X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis.

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.     

Ionophore A23187 can mimick the changes in membrane permeability that occur during acetylcholine-stimulation of pancreatic acinar secretion

Acetylcholine (ACh) released from vagal terminals increases the permeability of the pancreatic acinar membrane to Na⁺ and Ca²⁺ ions. In this report, we compare the induced changes in intracellular Na⁺ and Ca²⁺ electrode potentials (E_Na and E_Ca) due to ACh-stimulation of acini with those observed during stimulation with the calcium ionophore, A23187, which mimicks the action of ACh on pancreatic secretion. Stimulation with ACh concentrations varying from 10^–8 to 10^–5 M and with A23187 concentrations of 10^–6 and 10^–5 M caused parallel increases in cytosolic Ca²⁺ and Na⁺ ([Ca]_i, [Na]_i). The magnitude of the increases in [Ca]_i and [Na]_i due to A23187-stimulation further indicate that when presented with a calcium challenge the acinar cells continue to regulate [Ca]_i close to physiological levels and suggest that the observed increases in ionized calcium could reflect much larger increases in complexed Ca²⁺. ACh-stimulation following removal of either extracellular Na⁺ or Ca²⁺ ions, eliminated the intracellular increases found when the removed ions is present, but did not affect the increases usually found with the other ion. The independence of the permeability changes to either the presence of Ca²⁺ or Na⁺ indicates the ACh-induced currents carried by Na⁺ and Ca²⁺ are also independent. The selective translocation of Na⁺ and Ca²⁺ during acetylcholine-stimulation in a manner analogous to the changes observed when ionophore A23187 was used as stimulus, indicates the ability of the activated acinar membrane to function as an ionophore.     

Stimulation-induced calcium signalling and ion transport in rat pancreatic acini

In the present study we have characterized receptor-mediated Ca²⁺ signalling patterns as well as Ca²⁺-mediated ion transport mechanisms in collagenase isolated rat pancreatic acini. Measurements of the initial Ca²⁺ response to maximal carbachol stimulation revealed a rapid increase in [Ca²⁺]_i, which, in general, occurred synchronously throughout the cells. Less frequently, not all cells in the acinus responded to carbachol, but did respond to subsequent stimulation with bombesin, indicating that not all cells possess receptors for all the applied agonists. In view of the heterogeneity in the agonist-evoked Ca²⁺ responses, ionomycin was used to assess the role of Ca²⁺ in activating K⁺, Na⁺ and Cl^- transport mechanisms. Ionomycin induced a rise in [Ca²⁺]_i, thereby increasing Cl^- permeability as well as stimulating K⁺ efflux, probably through non-specific cation channels. However, the resting K⁺ efflux was insensitive to blockers of non-specific cation channels, indicating the existence of a selective resting K⁺ conductance. Ionomycin also stimulated influx of Na⁺, which in part was mediated by non-specific cation channels. The changes in ion fluxes measured in the present study revealed that when [Ca²⁺]_i is raised in rat pancreatic acini, they gain Na⁺ and Cl^- and lose K⁺, with non-specific cation channels being essential for this process.     

Effect of contraction on lymphatic,venous, and tissue electrolytes and metabolites in rabbit skeletal muscle

Summary The effect of muscle contraction on lymphatic and plasma [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], [P_i], [lactate] ([Lac^–]); [creatine] ([Cr]), ideal osmolality (OSM), and [protein] was evaluated in femoral venous blood and lymph specimens sampled from the calf muscles of rabbits before, in the course of, and after contractions. In addition, total [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], and [H₂O] were analyzed in the muscle tissue. To facilitate lymph sampling both hind limbs were passively flexed and extended, in imitation of natural running movements, by an electrically driven crank. The muscles of one side also performed superimposed rhythmic isotonic contractions. Before contractions, lymphatic [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Lac^–], [Cr], and OSM did not significantly differ from corresponding femoral venous concentrations, [Cl^–], and [P_i] were significantly higher, [protein] significantly lower in the lymph than in the plasma. During contractions lymphatic [K⁺], OSM, [Lac^–], and [P_i] were raised significantly more in the lymph compared with the plasma concentrations. [Na⁺], [Cl^–], [Ca²⁺], and [Mg²⁺] showed only small changes in the course of contractions and thereafter, and they were altered in a similar way in the lymph and plasma. It was suggested that lymphatic and interstitial concentrations were in equilibrium. Comparing inactive with active muscles, the latter lost K⁺ but gained Na⁺, Cl^–, and H₂O, whereas minimal changes occurred in the [Ca²⁺] and [Mg²⁺]. The changes were discussed in connection with the hypothesis that electrolyte shifts might be involved in the activation of the muscular non-proprioceptive interstitial nerve endings which appear to play a role in reflexogenic cardiovascular and respiratory control.A preliminary report of this work has been given elsewhere [33]Supported by Deutsche Forschungsgemeinschaft     

