Voltage-gated Ca2+ current in pancreatic B-cells

Voltage-dependent inward Ba⁺⁺ and Ca⁺⁺ currents were recorded in cultured neonatal rat pancreatic islet cells using the whole-cell voltage clamp technique. Outward current was suppressed by internal Cs⁺ and ATP and external TEA. Inward currents activated rapidly and decayed to a variable extent. The current decay was particularly marked when using long duration or large depolarizing pulses. Currents were due to Ca⁺⁺ channel activation since they were abolished by omitting Ba⁺⁺ and Ca⁺⁺ or including Co⁺⁺.     

Mechanism of Ca++ Release from the Sarcoplasmic Reticulum: A Computer Model

The proposed model describes myocyte calcium (Ca⁺⁺) cycling, emphasizing the kinetics of sarcoplasmic reticulum (SR) Ca⁺⁺ release channels. The suggested SR channel regulating mechanism includes two types of Ca⁺⁺ binding sites: (1) low affinity sites with high binding rates, regulating the opening of Ca⁺⁺ channels and (2) high affinity sites with low binding rates, which regulate their closing. The amount of Ca⁺⁺ released from the SR and the peak value of Ca⁺⁺ ion concentration [Ca⁺⁺] in the cytoplasm were found to depend on the rate of the increase of [Ca⁺⁺], similar to Ca⁺⁺ induced Ca⁺⁺ release experiments. The model describes spontaneous release of Ca⁺⁺ from overloaded SR. The dependence of the control mechanism on the activating and inactivating sites is substantiated by simulations of ryanodine intervention, providing results similar to experimental results. Simulations under conditions of isolated SR vesicles produced Ca⁺⁺ release results similar to measured data. Consequently, it is suggested that the recovery of Ca⁺⁺ release channels represents the rate limiting factor in the process of mechanical restitution. © 1998 Biomedical Engineering Society. PAC98: 8722Fy, 8710+e     

Adrenaline and the plateau phase of the cardiac action potential

Summary Conduction block in heart cells by K⁺ rich, or Na⁺ depleted solutions can be overcome by adrenaline. In order to explain this phenomenon, the effect of adrenaline on the membrane resting and action potentials of cow Purkinje fibers was measured at various extracellular concentrations of Na⁺, K⁺ and Ca⁺⁺, in the presence of tetrodotoxin, Mn⁺⁺ and beta-receptor antagonists.It was found that adrenaline specifically increases the amplitude and duration of the plateau phase of the cardiac action potential. Plateu-like action potentials, without preceding Na⁺-spike, can be generated and conducted in an all-or-nothing way. In K⁺ rich solutions and under the influence of adrenaline, the depolarization proceeds in two steps. The first step corresponds to the Na⁺-spike. The second step or secondary depolarization corresponds to the plateau; it was not modified by changes of the membrane potential between –85 and –55 mV, or by reduction of extracellular Na⁺ ions, but was specifically blocked by Mn⁺⁺ ions and beta-receptor antagonists. Its amplitude increased by 17 mV for a tenfold change in extracellular Ca⁺⁺. Tetrodotoxin preferentially blocked the Na⁺-spike, but also slowed the rate of potential change during the secondary depolarization.The simplest explanation for the observed phenomena can be found in an increase of Ca⁺⁺ inward current under the influence of adrenaline. The existence of an inward Na⁺⁺ current, different in characteristics from the Na⁺ conductance during the fast upstroke, cannot be ruled out. Some data are in accord with a decrease in K⁺ conductance.     

Synaptic and non-synaptic mechanisms underlying low calcium bursts in the in vitro hippocampal slice

