The effect of substances releasing intracellular calcium ions on sodium-dependent calcium efflux from guinea-pig auricles.

1. 45-Ca efflux and resting tension were measured in isolated guinea-pig auricles under conditions known to change the intracellular free Ca ion concentration. 2. In the presence of [Na]o, caffeine (2mM) increases 45-Ca efflux, but does not produce a contracture, while in the absence of [Na]o and [Ca]o caffeine causes a contracture without increasing 45-Ca efflux. Adrenaline (10-minus5-10-minus 4M) with or without theophylline (0-5-1-0mM) has no effect on either 45-Ca efflux or resting tension. 3. In the presence of caffeine the rate of net efflux of Ca depends on [Na]o-2. Caffeine contractures of muscles in Na-free solution relax upon the addition of [Na]o. Relaxation is correlated with the increase in net efflux of Ca. 4. Cyanide (2mM) produces a variable increase in 45-Ca efflux without a concomitant contracture in Na-containing solutions, but in Na, Ca-free solutions a large contracture occurs without significant increase in 45-Ca efflux. 5. A large increase in 45-Ca efflux and a contracture were observed with the 'Ca-ionophore' X 537 A. 6. Changes in membrane potential (K-depolarization) in hypertonic solutions have no significant effect on Na-dependent 45-Ca efflux, which is an agreement with an electroneutral 2:1 Na-Ca exchange. 7. Cyanide and X 537 A both cause a considerable release of Ca ions from isolated guinea-pig heart mitochondria, while caffeine has no effect. 8. The results suggest a powerful role of the Na-Ca exchange system in reducing the intracellular Ca concentration after Ca release from intracellular stores.     

Multifunctional triblock copolymers for intracellular messenger RNA delivery

Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86-216?nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies.     

Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast

The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP₃) in yeast is rapidly transformed into IP₄ and IP₅ by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP₅ into IP₆. In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP₃ accumulation upon glucose addition was completely absent in the plc1 strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP₂ and generation of IP₃, seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP₃ signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.Communicated by S. Hohmann     

Evidence for active chloride transport in dogfish rectal gland.

To evaluate the characteristics of the transport process for Na and C1 by the rectal gland of the dogfish shark, experiments were performed with an in vitro perfused model. Changes in glandular secretion rate and net electrical potential difference (PD) were measured during independent alterations of the Na and C1 concentrations of the perfusate solution. The rate of net secretion was enhanced by increasing the concentration of either Na or Cl in the perfusate, demonstrating a substratedependent process. When tetramethyl-ammonium was substituted for Na, the rate of secretion fell in association with a rise in net PD from -8 mV to -17 mV (lumen negative). When SO4 was substituted for Cl, secretion rate also fell, but in association with a fall in net PD. Despite almost complete replacement of Cl with SO4, net PD was not reversed. Although the mechanism responsible for Na secretion remains unclear, these data suggest that chloride is transported by an active process in the dogfish rectal gland.     

Fall in intracellular pH mediated by GABAA receptors in cultured rat astrocytes.

The influence of muscimol (a specific gamma-aminobutyric acid-A (GABAA) receptor agonist) on intracellular pH (pHi) was studied in cultured rat astrocytes by means of fluorescence spectrophotometry with BCECF as the H+ indicator. In an HCO3(-)-free medium, muscimol had little effect on pHi. In a solution containing 22 mM HCO3-, muscimol produced a reversible, concentration-dependent fall in pHi with a maximum of about 0.1-0.15 units. The muscimol-induced fall in pHi was antagonized by an increase in the external K+ concentration, which suggest that the acidosis is an immediate consequence of a net efflux of HCO3- through GABAA receptor channels rather than an indirect effect caused by a change in membrane potential. The present results raise the possibility that astrocytes may participate in the regulation of extracellular pH at GABAergic synapses and contribute to activity-induced pH changes in nervous tissue.     