Calcium entry is regulated by Zn in relation to extracellular ionic environment in human airway epithelial cells

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). This elevation of [Ca²⁺]_i and activation of Ca²⁺-dependent Cl⁻ channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca²⁺]_i in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na⁺ and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca²⁺ signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca²⁺ entry was either inhibited or stimulated by Zn²⁺ depending on the extracellular Na⁺ concentration. In Na⁺-free environment, Zn²⁺ and other divalent cations elicited a biphasic Ca²⁺ signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca²⁺ entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.     

Ca2+ modulates an unspecific cation conductance in olfactory cilia of Xenopus laevis

Summary Olfactory neurones of Xenopus laevis were studied by the patch clamp technique under voltage-clamp conditions. Isolated receptor cells were obtained by dissociating the olfactory mucosa in a Ca²⁺-free solution. Usually some of the resulting isolated olfactory cells lost all of their cilia during the dissociation procedure. Comparing the currents of cells with cilia to those of cells without cilia, a marked difference was found. When all known voltage-gated currents except the Ca²⁺-current were blocked, cells without cilia showed the voltage-gated Ca²⁺-current alone whereas cells with cilia clearly had an additional conductance g_c. It could be activated in two ways, either by Ca²⁺ entry through Ca²⁺-channels or by Ca²⁺ entry through the Na/Ca-exchanger working in the reversed mode at positive membrane potentials. This ciliar conductance g_c had its reversal potential at 0 mV. Replacing extracellular Cl^- by isethionate on the one hand, and Na⁺ by Cs⁺ or N-methyl-D-glucamine on the other showed that g_c was permeable for cations but not for Cl^-. In conclusion, there appears to be a Ca²⁺-dependent unselective cation conductance on the cilia of olfactory neurones. The probable role of g_c as the last step in an IP₃/Ca mediated transduction path-way is suggested.     

Caffeine stimulates the reverse mode of Na+/Ca2+ exchanger in ferret ventricular muscle

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K‐free medium that blocked the Na⁺–K⁺ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na‐free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca²⁺]_o. Divalent cations Ni²⁺ and Cd²⁺, which have an inhibitory effect on the Na⁺/Ca²⁺ exchanger, produced dose‐dependent inhibition of caffeine responses with apparent Ki of 780 ± 19 and 132 ± 5 μM , respectively. Caffeine also caused dose‐dependent inhibition of Na‐free contractures (Ki=4.62 ± 1.5 mM ), and the reduction or removal of [Na⁺]_o exerted an inhibitory effect on caffeine contractures (Ki=73.5 ± 17.12 mM ). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na⁺/Ca²⁺ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange,the Na+-K+-2Cl− cotransport and cellular volume

To explore further the mechanisms that regulate the Na⁺/H⁺ antiport in human platelets, we examined the effect of Na⁺ pump inhibition by ouabain and K⁺ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca²⁺ and Na⁺, coupled with an alkaline shift in the cytosolic pH set point for the Na⁺/ H⁺ antiport. Inhibition of the Na⁺ pump by the removal of K⁺ from the medium increased the cytosolic Na⁺ but not the cytosolic Ca²⁺; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na⁺/H⁺ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na⁺-K⁺-2Cl^– cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na⁺ pump and the Na⁺/H⁺ antiport, mediated by the Na⁺/Ca²⁺ exchange and the cytosolic free Ca²⁺, and (b) a link between the Na⁺/H⁺ antiport and the Na⁺-K⁺-2Cl^– cotransport through cellular volume.This work was supported by grants from the National Heart, Lung, and Blood Institute (HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoral research fellow of the American Heart Association, New Jersey Affiliate     