Summary 1. The epileptiform activity generated by lowering extracellular [Ca⁺⁺] was studied in the CA1 subfield of rat hippocampal slices maintained in vitro at 32° C. Extracellular and intracellular recordings were performed with NaCl and KCl filled microelectrodes. 2. Synaptic potentials evoked by stimulation of the stratum radiatum and alveus were blocked upon perfusion with artificial cerebrospinal fluid (ACSF) containing 0.2 mM Ca⁺⁺, 4 mM Mg⁺⁺. Blockade of synaptic potentials was accompanied by the appearance of synchronous field bursts which either occurred spontaneously or could be induced by stimulation of the alveus. 3. Both spontaneous and stimulus-induced low Ca⁺⁺ bursts recorded extracellularly in stratum pyramidale consisted of a negative potential shift with superimposed population spikes. This extracellular event was closely associated with intracellularly recorded action potentials rising from a prolonged depolarization shift. Steady hyperpolarization of the cell membrane potential decreased the amplitude of the depolarizing shift suggesting that synaptic conductance were not involved in the genesis of the low Ca⁺⁺ burst. 4. Spontaneous depolarizing inhibitory potentials recorded in normal ACSF with KCl filled microelectrodes were reduced in size in low Ca⁺⁺ ACSF. However, small amplitude potentials could still be observed at a time when low Ca⁺⁺ bursts were generated by hippocampal CA1 pyramidal neurons. 5. Bicuculline methiodide, an antagonist of -aminobutyric acid (GABA), was capable of modifying the frequency of occurrence and the shape of synchronous field bursts. The effects evoked by bicuculline methiodide were, however, not observed when 81–100% of NaCl was replaced with Na-Methylsulphate. Hence, it was concluded that in low Ca⁺⁺ ACSF even though large release of transmitter such as those following electrical activation of stratum radiatum or alveus cannot be observed, small spontaneous release of the inhibitory transmitter GABA seems to persist. 6. Substitution of NaCl with Na-Methylsulphate also caused changes in the synchronous field bursts which were different from those observed following application of bicuculline methiodide. These findings suggest that in low Ca⁺⁺ ACSF, in addition to residual GABAergic Cl^- mechanisms, non-synaptic Cl^- conductances might play a role in controlling the excitability of hippocampal neurons.Supported by grants from the MRC of Canada (MA-8109) and Sick Children Foundation to MA     

Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat

Summary Extracellular calcium and potassium activities (a_Ca and a_K) as well as neuronal activity were simultaneously recorded with ion-sensitive electrodes in the somatosensory cortex of cats. Baseline a_Ca was 1.2–1.5 mM/1, baseline a _k 2.7–3.2 mM/1. Transient decreases in a_Ca and simultaneous increases in a_K were evoked by repetitive stimulation of the contralateral forepaw, the nucleus ventroposterolateralis thalami and the cortical surface. Considerable decreases in a_Ca (by up to 0.7 mM/1) were found during seizure activity. A fall in a_Ca preceded the onset of paroxysmal discharges and the rise in a_K after injection of pentylene tetrazol. The decrease in a_Ca led also the rise in a_K during cyclical spike driving in a penicillin focus. It is concluded that alterations of Ca⁺⁺ dependent mechanisms participate in the generation of epileptic activity.     

CaV2.1 channels are modulated by muscarinic M1 receptors through phosphoinositide hydrolysis in neostriatal neurons

In adult neostriatal projection neurons, the intracellular Ca²⁺ supplied by Ca_V2.1 (P/Q) Ca²⁺ channels is in charge of both the generation of the afterhyperpolarizing potential (AHP) and the release of GABA from their synaptic terminals, thus being a major target for firing pattern and transmitter release modulations. We have shown that activation of muscarinic M₁-class receptors modulates Ca_V2.1 channels in these neurons in rats. This modulation is reversible, is not membrane delimited, is blocked by the specific M₁-class muscarinic antagonist muscarine toxin 7 (MT-7), and is neither mediated by protein kinase C (PKC) nor by protein phosphatase 2B (PP-2B). Hence, the signaling mechanism of muscarinic Ca_V2.1 channel modulation has remained elusive. The present paper shows that inactivation of phospholipase C (PLC) abolishes this modulation while inhibition of phosphoinositide kinases, PI-3K and PI-4K, prevents its reversibility, suggesting that the reconstitution of muscarinic modulation depends on phosphoinositide rephosphorylation. In support of this hypothesis, the supply of intracellular phosphatidylinositol (4,5) bisphosphate [PI(4,5)P₂] blocked all muscarinic modulation of this channel. The results indicate that muscarinic M₁ modulation of Ca_V2.1 Ca²⁺ channels in these neurons involves phosphoinositide hydrolysis.     