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pH_i), and HCO ₃ ⁻ -dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pH_i was 6.96 in both nominally CO₂/HCO ₃ ⁻ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ₃ ⁻ (6.96±0.18;n=48) (at pH 7.4). Inhibition of the Na⁺/H⁺ exchange by amiloride (2 mM) caused a significant decrease of pH_i in nominally CO₂/HCO ₃ ⁻ -free saline. Addition of CO₂/HCO ₃ ⁻ in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na⁺ also caused a fall of pH_i, and addition of CO₂/HCO ₃ ⁻ in Na⁺-free saline evoked a further fall of pH_i, while the outward current was reduced or even reversed. The stilbene 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.3 mM) reduced the pH_i recovery from the CO₂/HCO ₃ ⁻ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ₃ ⁻ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ₃ ⁻ -containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO₂/HCO ₃ ⁻ . The results suggest that the CO₂/HCO ₃ ⁻ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ₃ ⁻ cotransporter, which helps to regulate pH_i in these cells. In addition, a prominent Na⁺/H⁺ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steadystate pH_i.     

The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones

1. Intracellular pH (pH(i)), Cl(-) and Na(+) levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH(i) recovery from internal acidification investigated.2. Reducing the external HCO(3) (-) concentration greatly inhibited the rate of pH(i) recovery from HCl injection.3. Reducing external Cl(-) did not inhibit pH(i) recovery, but reducing internal Cl(-), by exposing the cell to sulphate Ringer, inhibited pH(i) recovery from CO(2) application.4. During pH(i) recovery from CO(2) application the internal Cl(-) concentration decreased. The measured fall in internal Cl(-) concentration averaged about 25% of the calculated increase in internal HCO(3) (-).5. Removal of external Na inhibited the pH(i) recovery from either CO(2) application or HCl injection.6. During the pH(i) recovery from acidification there was an increase in the internal Na(+) concentration ([Na(+)](i)). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na(+)](i) that occurred during pH(i) recovery from an injection of HCl was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH(i) recovery after HCl injection were not additive.9. It is concluded that the pH(i) regulating system involves tightly linked Cl(-)-HCO(3) (-) and Na(+)-H(+) exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO(3) (-) and the movement outward of Cl(-) and H(+) ions.     

Evidence that hepatocytes can phagocytize exogenous substances.

Although the phagocytic action of Kupffer cells is well known, such a phenomenon has not been well documented for hepatocytes. Following the injection of a suspension of egg lecithin-coated silicon particles (0.5-1.5 microns in diameter) into the portal vein of rats, Kupffer cells showed minimal phagocytotic action, which was in contrast to the hepatocytes which displayed numerous phagocytized silicon particles. By comparison, when noncoated silicon particles of the same diameter as those that were coated were injected into the portal vein, the opposite observation was made. There was no uniformity in the ability of the hepatocytes to phagocytize either coated or noncoated particles from one lobule to another. Some showed active phagocytosis, while in others no evidence of such a process was observed. These data provide strong evidence for the selective phagocytic action of liver hepatocytes.     

Can magnesium act as a second messenger? Current data on translocation induced by various biologically active substances.

Free intracellular Mg2+([Mg2+]i) can potentially integrate the signals from hormones, cellular metabolism and organismal ion homeostasis and affect the activities of ion channel and other effectors. Interest in [Mg2+]i has been heightened by recent reports that small changes in [Mg2+]i in the physiological range can significantly modulate important cellular functions. In addition, a variety of new evidence shows that [Mg2+]i instantaneously changes following stimulation with various biologically active substances. These observations suggest that [Mg2+]i may act as a second messenger.     