Effect of a Transient Period of Ischemia on Myocardial Cells: I. Effects on Cell Volume Regulation

The effect of temporary periods of ischemia on the electrolytes and water of myocardial cells were studied in groups of mongrel dogs. Myocardial tissue exposed to 40 minutes of ischemia induced by occlusion of the circumflex branch of the left coronary artery developed no changes in water or electrolytes when compared to nonischemic left ventricle of the same or sham-operated animals, even though this period of ischemia is known to produce irreversible injury to many of the damaged cells. However, reperfusion of the affected myocardium with arterial blood for only 2 minutes resulted in striking increases in tissue H₂O, Na^-, Cl^- and Ca^2-. These changes in electrolytes increased in severity with longer periods of reflow, and tissue K⁺ was decreased significantly after 10 minutes of reflow had passed. Analysis of the results suggested that the tissue edema was primarily the result of cellular swelling. Myocardium exposed to 15 minutes of ischemia followed by 2 minutes of reflow showed no significant changes aside from a slight increase in Na⁺. These studies demonstrate that defects in cell volume regulation occur early in severe ischemic injury.     

Effects of blockers of Ca2+ channels and other ion channels on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate

The inhibitory effects of various ion channel blockers were examined on in vitro excystment of Paragonimus ohirai metacercariae induced by a bile salt, sodium cholate. At a concentration of 10 µM, bepridil, a non-selective Ca²⁺ channel blocker, completely inhibited in vitro excystment, whereas TEA, lidocaine, and R(+)-IAA-94, channel blockers against K⁺, Na⁺ and Cl^– ions, respectively, benzamil, an Na⁺/H⁺ and Na⁺/Ca²⁺ ion exchanger blocker, and R(+)-DIOA, a [K⁺, Cl^–] cotransporter inhibitor, did not. Considering the previous result that Ca²⁺ ionophores are also efficient inducing factors for in vitro excystment of P. ohirai metacercariae and the present result, bile salts appear to induce the excystment of P. ohirai metacercariae through evoking the Ca²⁺ channels of target cells within the metacercarial juveniles.     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Intracellular ion activities and equilibrium potentials in motoneurones and glia cells of the frog spinal cord

Intra- and extracellular ion activities were measured with ion sensitive microelectrodes in motoneurones and glia cells of the spinal cord of the frog. These data were corrected for cross sensitivities of the ion exchangers to intracellular interfering ions, and equilibrium potentials for K⁺, Na⁺, Ca²⁺ and Cl^? (E _K,E _Na,E _Ca andE _Cl) were calculated. In motoneurones with membrane potentials exceeding ?60mV the following mean equilibrium potentials were determined. $$\begin{gathered} E_{Na} = + 29.4mV, E_K = - 87.9mV, E_{Ca} = + 52.6mV, \hfill \\ E_{Cl} = - 34.1mV \hfill \\ \end{gathered}$$ . The corresponding values for glia cells were: $$\begin{gathered} E_{Na} = + 40.5mV, E_K = - 84.0mV, E_{Ca} = + 35.7mV, \hfill \\ E_{Cl} = - 59.7mV \hfill \\\end{gathered}$$ . The intracellular ionic milieu is probably disturbed by the impalement of the cells. This transiently decreases the intracellular K⁺ and increases intracellular Na⁺. These effects were estimated and their origin is discussed. The results of the experiments suggest a non-passive transmembrane distribution of K⁺, Na⁺ and Ca²⁺ in motoneurones and glia cells, a non-passive transmembrane distribution of Cl^? in motoneurones, and a passive transmembrane distribution of Cl^? in glia cells.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Muscarinic-receptor activation stimulates oscillations in K+ and Cl− currents which are acutely dependent on extracellular Ca2+ in avian salt gland cells