NPPB block of Ca++-activated Cl− currents in Xenopus oocytes

NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), a mentoer of the novel class of Cl^– channel blockers related to diphenylamine-2-carboxylate, was studied for its effect on the Ca⁺⁺-activated Cl^– current (I_Cl(Ca)) in frog (Xenopus laevis) oocytes. I_Cl(Ca) was activated by bath application of 5 mM Ca⁺⁺ to oocytes that had been Ca⁺⁺ permeabilized with the Ca⁺⁺ ionophore A23187. In order to prevent the incativation of I_Cl(Ca)) that occurs with repetitive applications of Ca⁺⁺, oocytes were also treated with H-7 (1,5-isoguinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases. NPPB reversibly blocked both the fast transient (I_fast) and slow (I_slow) components of I_Cl(Ca) with inhibition constants or 22 M and 68 M, respectively. NPPB block of I_Cl(Ca) was voltage dependent and potentiated by depolarization of the oocyte membrane. Our results indicate that NPPB is a potent blocker of the oocyte endogenous I_Cl(Ca) and may prove useful in the study of exogenously expressed Cl channels.     

The weak bases NH3 and trimethylamine inhibit the medium and slow afterhyperpolarizations in rat CA1 pyramidal neurons

The weak bases NH₃ and trimethylamine (TMeA), applied externally, are widely used to investigate the effects of increasing intracellular pH (pH_i) on neuronal function. However, potential effects of the compounds independent from increases in pH_i are not usually considered. In whole-cell patch-clamp recordings from rat CA1 pyramidal neurons, bath application of 1–40 mM NH₄Cl or TMeA HCl reduced resting membrane potential and input resistance, inhibited the medium and slow afterhyperpolarizations (AHPs) and their respective underlying currents, mI_ahp and sI_ahp, and led to the development of depolarizing current-evoked burst firing. Examined in the presence of 1 M TTX and 5 mM TEA with 10 mM Hepes in the recording pipette, NH₃ and TMeA increased pH_i and the magnitudes of depolarization-evoked intracellular [Ca²⁺] transients, Ca²⁺-dependent depolarizing potentials, and inward Ca²⁺ currents but reduced the slow AHP and sI_ahp. When internal H⁺ buffering power was raised by including 100 mM tricine in the patch pipette, the effects of NH₃ and TMeA to increase pH_i and augment Ca²⁺ influx were attenuated whereas the reductions in the slow AHP and sI_ahp (as well as membrane potential and input resistance) were maintained. The findings indicate that increases in pH_i contribute to the increases in Ca²⁺ influx observed in the presence of NH₃ and TMeA but not to the reductions in membrane potential, input resistance or the magnitudes of AHPs. The results have implications for the interpretation of data from experiments in which pH_i is manipulated by the external application of NH₃ or TMeA.     

On the cellular mechanism of the inotropic action of acetylcholine on isolated rabbit and dog atria

Summary The experiments undertaken in an attempt to localize the site of the inotropic action of acetylcholine on cardiac muscle were performed on canine and rabbit isolated left auricles. Simultaneous recordings were made of intracellular action potentials and contractile strength of electrically driven atria perfused with Tyrode solution.Acetylcholine added to the bath at the beginning of a pause in stimulation decreased only slightly the strength of the first post-rest contraction, while the corresponding and subsequent action potentials displayed complete loss of the plateau, associated with a marked reduction of contractile force of the second and subsequent post-rest beats. It is concluded that acetylcholine does neither inhibit intracellular redistribution and release of Ca⁺⁺ ions, nor Ca⁺⁺ reacting with the contractile system, contractile proteins being also not affected.Acetylcholine and Mn⁺⁺ ions have been found to exert strikingly similar and additive effects on the configuration of action potentials. Both those factors inhibited the action potentials that can be elicited in atria exposed to a high potassium concentration and adrenaline, and which are believed to be caused by a calcium current.The findings of our experiments can be explained by a direct action of acetylcholine on the Ca⁺⁺ transmembrane transport, which does not appear to be at variance with the observation that acetylcholine increases the potassium membrane conductance. Indirect influence of acetylcholine through an effect of this increased potassium conductance on Ca⁺⁺ inward current is discussed.     