Anthracycline-associated cardiac and renal damage in rabbits. Evidence for mediation by vasoactive substances

We tested the hypothesis that anthracycline-induced cardiac and renal damage is mediated by vasoactive substances. A 1-minute exposure to 5 micrograms per ml. of doxorubicin (DXR, Adriamycin) produced cardiac histamine release in isolated rabbit hearts. Under conditions in which histamine uptake and metabolism were impaired, the administration of DXR, 2 mg. per kg., over 1 minute was associated with elevations in arterial histamine and catecholamines. The chronic weekly administration of DXR produced severe cardiac and renal damage. The administration of combined histaminic and adrenergic blockade with diphenhydramine, cimetidine, phentolamine, and propranolol (DCPP) pre- and immediately post-DXR resulted in near total protection against DXR-mediated cardiac damage and prevented the majority of the renal lesions. The combined administration of diphenhydramine, cimetidine, phentolamine, and propranolol did not appear to be acting by mechanisms other than blockade of vasoactive amine receptors as cardiac uptake of DXR and the DXR antitumor response were not altered by diphenhydramine, cimetidine, phentolamine, and propranolol. This study demonstrates that anthracycline-associated cardiac and renal toxicity may be mediated by vasoactive substances and that anthracycline cardiomyopathy is potentially preventable.     

A new double-barrelled,ionophore-based microelectrode for chloride ions

A new Cl^– selective microelectrode based on the ionophore 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride is presented which discriminates better against HCO ₃ ^– and several organic anions than electrodes containing the Corning 477913 ion-eschanger. Using a redesigned construction procedure, fine-tip double-barrelled microelectrodes were produced which had slopes of –52.4±0.6 mV (SE,n=24), resistances of about 7·10¹¹ and a selectivity coefficient logK _{Cl, HCO3} ^pot of –1.40±0.03. Some electrodes showed a small unexplained sensitivity to pH>7.6. When used to puncture cells of isolated S3 segments of rabbit renal proximal tubule during perfusion with HCO ₃ ^– Ringer solution, the electrodes gave a membrane potential of –69.8±1.5 mV and an intracellular Cl^– activity, [Cl^–]_i, of 35.3±2.6 mmol/l. Upon switching bath and lumen perfusions to Cl^–-free solutions the residual [Cl^–]_i dropped to 1.20±0.03 mmol/l, while in similar measurements with ion-exchanger electrodes the residual [Cl^–]_i dropped only to 10.9±0.5 mmol/l. These observations demonstrate the superiority of the new electrode and prove that previously determined high [Cl^–]_i values in Cl^–-free ambient solutions reflect interference problems rather than non-exchangeable intracellular chloride.     

Manganese ions as intracellular contrast agents: proton relaxation and calcium interactions in rat myocardium

Paramagnetic manganese (Mn) ions (Mn(2+)) are taken up into cardiomyocytes where they are retained for hours. Mn content and relaxation parameters, T(1) and T(2), were measured in right plus left ventricular myocardium excised from isolated perfused rat hearts. In the experiments 5 min wash-in of MnCl(2) were followed by 15 min wash-out to remove extracellular (ec) Mn(2+) MnCl(2), 25 and 100 micro M, elevated tissue Mn content to six and 12 times the level of control (0 micro M MnCl(2)). Variations in perfusate calcium (Ca(2+)) during wash-in of MnCl(2) and experiments including nifedipine showed that myocardial slow Ca(2+) channels are the main pathway for Mn(2+) uptake and that Mn(2+) acts as a pure Ca(2+) competitor and a preferred substrate for slow Ca(2+) channel entry. Inversion recovery analysis at 20 MHz revealed two components for longitudinal relaxation: a short T(1 - 1) and a longer T(1 - 2). Approximate values for control and Mn-treated hearts were in the range 600-125 ms for T(1 - 1) and 2200-750 ms for T(1 - 2). The population fractions were about 59 and 41% for the short and the long component, respectively. The intracellular (ic) R(1 - 1) and R(2 - 1) correlated best with tissue Mn content. Applying two-site exchange analyses on the obtained T(1) data yielded results in parallel to, but also differing from, results reported with an ec contrast agent. The calculated lifetime of ic water (tau(ic)) of about 10 s is compatible with a slow water exchange in the present excised cardiac tissue. The longitudinal relaxivity of Mn ions in ic water [60 (s mM)(-1)] was about one order of magnitude higher than that of MnCl(2) in water in vitro [6.9 (s mM)(-1)], indicating that ic Mn-protein binding is an important potentiating factor in relaxation enhancement.     

