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.     

Cardiac calcium currents at the level of single channels

Properties of cardiac Ca channels have come into sharper focus with the advent of single cell preparations and suction pipette recording methods. We briefly summarize our present picture of the gating and permeation properties of the conventional, dihydropyridine-sensitive type of Ca channel (L-type). Distinctive features of a second type of voltage-gated Ca channel (T-type) are discussed.     

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.     

Patch clamp technique and biophysical study of membrane channels

Summary The present work describes the patch clamp technique, which first allowed the recording of single channel currents in biological membranes. In particular, it describes procedures for preparation and applications of the four different patch clamp configurations. Briefly, the cell-attached configuration is widely used for investigating channel modulation by transmitters acting via second messengers. The cell-free configurations (inside-out and outside-out), complementary to one another with respect to the orientation of the membrane surface, are particularly indicated for the study of the biophysics (kinetics, conductivity, selectivity, mechanism of permeation and block) of ionic channels. Finally, the whole-cell configuration which, because of the remarkable feature that it allows voltage clamp of very small cells, has given access to a number of physiologically important preparations never studied before.     

Patch clamp technique and biophysical study of membrane channels

The present work describes the patch clamp technique, which first allowed the recording of single channel currents in biological membranes. In particular, it describes procedures for preparation and applications of the four different patch clamp configurations. Briefly, the cell-attached configuration is widely used for investigating channel modulation by transmitters acting via second messengers. The cell-free configurations (inside-out and outside-out), complementary to one another with respect to the orientation of the membrane surface, are particularly indicated for the study of the biophysics (kinetics, conductivity, selectivity, mechanism of permeation and block) of ionic channels. Finally, the whole-cell configuration which, because of the remarkable feature that it allows voltage clamp of very small cells, has given access to a number of physiologically important preparations never studied before.     

Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells

Dispersal of the constituent cells of mammalian visceral and vascular smooth muscles has permitted recordings both of membrane currents under whole-cell voltage clamp, and of currents through single ionic channels using the patch-clamp technique. A rectangular depolarizing step applied to a single cell under voltage clamp yielded a net inward current followed by a net outward current in normal physiological solution. In isolated, 'inside-out' patches of cell membrane a calcium- and potential-sensitive K channel (100 pS conductance) and a calcium-insensitive, potential-sensitive K+ channel (50 pS conductance) with slow kinetics have so far been identified and characterized.     

Preparation of isolated single cardiac cells from adult frog atrial tissue

Summary Isolated cardiac cells from bullforg atrial tissue can be readily prepared by digestion of intact fragments of atrial tissue with trypsin and collagenase. These isolated cells have dimensions of about 5 m in width and range in length from 300 m to over 500 m. Such isolated cells may prove useful for the investigation of contractile activity of cardiac muscle at the single cell level and at the sarcomere level within the single cell.This investigation was supported by U.S. Public Health Service, National Institutes of Health Grant No. HL 12426 and a Kansas Heart Association Grant-in-Aid.We wish to thank Dr.J. Sommers of Duke University Medical Center for doing electron microscopy of the isolated cells to confirm the single cell nature of the preparations.     

Single cell biology beyond the era of antibodies: relevance,challenges, and promises in biomedical research

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.     

Effects of hyperpolarizing currents on the action potentials of nodal and perinodal sino-atrial cells of the rabbit heart]

A technique of focal polarization allowed us to confirm that the action potentials of the true rabbit heart pacemaker cells possess a rapid sodium channel normally inactivated because of the low value of the take-off potential of this tissue. The latent pacemaker cells are regular sinocaval cells depolarized as a consequence of the proximity of true pacemaker cells.     

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.     

Chemically mediated cell-to-cell contractile activation in isolated frog atrial cardiac cells

Summary A dying single frog atrial cardiac cell liberates an unknown substance which diffuses away from the dying cell and activates contractile activity in other isolated intact single cardiac cells within the vicinity of the dying cell.This investigation was supported by US Public Health Service, National Institutes of Health Grant HL 18943 and a Kansas Heart Association Grant-In-Aid.We wish to thank Dr Alan Chapman of the Department of Anatomy for doing the scanning electron microscopy of the cardiac cells.     

Ureteral pacemaker potentials recorded with the sucrose gap technique.

Ureteral contractions occur at intervals which are integral multiples of the period of pacemaker potentials recorded in vitro from the renal pelvis with a sucrose gap, suggesting that a gating mechanism in the pyeloureter regulates the rate at which the pacemaker initiates contractions.     

Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells

Summary Dispersal of the constituent cells of mammalian visceral and vascular smooth muscles has permitted recordings both of membrane currents under whole-cell voltage clamp, and of currents through single ionic channels using the patch-clamp technique. A rectangular depolarizing step applied to a single cell under voltage clamp yielded a net inward current followed by a net outward current in normal physiological solution. In isolated, inside-out patches of cell membrane a calcium- and potential-sensitive K channel (100 pS conductance) and a calcium-insensitive, potential-sensitive K⁺ channel (50 pS conductance) with slow kinetics have so far been identified and characterized.     

HCN channels: Structure, cellular regulation and physiological function

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. HCN channels are unique among vertebrate voltage-gated ion channels, in that they have a reverse voltage-dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN channels are encoded by four genes (HCN1–4) and are widely expressed throughout the heart and the central nervous system. The current flowing through HCN channels, designated I_h or I_f, plays a key role in the control of cardiac and neuronal rhythmicity (“pacemaker current”). In addition, I_h contributes to several other neuronal processes, including determination of resting membrane potential, dendritic integration and synaptic transmission. In this review we give an overview on structure, function and regulation of HCN channels. Particular emphasis will be laid on the complex roles of these channels for neuronal function and cardiac rhythmicity. Received 22 August 2008; received after revision 22 September 2008; accepted 24 September 2008     

Identification of a pre-hibernating state in myocardium from nonhibernating chipmunks

Summary During the hibermating season, the amplitude of the cardiac action potential plateau of nonhibernating chipmunks was reduced. Replacing external Ca by Sr inhibited the electromechanical responses of these preparations. Similar properties were observed in hibernating animal preparations, suggesting that changes in cardiac function are already triggered before hibernation begins.15 December 1986     

Phospholipid composition of cardiac (Na+ + K+)-ATPases from various species

There is a difference in phospholipid composition of cardiac (Na+ + K+)-ATPase preparations between species which are sensitive to ouabain and those which are not. Sphingomyelin is higher and phosphatidylcholine is lower in the enzymes from sensitive species than in those from insensitive ones. Lysophosphatidylcholine is detectable only in the latter preparations.     

Identification of a pre-hibernating state in myocardium from nonhibernating chipmunks

During the hibernating season, the amplitude of the cardiac action potential plateau of nonhibernating chipmunks was reduced. Replacing external Ca by Sr inhibited the electromechanical responses of these preparations. Similar properties were observed in hibernating animal preparations, suggesting that changes in cardiac function are already triggered before hibernation begins.     

Preparation of isolated single cardiac cells from adult frog atrial tissue.

Isolated cardiac cells from bullfrog atrial tissue can be readily prepared by digestion of intact fragments of atrial tissue with trypsin and collagenase. These isolated cells have dimensions of about 5 mum in width and range in length from 300 mum to over 500 mum. Such isolated cells may prove useful for the investigation of contractile activity of cardiac muscle at the single cell level and at the sarcomere level within the single cell.     

Branchial vascularization in the eel: action of acetylcholine and adrenaline on the distribution of polymerizable resin in the different vascular compartments]

The current findings from gill vascular cast preparations in the eel emphasize the division in each primary lamella of the afferent vasculature into two efferent pathways: an arterial pathway (via the secondary lamellae and the efferent branchial artery to the dorsal aorta), a venous pathway (via the central lamellar compartment and the branchial vein to the sinus venosus). By the same technique two antagonist mechanisms have been shown presumably controlling the blood flow in both pathways. 1. Acetylcholine increases the filling of the central lamellar compartment by constricting the efferent arterial sphincters and therefore increases the venous return. 2. Epinephrine impairs the filling of the central lamellar compartment (by acting on alpha receptors) and dilates the arterial pathway (by acting on beta receptors). Therefore the stimulation of these two synergic receptors by epinephrine increases the systemic blood flow.     

Impulse propagation from the SA-node to the ventricles

Normally the pacemaker of the mammalian heart is located in the sinus node. In the rabbit the sinus node can be subdivided into two regions, the center of the node where the impulse originates and the border zone through which the impulse is conducted towards the atrium. Conduction properties of both regions were investigated. It appeared that conduction velocity increases and refractoriness decreases when one goes from the nodal center towards the atrium. The tissue mass of the atrium is large in comparison to the sinus node and normally the resting membrane potential of atrial fibers is more negative than that of nodal fibers; consequently, a potential difference exists causing a current flow between both areas. Evidently this hyperpolarizing current flow depresses impulse formation in the border zone fibers which have better intrinsic pacemaker properties than fibers in the nodal center. If the impulse has reached the atrium it is conducted with a relatively high safety factor and will reach the AV node in principle without difficulty. The AV node, if deprived of sinus nodal dominance, develops spontaneous activity originating from the lower nodal fibers. Also in this structure, electrotonic depression by surrounding tissue causes deceleration of the pacemaker.