INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

N,N-dimethyl-D-erythro-sphingosine increases intracellular Ca2+ concentration via Na+-Ca2+-exchanger in HCT116 human colon cancer cells

N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca²⁺ increase concentration ([Ca²⁺]_i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca²⁺]_i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca²⁺]_i in colon cancer cells is composed of Ca²⁺ release from intracellular Ca²⁺ stores and subsequent Ca²⁺ influx. The Ca²⁺ release is not related to modulation of inositol 1,4,5-trisphosphate (IP₃) receptor or ryanodine receptor. On the other hand, the Ca²⁺ influx is mediated largely through Ca²⁺ channels sensitive to verapamil, nifedipine, Ga³⁺, and La³⁺. Furthermore, we found that the response is inhibited by bepridil and Ni²⁺, specific inhibitors of Na⁺-Ca²⁺-exchanger, suggesting involvement of Na⁺-Ca²⁺ exchanger in the DMS-induced [Ca²⁺]_i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes.     

小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

STORE-OPERATED Ca2+ CHANNELS AND MICRODOMAINS OF Ca2+ IN LIVER CELLS

• 1 Oscillatory increases in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) play essential roles in the hormonal regulation of liver cells. Increases in [Ca²⁺]_cyt require Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ entry across the plasma membrane.
• 2 Store‐operated Ca²⁺ channels (SOCs), activated by a decrease in Ca²⁺ in the ER lumen, are responsible for maintaining adequate ER Ca²⁺. Experiments using patch‐clamp recording and the fluorescent Ca²⁺ reporter fura‐2 indicate there is only one type of SOC in rat liver cells. These SOCs have a high selectivity for Ca²⁺ and properties essentially indistinguishable from those of Ca²⁺ release‐activated Ca²⁺ (CRAC) channels.
• 3 Although Orai1, a CRAC channel pore protein, and stromal interaction molecule 1 (STIM1), a CRAC channel Ca²⁺ sensor, are components of liver cell SOCs, the mechanism of activation of SOCs, and in particular the role of subregions of the ER, are not well understood.
• 4 Recent experiments have used the transient receptor potential vanilloid 1 (TRPV1) non‐selective cation channel, ectopically expressed in liver cells, and a choleretic bile acid to deplete Ca²⁺ from different ER subregions. The results of these studies have provided evidence that only a small component of the ER is required for STIM1 redistribution and the activation of SOCs.
• 5 It is concluded that different Ca²⁺ microdomains in the ER and cytoplasmic space are important in both the activation of SOCs and in the signalling actions of Ca²⁺ in liver cells. Future experiments will investigate the nature of these microdomains further.

     

Cellular Ca2+ Dynamics in Urinary Bladder Smooth Muscle From Transgenic Mice Overexpressing Na+-Ca2+ Exchanger

The rise of Ca²⁺ concentration ([Ca²⁺] _i ) by reducing external Na⁺ in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na⁺/Ca²⁺ exchanger type-1.3 (NCX1.3^tg/tg) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3^tg/tg. The Ca²⁺ release by caffeine in UBSMCs was comparable between NCX1.3^tg/tg and WT, but [Ca²⁺]_i decay was faster in NCX1.3^tg/tg. Contractions induced by acetylcholine, 60 mM K⁺, or electrical stimulation were significantly smaller in UB segments of NCX1.3^tg/tg. NCX worked in Ca²⁺-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3^tg/tg.     