Topography of the respiratory and circulatory responses to acetylcholine and nicotine on the ventral surface of the medulla oblongata

The effects of different [Ca²⁺]₀ in the presence of variable [Na⁺]₀ (100 to 37 mM) on the slow action potentials were studied in isolated rat atria in the presence of 25 mM K⁺ plus 10^–6 M isoproterenol. Two modes of electrical stimulation were used, sustained stimulation at 0.5 Hz (steady-state mode), and stimulation by a single stimulus after rest periods of 2 to 5 min (1st response mode). With the first type of stimulation, and for [Ca²⁺]₀ between 0.5 and 10 mM and [Na⁺]₀ between 100 and 65 mM, the slow action potential overshoot increased linearly with the logarithm of the [Ca²⁺]₀ (28.4 mV per 10-fold change in [Ca²⁺]₀). However, elevation of [Ca²⁺]₀ above 10 mM caused depression of the overshoot. This overshoot depression by high [Ca²⁺]₀ was accentuated if [Na⁺]₀ was decreased to 37 mM. With the 1st response mode of stimulation, the overshoot — log [Ca²⁺]₀ relationship was linear within a wider [Ca²⁺]₀ range (0.5 to 25 mM), and was less sensitive to further decreases in [Na⁺]₀. It is suggested that rat atria slow action potentials are generated by selective influx of Ca²⁺ but not Na⁺, and that the depression of amplitudes observed a high [Ca²⁺]₀ and low [Na⁺]₀ is due to a decrease in the Na²⁺ exchange mechanism which results in a higher [Ca²⁺]_i, and not to a decrease in the inward Na⁺ current. Adenosine produced a parallel downward displacement of the overshoot to log [Ca²⁺]₀ relationship. This adenosine effect was concentration dependent, independent of [Ca²⁺]₀ and the frequency of stimulation. In contrast, the effects of 0.4 mM La³⁺ were dependent on the [Ca²⁺]₀ and on the frequency of stimulation. Adenosine also produced a downward shift of the relationship between maximal rate of rise of the slow action potential and membrane resting potential in such a manner that its effects cannot be attributed to changes in inactivation potential of the slow channels. Hence, adenosine and La³⁺ depress the slow Ca²⁺ action potentials by two different mechanisms. Adenosine may act by 1) decreasing the number of functional slow channels, 2) decreasing the conductance of the individual channels, or 3) altering the kinetic properties of these channels. La³⁺ may act by competing with Ca²⁺ for membrane binding sites. These membrane binding sites appear to be characterized by frequency dependence.Supported by Grants NHLBI 10384 and AHA 74-942.Dr. Luiz Belardinelli is the recipient of a fellowship No. 1112. 1273/75 from Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq) and Fundacao Universitaria de Cardiologia do Rio Grande do Sul, Brasil.     

Alpha-Adrenozeptoren am Myokard: Vorkommen und funktionelle Bedeutung

Summary Alpha-adrenoceptors mediating positive inotropic effects are well established in the heart of various species including human heart. The mechanism by which alpha-adrenoceptor stimulation increases force of contraction is not known. cAMP is unlikely to be involved as a mediator. Evidence has been presented that an increase in magnitude and duration of the slow Ca⁺⁺ inward current may be partly responsible for the positive inotropic efffect. In addition, stimulation of alpha-adrenoceptors may increase Ca⁺⁺ sensitivity of the contractile proteins. Stimulation of alpha-adrenoceptors by endogenous catecholamines may serve as a reserve mechanism under various conditions of impaired beta-adrenergic influence, e.g. hypothyroidism, bradycardia or ischemia. Furthermore, alpha-adrenoceptors may be involved in the genesis of reperfusion arrhythmias in ischemic heart.

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Inhibitory effect of angiotensin on renal sodium reabsorption