Evidence for intracellular histamine liberation in isolated rat mast cells

The beta-adrenoceptor blocking drug exaprolol liberated histamine from isolated rat mast cells in a dose- and time-dependent way. Histamine was liberated within seconds and was not followed by a parallel granule liberation. The inhibition of histamine liberation was induced with low temperature, low pH, high concentration of Ca²⁺, TTD, suramin and EDTA. Subcellular distribution of³H-exaprolol demonstrated a quantitative relationship between histamine depletion against exaprolol uptake in isolated rat mast cell granules. A nonspecific mechanism of action in the effect of exaprolol on mast cells is discussed. It is proposed that the drug acts on mast cells due to the direct and indirect ion exchange mechanism resulted in disproportion between histamine and granule liberation.     

Influence of changes in external potassium and chloride ions on membrane potential and intracellular potassium ion activity in rabbit ventricular muscle

1. The membrane responses of rabbit papillary muscles to rapid changes in [K](o) and [Cl](o) were measured with open-tipped micropipettes and with closed micropipettes made from K-selective glass.2. The muscle cells behaved primarily as a K electrode, and responses to changes in [K](o) with constant [Cl](o) or with constant [K](o) x [Cl](o) were substantially the same.3. When [Cl](o) was changed at a constant [K](o) the membrane potentials changed rapidly and symmetrically by a small value and remained constant for 30 min.4. Measurement of potential with K(+)-selective micro-electrodes in these experiments showed no change in intracellular K activity. In addition to permitting calculation of K permeability, these measurements reassured us that the K(+)-selective electrodes were well insulated and not influenced by electrical shunts at the impalement site.5. Although the membrane response to changes in [Cl](o) was small, it was possible to calculate that the permeability ratio (P(Cl)/P(K)), was 0.11. The Cl and K conductances were about 0.015 mmho/cm(2) and 0.09 mmho/cm(2) respectively, resulting in a conductance ratio (g(Cl)/g(K)) of about 0.17.6. The time course of depolarization by increase in [K](o) was rapid (half-time 5 sec), but repolarization on return to lower [K](o) was much slower (half-time 50 sec). The depolarization time course was easily fitted by the potential change calculated by assuming the need for K diffusion into the extracellular spaces and taking account of the logarithmic relation between membrane potential and [K](o). These calculations did not fit the time course of repolarization, which was slowed in the fashion expected from an inward-rectifying membrane.7. The influence of [K](i) on membrane potential was investigated by changes in tonicity of the external solution. Hypotonic solution produced a change in intracellular K activity close to that produced by ideal water movement. However, in hypertonic solution, intracellular K activity did not rise as much as predicted, suggesting a change in intracellular activity coefficient.     

Chelation of intracellular zinc ions affects human sperm cell motility.

The effects of two different zinc chelators, diethyldithiocarbamate (DEDTC) and calcium ethylenediaminetetraacetic acid (EDTA), in full semen samples and 'swim-up' samples were investigated. DEDTC, which crosses cell membranes, and EDTA, which does not cross cell membranes, were added to semen samples in different concentrations. Sperm cell motility parameters were assessed by computer-assisted semen analysis (CASA). It was found that very small concentrations (0.01 mM) of DEDTC immobilized the sperm cells within 80 min, while EDTA had no depressing effect at the concentrations used. In full semen samples EDTA enhanced straight line velocity (VSL) at concentrations of 1.0 and 0.5 mM; this effect was not found at higher concentrations. It is suggested that intracellular mitochondrial zinc ions play a crucial role for sperm cell motility, while loosely bound or free zinc ions in the seminal plasma exert a secondary role on human sperm cell motility.     