By utilizing the perforated-patch variant of the whole-cell patch-clamp recording technique, in order to maintain the integrity of the normal cellular buffering systems, we demonstrate that carbachol (CCh) stimulates simultaneous oscillations in a Ca²⁺- and voltageactivated K⁺ current and a linear Ca²⁺-activated Cl^– current in an exocrine avian salt gland cell preparation. Similar conductance changes, although sustained rather than oscillatory, are stimulated by the Ca²⁺ ionophore A23187. The outward K⁺ current can be inhibited by tetraethylammonium chloride (TEA) whereas the Cl^– current is inhibited by the Cl^– channel blockers 5-nitro-2-(3-phenylpropylamino) (NPPB) and N-phenylanthranilic acid (DPC). The oscillations in current stimulated by CCh are acutely dependent on extracellular Ca²⁺ and are not affected by the application of low doses of caffeine. In addition, the application of caffeine at all doses fails to mimic the current transients stimulated by CCh. As both caffeine and A23187 are unable to stimulate oscillations under the perforated-patch conditions we suggest that in avian salt gland cells the primary oscillatory mechanism probably involves a one-pool mechanism of Ca²⁺ release which is intimately related to the activation of a Ca²⁺ influx pathway.     

Daily changes in foetal urine and relationships with amniotic and allantoic fluid and maternal plasma during the last two months of pregnancy in conscious, unstressed ewes with chronically implanted catheters

1. The fluid sacs and bladders of ten foetuses and the allantoic sacs of five foetuses were catheterized between 79 and 96 days gestational age and daily samples were withdrawn until lambs were born naturally at ~147 days. Maternal jugular plasma obtained daily allowed the nutritional status of each ewe to be regulated and monitored. All lambs were observed for 7 weeks, and at post-mortem no abnormalities were seen in those operated upon in utero.

2. The osmolality, [Na⁺], [K⁺], [Cl^-], [glucose], [fructose], [urea], [amino acid] and pH of all samples were measured.

3. Foetal surgery seemed to affect the actual concentrations of some solutes, but gestational trends in foetal fluid composition were unaltered.

4. Until about 7 days before birth the foetal urine osmolality, [Na⁺], [Cl^-] and [fructose] decreased, its [urea], [amino acid] and pH remained relatively constant, and from about 120 days gestational age the [K⁺] increased. During the last 7 days there was a marked increase in the osmolality and the concentrations of all these solutes, and a decrease in pH.

5. Entry of foetal urine into the fluid sacs tended to decrease the osmolality, [Na⁺], [K⁺], [Cl^-] and [glucose] of both foetal fluids and the [amino acid] of allantoic fluid, and tended to increase the [fructose] and [urea] of both fluids and the [amino acid] of amniotic fluid.

6. Changes in urine composition suggested large daily variations in the secretion of foetal antidiuretic hormone and also a rapid increase in its secretion during the last 7 days, and particularly the last 2-4 days before birth.

7. Changes in the [Na⁺]/[K⁺] ratios of foetal urine and allantoic fluid were parallel during post-operative recovery, during the course of pregnancy and immediately before birth, and this was consistent with a simultaneous action of foetal plasma corticosteroids on the foetal kidneys and chorioallantois.

8. Variations in the [fructose] of foetal urine and allantoic fluid were parallel to changes in their [Na⁺]/[K⁺] ratios and suggested an involvement of foetal corticosteroids in the regulation of the [fructose] of foetal plasma.

9. Further evidence has been presented supporting the hypothesis that maternal induced foetal hypoglycaemia effects a relative increase in the secretion of foetal corticosteroids having an action on the chorioallantois. Also, high concentrations of maternal plasma corticosteroids may decrease the permeability of the placenta to glucose.

     

Active transport of ions by the gastric mucosa of the rabbit foetus

1. The short-circuit current and absolute fluxes of Na⁺ and Cl^- across the gastric mucosa of the 28-day rabbit foetus have been measured in vitro.

2. Substitution of Na⁺ in the solution bathing the mucosal surface by choline ion or K⁺ resulted in a 70% decrease in short-circuit current which was reversed when Na⁺ was restored to the mucosal solution. The portion of the short-circuit current dependent on the presence of Na⁺ in the mucosal solution was found to be equivalent to the net flux of Na⁺ from mucosa to serosa.

3. The net flux of Cl^- from serosa to mucosa was compared with the short-circuit current persisting when Na⁺ had been replaced in the mucosal solution. Averaged results from sixteen experiments indicated that the net flux of Cl^- was equivalent to 166% of the Na⁺ independent short-circuit current.

