammalian heart

Summary The O_2– and Ca²⁺-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H⁺ and e^– and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca²⁺ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca²⁺ or by raising [Ca²⁺]_i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca²⁺-paradox by the membrane perturbation associated with removal of extracellular Ca²⁺; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca²⁺]_i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca²⁺-paradox which continues under anoxia. Massive entry of Ca²⁺ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.     

Calcium channel modulation by beta-adrenergic neurotransmitters in the heart

Calcium ions play a crucial role in the regulation of the heart beat. During each action potential Ca2+ ions flow into the cell and are directly and indirectly involved in generation of pacemaker potentials and of contractile force. Adrenergic and cholinergic neurotransmitters modulate Ca2+ influx. The most detailed analysis has been made on the mechanism of the beta-adrenergic effect on calcium channels in cardiac cell membranes. This is briefly summarized in a personal account, while for more detailed information the reader is referred to more extensive recent reviews.     

Dantrolene and the effect of temperature on the spontaneous release of transmitter at the frog neuromuscular junction

The characteristic effect of temperature on m.e.p.p. frequency at the amphibian neuromuscular junction is unaltered by the presence of Dantrolene (an agent that is believed to reduce the efflux of Ca2+ from intracellular stores) or by changes in [Ca2+)o. It is concluded that temperature affects the release system directly, with a transition temperature at about 16 degrees C.     

Evidence for suppression of Na-dependent Ca2+ efflux from rat brain synaptosomes by ovarian steroids in vivo

The possibility that intracellular Ca2+, which mediates neurotransmitter release, regulation of membrane permeability, microtubule polymerization and axonal transport, is influenced by gonadal steroids via a Na-Ca exchange mechanism was examined. The resting Ca2+ uptake into synaptosomes was measured using crude synaptosomal pellets (P2 fraction), isolated from the brain stem, mesencephalic reticular formation (MRF), nucleus caudatus (NC) and the hippocampus of intact, long-term ovariectomized (OVX) and OVX plus progesterone (P) or estradiol-17 beta benzoate (EB) treated adult female rats. Irrespective of the brain structure investigated, the uptake was 1) markedly increased in synaptosomes from OVX animals in comparison to intact controls, and 2) reduced to near control values in synaptosomes from OVX rats treated s.c. with a single dose of 2 mg P or 5 micrograms EB. Since Ca2+ influx into synaptosomes was shown earlier to depend on external sodium concentration, which was the same in all experiments described in this work, the results obtained indicate that ovarian steroids modulate basal synaptic activity in the rat brain by suppressing Na-dependent Ca2+ efflux from the nerve cell.     

Calcium and sodium distribution and movements in smooth muscle

Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K, Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the major intracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca2+, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise in total cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca2+, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.