Zusammenfassung Am isolierten Frosch-Sartorius wurde die isometrische Spannungsentwicklung und die Konzentration an Phosphorylkreatin (PKr) in isotonischer KCl-Lösung in Funktion der Zeit gemessen. Dabei sollte die Ursache für das spontane Einsetzen der Erschlaffung und der Regeneration der Bestände an PKr während der Dauerdepolarisation aufgeklärt werden.Vorbehandlung des Muskels in Ringer-Lösung mit 4,5 mM Ca⁺⁺ (statt normal 1,8 mM) unterdrückte die schnelle Phase der KCl-Kontraktur und führte zur Verlängerung der Kontrakturdauer. Der Wiederanstieg der Konzentration an PKr vollzog sich dabei langsamer als in KCl-Lösung mit normalem Ca⁺⁺-Gehalt.Kühlung des Muskels von 20° C auf 0° C bewirkte eine auffällige Erschlaffungs-verzögerung in isotonischer KCl-Lösung bei gleichzeitiger Hemmung der Regeneration der Bestände an PKr.In Gegenwart bekannter in vitro-Inhibitoren der Ca⁺⁺-Aufnahme in die Vesikel des sarkoplasmatischen Reticulums (wie SCN^–, Thymol und Coffein) kam es zu einer Verstärkung der KCl-Kontraktur hinsichtlich Höhe und Dauer sowie zur Ausbildung eindeutiger Verkürzungsrückstände. Die Regeneration der Bestände an PKr verlief dabei stark verzögert und war bei gleichzeitiger Anwesenheit von SCN^– und Thymol vollständig blockiert.Die Untersuchungen zeigen, daß bei funktioneller Ausschaltung der Oberflächenmembran durch K⁺-Depolarisation das Einsetzen der Erschlaffung und der Regeneration der Bestände an PKr abhängt von der Ca⁺⁺-Rückbindung in die Vesikel des sarkoplasmatischen Reticulums. Der Prozeß der Erschlaffung würde demnach kontrolliert durch die Funktion intracellulärer Membranstrukturen und nicht durch den elektrischen Zustand der Oberflächenmembran.Die Frage der Funktion einzelner Membranabschnitte sowie die Frage der morphologischen Komponenten des intracellulären Ca⁺⁺-Turnovers werden im Hinblick auf die Kompartmentierung der Muskelzelle diskutiert.Wesentliche Teile der vorliegenden Arbeit wurden von Fräulein Ch. Petzoldt der Medizinischen Fakultät Freiburg i. Br. als Dissertation vorgelegt.     

Eye-head coupling in humans

Summary One component of the dynamic response of muscle spindles is characterized by a phase lead and frequency dependent sensitivity in response to sinusoidal stretches at frequencies around 1 Hz. Possible mechanisms producing this component, designated the mid-frequency dynamics, were investigated by testing the hypotheses that they arise from the mechanical behavior of the intrafusal muscle and alternatively from within the sensory terminals. Destruction of the myofibrillar structure of the intrafusal muscle fibers did not alter the mid-frequency dynamics, indicating that they do not arise from viscoelastic properties of the intrafusal muscle. An Arrhenius plot of the temperature dependence of the mid-frequency dynamics yielded an equivalent activation energy of 6.5 Kcal/M in the temperature range 23–42° C and a 3-fold higher activation energy at lower temperatures. These observations are consistent with a dynamic process associated with a membrane-bound bio-chemical process. The addition of Ca⁺⁺ and Ca⁺⁺-activated-K⁺ (K(Ca)) channel blockers (ZnCl₂, Apamin and TEA) to the bathing solution altered the response dynamics by reducing the mid-frequency phase lead. The results suggest a negative feedback on the membrane potential generated by K⁺⁺ efflux following a Ca⁺⁺ influx that opens K(Ca) channels. A quantitative model fit to the experimental data yields a time constant of about 80 ms representing the limiting process associated with activation of the K(Ca) channels in this system. The results indicate that the mechanism underlying the mid-frequency dynamics includes at least two processes: one, not identified in this study, generates the phase lead and another, involving Ca⁺⁺ and K(Ca) channels, provides a negative feedback that modifies the phase lead.     

An early outward conductance modulates the firing latency and frequency of neostriatal neurons of the rat brain

Summary An in vitro slice preparation was used to obtain intracellular recordings of neostriatal neurons. Indirect evidence for the presence of an early outward conductance in neostriatal neurons is presented. With near threshold stimulation neostriatal neurons fired very late during the pulse. The long firing latency was associated with a slow (ramp-like) depolarization. In the presence of TTX the slow depolarization was lost and outward-going rectification dominated the subthreshold response. This finding demonstrated that both, outward and inwardgoing conductances play a role during the ramp-like depolarization. Outward-going rectification during depolarizing responses could be further augmented if the depolarizing stimulus was preceded by a conditioning hyperpolarization. A conditioning hyperpolarization prolonged the firing latency and slowed the firing frequency. A conditioning depolarization had opposite effects. After TTX treatment, the response showed a hyperpolarizing sag when depolarizing stimulation was preceded by conditioning hyperpolarization. 4-AP (0.5–2.5 mM) blocked the effects of the conditioning hyperpolarization on the firing latency and on the voltage trajectory. 4-AP also disclosed a slow depolarization which could produce neuronal firing very early during the pulse. This depolarization was TTX-sensitive and Co⁺⁺-insensitive. In contrast to 4-AP, TEA (20 mM) did not produce a reduction in the firing latency but disclosed a membrane oscillatory behavior most probably produced by the interplay of these opposing conductances: the slow inward (probably Na⁺) and the transient outward (probably K⁺). Repetitive firing during 4-AP treatment was of the phasictonic type with an initial burst riding on the initial Co⁺⁺-insensitive slow depolarization and a somehow irregular train of spikes during the remainder of the stimulation. Action potentials during 4-AP treatment were followed by an afterdepolarization which dominated the initial part of the interspike interval.     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