Differential requirements for Ca2+ concentrations for catecholamine release and biosynthesis in isolated bovine adrenal chromaffin cells

Differential effects of [Ca²⁺] on catecholamine release and biosynthesis in isolated bovine adrenal chromaffin cells were investigated. Carbamylcholine, an agonist of the nicotinic and muscarinic acetylcholine receptor, or Na⁺ deprivation in the incubation medium, stimulated catecholamine release and biosynthesis in these cells. The concentrations of extracellular [Ca²⁺] which stimulate catecholamine biosynthesis were less than those which stimulate catecholamine release. An increase in intracellular levels of free Ca²⁺ ([Ca²⁺]_i) induced by Na⁺ deprivation was dependent on extracellular [Ca²⁺]. These results indicate that, in bovine adrenal chromafn cells, catecholamine biosynthesis is regulated by lower levels of [Ca²⁺]_i than is catecholamine release.     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

     

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein

We examined the nature of the palytoxin (PTX)-induced channel and its relevance to the Ca²⁺ mobilizing effect of the toxin on smooth muscle cells isolated from rabbit portal vein using whole-cell voltage-clamp and microfluorimetric techniques. PTX (1 nM) induced a sustained, irreversible inward current at a holding potential of –40 mV. The PTX-induced current reversed at 0.5 ± 0.6 mV, and the PTX-induced channel permitted the passage of Na⁺, K⁺, Cs⁺ and, to a lesser extent, Li⁺, but not choline⁺ or Ca²⁺. During the sustained phase of the current, superfusion of Ni²⁺ (5 mM), La³⁺ (0.5 mM) or 2,4-dichlorobenzamil (2,4-DCB, 25 μM) reduced the current amplitude and decreased the slope conductance without changing the reversal potential. In 5 of 7 experiments, ouabain transiently increased the PTX-induced inward current and shifted the reversal potential in a positive direction. Subsequently, ouabain inhibited the current in every cell. PTX (10 nM) induced a sustained rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was resistant to verapamil but suppressed by omission of extracellular Ca²⁺. When external Na⁺ was replaced by choline⁺, PTX did not increase [Ca²⁺]_i. Pretreatment with 2,4-DCB prevented the elevation of [Ca²⁺]_i due to PTX. These results suggest that PTX does not directly stimulate Ca²⁺ entry but induces entry through Na⁺-Ca²⁺ exchange as a consequence of increased cytosolic Na⁺. Ni²⁺, La³⁺, 2,4-DCB and ouabain were shown to act as blockers of the PTX-induced channel. Ouabain may also inhibit Na⁺ pump current activated by cytosolic Na⁺. Received: 15 May 1996 / Accepted: 28 August 1996     

G02 AND Gi3 PROTEINS MEDIATE THE ACTION OF SOMATOSTATIN ON MEMBRANE Ca2+ AND K+ CURRENTS IN OVINE PITUITARY SOMATOTROPHS

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is regulated by the influx of Ca²⁺ via membrane Ca²⁺ channels. Somatostatin reduces the influx of Ca²⁺ through two separate mechanisms, namely a direct action on Ca²⁺ channels and an indirect action on membrane potentials through the activation of K⁺ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca²⁺ or K⁺ currents. 4. A significant reduction in Ca²⁺ currents and an increase in K⁺ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca²⁺ and K⁺ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-λ-s (200 μmol/L) abolished the recovery phase of K⁺ current response to SRIF after its removal, whereas GDP-β-s (200 μmol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca²⁺ current response to SRIF. 6. Intracellular dialysis of antibodies to αo, α_{1_3}, ai_1-2 and ai₃summits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-ai_1-3 or anti-@aLi₃ antibodies significantly attenuated the increase in the K⁺ current in response to 10 nmol/L SRIF, whereas neither anti-αo nor anti-αi_2 antibodies diminished the effect of SRIF on the K⁺ current. 8. Dialysis of anti-αo antibodies significantly attenuated the reduction in the Ca²⁺ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-αi-2 nor anti-αi3 antibody dialysis diminished the effect of SRIF on the Ca²⁺ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi₃ AS significantly diminished the inhibitory effect of SRIF on the Ca²⁺ current. This effect of ao ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for αo₁ (αo₁ AS) or αo₂(α0₂ AS) were dialysed into somatotrophs and only αo₂ AS significantly attenuated the inhibition of SRIF on the Ca²⁺ current. 10. It is concluded that the G_i3 protein mediates the effect of SRIF on the K⁺ current and that the G02 protein mediates the effect of SRIF on the Ca²⁺ current in primary cultured ovine somatotrophs.     