4. The results indicated that the component of short-circuit current associated with acid secretion was independent of the presence of Na⁺ in the mucosal solution.

5. The small scale of the experiments and the secretion of mucus by the preparation did not permit successful simultaneous measurement of H⁺ secretion and short-circuit current.

6. Replacement of Cl^- by SO₄^2- or glucuronate in the solutions on both sides did not result in a reversal or decrease in magnitude of the Na⁺ independent short-circuit current, even after allowing time for the tissue to become depleted of Cl^-. It is suggested that a non-specific active anion transport was occurring.

     

Biphasic rises in cytosolic free Ca2+ in association with activation of K+ and Cl− conductance during the regulatory volume decrease in cultured human epithelial cells

During exposure to a hypotonic solution (55% osmolarity), cultured human epithelial (Intestine 407) cells exhibit a regulatory volume decrease after osmotic swelling. This process is known to involve parallel activation of volume-regulatory K⁺ and Cl^– conductances. Biphasic increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) were observed by microspectrofluorometry, in fura-2-loaded cells upon hypotonic stress. Electrophysiological studies with Ca²⁺-selective and conventional microelectrodes indicated that a biphasic [Ca²⁺]_i increase was associated with a biphasic hyperpolarization, whereas an interposing [Ca²⁺]_i decrease coincided with a transient depolarization. A Ca²⁺ ionophore, ionomycin, produced a sustained Ca²⁺ increase and a prolonged hyperpolarization which was sensitive to the K⁺ channel blocker, quinine. A subsequent hypotonic challenge gave rise to a depolarization, which was sensitive to a stilbene-derivative Cl^– channel blocker, without inducing further changes in [Ca²⁺]_i. Normal cell volume regulation in a hypo-osmotic medium could take place even in the presence of ionomycin. It is concluded that a biphasic [Ca²⁺]_i increase is closely associated with activation of the volume-regulatory K⁺ conductance, and that the interposing [Ca²⁺]_i decrease is neither a causative factor for activation of the volume-regulatory Cl^– conductance nor a prerequisite for regulatory volume decrease in epithelial cells exposed to a hypotonic solution.     

Calcium uptake and release modulated by counter-ion conductances in the sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum (SR) plays the central role in regulating the free myoplasmic Ca²⁺ level for the contractile activation of skeletal muscle. The initial stages of the voltage-controlled Ca²⁺ release mechanism are known in molecular detail. However, there is still very little known about the later stages of Ca²⁺ uptake and total Ca²⁺ turnover in the contraction–relaxation cycle under normal physiological conditions or under conditions influenced by fatigue or disease. Ca²⁺ uptake and release are both accompanied by ‘counter-ion’ movements across the SR membrane which prevent or reduce the generation of SR membrane potentials and balance for electroneutrality in the SR lumen. The SR membrane is permeable for the cations K⁺, Na⁺, H⁺ and Mg²⁺ and the anion Cl^-. Using electron-probe X-ray microanalysis, it has been shown that during tetanic stimulation the Ca²⁺ release was mainly balanced by uptake of K⁺ and Mg²⁺, leaving a charge deficit that was assumed to be neutralized via H⁺ ion or organic counter-ion movement. The low time resolution of electron-probe X-ray microanalysis leaves the possibility of other transient concentration changes in the SR, e.g. for Cl^- ions. Possible physiological roles of the SR counter-ion conductances can be tested using skinned muscle fibre preparations with intact sarcoplasmic reticulum and removed or chemically permeabilized outer sarcolemma. In skinned fibres, the SR K⁺ conductance can be effectively reduced with SR K⁺ channel blockers such as 4-aminopyridine, tetraethylammonium and decamethonium. Interestingly, these blockers increase Ca²⁺ loading as well as Ca²⁺ release, whereas other less specific blockers, such as 1.10-bis-quanidino-n-decane, seem to reduce Ca²⁺ release, possibly also via blocking Ca²⁺ release channels. Thus, it seems very important also to test the effects of counter-currents carried by K⁺, Mg²⁺, H⁺ or Cl^- ions on intact and voltage-clamped single-fibre preparations.     

Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl^– channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^– from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na⁺-K⁺-2Cl^– cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^– cotransporters.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.