The apamin-sensitive potassium current in frog skeletal muscle: its dependence on the extracellular calcium and sensitivity to calcium channel blockers

Slow outward potassium currents were recorded in isolated frog skeletal muscle fibres using the double mannitol-gap voltage-clamp technique.Detubulated fibres failed to generate a slow outward current, and apamin had no effect on the remaining current.The maximum blocking effect of organic and inorganic Ca²⁺-channel blockers on the slow outward channels of intact fibres was larger than that of apamin. Apamin failed to induce an additional block when applied after Ca²⁺-channel blockers.In a low-Ca²⁺ solution (OCa, EGTA 1 mM) the slow outward current was slightly increased and the blocking effect of apamin was enhanced. A Ca²⁺-rich solution (Ca²⁺×10) increased the slow outward current and the blocking effect of apamin was drastically reduced.It is concluded that the apamin-sensitive current which is a component of the slow outward K⁺ current is located in the tubular membrane. Its activation seems barely dependent on the Ca²⁺ influx via the slow inward Ca²⁺ current. Apamin-receptor binding appears to be dependent on the extracellular Ca²⁺ concentration. Blockade of slow outward current by Ca²⁺-channel blockers is likely to be the result of a direct action on the slow K⁺ permeability rather than a consequence of Ca²⁺ channel inhibition.     

Intracellular calcium ions as regulators of renal tubular sodium transport

Summary This review addresses the putative role of intracellular calcium ions in the regulation of sodium transport by renal tubules. Cytoplasmic calcium-ion activities in proximal tubules of Necturus are less than 10^–7 M and can be increased by lowering the electrochemical potential gradient for sodium ions across the peritubular cell membrane, or by addition of quinidine or ionomycin to peritubular fluid. Whereas lowering of the peritubular Na concentration increases cytosolic [Ca⁺⁺] and [H⁺], ionomycin, a calcium ionophore, raises intracellular [Ca⁺⁺] without decreasing pH_i. The intracellular calcium-ion level is maintained by transport processes in the plasma membrane and membranes of intracellular organelles, as well as by calcium-binding proteins. Calcium ions inhibit net transport of sodium by reducing the rate of sodium entry across the luminal cell membrane. In the collecting tubule this inhibition is caused, at least in part, by an indirect reduction in the activity of the amiloride-sensitive sodium channel.Supported by NIH grant PHS-AM-11489 and a New York Heart Association Established Fellowship (G.F.). J.M. Yang's works was in partial fullfillment of his Ph.D. thesis requirements; he is supported by a fellowship from the Chung Shan Institute of Science and Technology, Taiwan     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

The stimulus-secretion coupling of glucose-induced insulin release

The short-term influence of low concentrations of extracellular K⁺ upon insulin release was investigated in the isolated perfused rat pancreas. In the absence of glucose, the removal of extracellular K⁺ provoked a slow and transient stimulation of insulin release. This phenomenon was enhanced and prolonged at either low (2.8 mM) or high (16.7 mM) concentrations of glucose, and was abolished in the absence of extracellular Ca²⁺. Removal of extracellular K⁺ also accelerated and augmented the initial phase of glucose-induced insulin release. The late phase of glucose-induced insulin release was rapidly augmented by removal of extracellular K⁺, but inhibited in response to a partial decrease in extracellular K⁺ concentration. The release of insulin evoked by glucose in the absence of K⁺ differed from that seen in the presence of K⁺ by a progressive decline in secretory rate during prolonged stimulation, and by a lesser sensitivity towards a shortage in extracellular Ca²⁺. During constant exposure to glucose, the restoration of a normal K⁺ concentration provoked a short-lived offresponse, followed by a 8 min-period of inhibited insulin release prior to normalization of the secretory activity. These data indicate that K⁺ deprivation, apart from its long-term untoward effects upon islet function, causes a transient stimulation or facilitation of insulin release, possibly by mobilizing Ca²⁺ from intracellular stores.