Increase in force of contraction by activation of the Na+'ox/Ca2+'ox-exchangerin human myocardium

Aims The aim of the present study was to investigate whether agents which enhance force of contraction via increasing intracellular Na⁺, i.e. cAMP-independently, remain effective in failing human myocardium.Methods Cumulative concentration-response curves with (±)BDF 9148 (0.01–10 μmol l⁻¹ ), a Na⁺-channel activator, and ouabain (0.01–0.1 μmol l⁻¹ ), a Na⁺/K⁺-ATPase inhibitor, were performed on electrically driven left ventricular human papillary muscle strips (1 Hz, 37° C; dilative cardiomyopathy, NYHA IV, heart transplantation, n=16; nonfailing, donor hearts, n=5). The β-adrenoceptor agonist isoprenaline (0.001–1 μmol l⁻¹ ) and Ca²⁺ (1.8–15 mmol l⁻¹ ) were studied for control. In addition, Ca²⁺ response curves were obtained on skinned fibre preparations from left ventricular myocardium (NYHA IV, n=7) in the presence of BDF 9148 (1 μmol l⁻¹ ) or a high Na⁺ concentration (50 mmol l⁻¹ ) to investigate a possible direct or indirect interaction of (±)BDF 9148 with the myofilaments.Results While isoprenaline was significantly less effective in increasing force of contraction in failing human myocardium than in nonfailing myocardium (P<0.01), in NYHA IV, (±)BDF 9148 and ouabain were as effective as in nonfailing human tissue. In failing and nonfailing myocardium, (±)BDF 9148 and ouabain exerted positive inotropic effects similar to those of Ca²⁺. However, the potency for (±)BDF 9148 to increase force of contraction was higher in NYHA IV than in nonfailing human myocardium (P<0.05). Neither (±)BDF 9148 (1 μmol l⁻¹ ) nor an increased concentration of Na⁺ (50 mmol l⁻¹ ) altered the Ca²⁺ sensitivity or maximal developed tension of the contractile apparatus in experiments on chemically skinned left ventricular fibres.Conclusions The enhanced sensitivity of the failing human myocardium towards Na⁺-channel modulation is not due to a direct or indirect interaction of (±)BDF 9148 with cardiac myofilaments but may be due to an altered Na⁺-homeostasis in human heart failure.     

DYSREGULATION OF Ca2+ MOVEMENT IN PLATELETS FROM PATIENTS WITH ACUTE ISCHAEMIC STROKE

• 1 Platelets play a pivotal role during acute ischaemic stroke. An increase in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) triggers intracellular signal transduction, leading to platelet aggregation and thrombosis. In the present study, we examined the differences between platelets from acute ischaemic stroke patients and at‐risk controls in terms of the increase in platelet [Ca²⁺]_i.
• 2 Thirty‐one patients with acute ischaemic stroke and 27 at‐risk controls were enrolled in the present study. Platelet [Ca²⁺]_i was measured using the fluorescent dye fura‐2 after stimulation with 100 µmol/L arachidonic acid (AA), 10 µmol/L ADP, 1 µmol/L platelet‐activation factor (PAF) and 0.1 U/mL thrombin.
• 3 Basal [Ca²⁺]_i was higher in the stroke group compared with at‐risk controls, irrespective of the presence or absence of extracellular Ca²⁺. In Ca²⁺‐containing medium, both PAF and ADP, but not AA and thrombin, significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. However, in Ca²⁺‐free medium, only PAF significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. Basal [Ca²⁺]_i and PAF‐induced platelet [Ca²⁺]_i increases were still higher in the stroke group at the subacute stage than in the at‐risk controls.
• 4 The results of the present study provide direct evidence that Ca²⁺ signalling in platelets from acute ischaemic stroke patients was altered in response to particular stimuli. The dysregulation of Ca²⁺ movement in platelets may persist up to the subacute stage of ischaemic stroke.