Sodium- and bicarbonate-independent regulation of intracellular pH in culture mouse astrocytes

The intracellular pH (pHi) of cultured mouse astrocytes was measured with double-barrelled pH-sensitive microelectrodes. In bicarbonate-buffered saline pHi was 7.05 and in HEPES-buffered saline 6.68. In both solutions H+ was not in electrochemical equilibrium; pHi was 0.7-1 pH unit more alkaline than expected from passive H+ distribution. Cells were acidified by applying NH4+ and the subsequent regulation of pHi was studied in bicarbonate-free saline. The mean rate of pHi recovery was 0.2 pH units min-1 which was not changed by amiloride or by removal of external Na+. Thus, the cells recovered from an acid load independently of Na(+)-H+ exchange, Na(+)-HCO3- cotransport or any other bicarbonate- or Na(+)-dependent mechanism.     

Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle.

1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a(i) (Cl)) of frog sartorius fibres at 25 degrees C.2. The internal pH (pH(i)) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).3. In normal bicarbonate-free solution (containing 2.5 mM potassium), the average chloride equilibrium potential, E(Cl) (calculated from a(i) (Cl) and the measured chloride activity of the external solution (a(o) (Cl)) was 87.7 +/- 1.7 mV (mean +/- S.E.; n = 16) in fibres where the average membrane potential, E(m), was 88.3 +/- 1.5 mV (mean +/- S.E.; n = 16). In experiments where a(i) (Cl) was varied between about 1 and 10 mM (which corresponds to values of E(m) between about -105 and -50 mV) E(Cl) was within 1-3 mV of E(m) at equilibrium. These measurements of a(i) (Cl) were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a(i) (Cl) below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.4. In solutions containing 15 mM bicarbonate and gassed with 5% CO(2), pH(i) was 6.9, corresponding to an internal bicarbonate concentration of 7.6 mM. E(Cl) measured in this solution was some 4 mV positive to E(m). Most of the difference between E(Cl) and E(m) could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a(Cl).5. If bicarbonate/CO(2) in the external solution was replaced by HEPES/pure O(2) at constant pH, then pH(i) rose from 6.88 +/- 0.02 (mean +/- S.E.) to 7.05 +/- 0.02. A change in external pH of 1 unit caused pH(i) to change by about 0.02 unit and the intracellular buffering power was calculated to be about 35.6. In solution made hypertonic by the addition of sucrose, E(m) changed little or depolarized and E(Cl) and E(m) remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E(Cl) became negative to E(m). Conversely in low chloride solution E(Cl) became positive to E(m).7. When the chloride permeability (P(Cl)) was reduced by the use of acid solution, E(Cl) moved positive to E(m) indicating an accumulation of internal chloride. When P(Cl) was increased again by returning to more alkaline solution, E(m) depolarized to E(Cl).8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P(Cl) is large.     

Evidence that maternal ventral skin substances promote suckling in infant rats.

Washing a lactating rat's ventral skin with acetone, alcohol and water markedly reduces the effectiveness with which her 2-week-old infants can initiate suckling on the anesthetized mother. Control experiments with detailed thermal and behavioral measures give evidence tending to rule out lowered skin temperature, altered thermal gradients, changed tactile characteristics of abdominal fur or nipples and aversion to remaining traces of the organic solvents as responsible for the infants' failure to find and attach to nipples. The data suggest that some substance(s) on the skin of the mother's ventrum act as olfactory and/or gustatory cues for the infants' orientation and attachment to nipples.     

Evidence for apical chloride channels in rabbit mandibular salivary glands

Double-barrelled, chloride-selective microelectrodes were used to study mandibular gland acinar cells at rest and during cholinergic stimulation. At rest, intracellular chloride activity was five times the expected equilibrium activity. During sustained stimulation with acetylcholine, chloride activity fell to three times the expected equilibrium activity. Thus, the gradient for chloride exit was reduced in the stimulated cell. These results lead to the conclusion that stimulation increases the permeability of the acinar cell to chloride. Experiments in which extracellular chloride was removed provided evidence that the permeability increase was due to opening of chloride channels located principally in the apical membrane of the acinar cell.