     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

BACKGROUND AND PURPOSE

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.

EXPERIMENTAL APPROACH

Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

KEY RESULTS

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na⁺-K⁺-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²⁺-dependent) basolateral K⁺ channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²⁺]_i, an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²⁺. Initial falls in [Ca²⁺]_i were not mediated by a sequestration of Ca²⁺ in intracellular Ca²⁺ storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²⁺]_i were inhibited by 2′,4′-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺-exchanger, and attenuated in Na⁺-free buffer, suggesting a transient stimulation of Ca²⁺ outflow by this transporter, directly demonstrated by Mn²⁺ quenching experiments.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive and Ca²⁺-dependent basolateral K⁺ conductances, the basolateral Na⁺-K⁺-pump as well as Ca²⁺ transporters were involved in the action of H₂S in regulating colonic ion secretion.     

Sodium-Calcium exchange: A possible target for drug development

The Na⁺-Ca²⁺ exchange system is a carrier-mediated transport process which couples the transmembrane movement of Ca²⁺ ions to the movement of Na⁺ ions in the opposite direction. It functions primarily as a Ca²⁺ extrusion process in cardiac cells and is thought to be an important mechanism for altering myocardial contractility through changes in intracellular [Na⁺]. We khave investigated the properties of the cardiac Na⁺-Ca²⁺ exchange system using a subcellular preparation of membrane vesicles derived from the cardiac sarcolemma. Vesicle studies have been useful in investigating the kinetics of Na⁺-Ca²⁺ exchange activity and in establishin the stoichiometry of the exchange process as 3 Na⁺ per Ca²⁺. The kinetec results are most easily interpreted in terms of a model for the Na⁺-Ca²⁺ exchange carrier which features two types of cation binding sites: a divalent site for which Ca²⁺ and 1-2 Na⁺ ions compete and a second, monovalent site which binds the third Na⁺ involved in Na⁺-Ca²⁺ exchange. Na⁺-Ca²⁺ exchange activity in vesicles is stimulated by a variety of agents or treatments, including limited proteolysis, phospholipase treatment, redox reagents, anionic amphiphiles, and intravesicular Ca²⁺, all of which lower the apparent K_m for Ca²⁺. The physiological significance of these modes of regulation of exchange activity is at present uncertain. Progress in identifying and purifying the exchange carrier has been hampered by the lack of specific high-affinity probes that could be used in labelling studies. Indirect estimates of the number of exchange carriers in sarcolemmal vesicles suggest that there are 10-20 pmol of exchanger per mg of membrane protein and that the exchange system has a maximal turnover of approximately 10³ sec^?1. Studies are under way in several laboratories to clone a cDNA for the exchange carrier using oocytes of Xenopus laevis as an expression system.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.     

Neuropeptide Y as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Several physiological stimuli cause a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes. This increased [Ca²⁺]_i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or non-adrenergic neurons, on Ca²⁺ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with ⁴⁵CaCl₂ for 1 h. Then, the fractional release of ⁴⁵Ca²⁺ from the cells was measured. NPY stimulated the efflux of ⁴⁵Ca²⁺ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10^–8 M to 10^–6 M). NPY (10^–6 M)-induced Ca²⁺ efflux was 2.0 ± 0.16% of the total cellular content. The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY, but not by Y₂ receptor agonist, NPY_13–36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY_18–36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca²⁺ efflux from cardiomyocytes is mediated through Y₁ receptors. It was also observed that NPY caused a rise in [Ca²⁺]_i to almost 150 nM. NPY-stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. Moreover, NPY caused a ²²Na⁺ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na⁺/Ca²⁺ exchange inhibitors. In addition, isoproterenol also caused ⁴⁵Ca²⁺ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y₁ receptors with which NPY may couple during Na⁺/Ca²⁺ exchange. Received: 21 May 1997 / Accepted: 26 August 1997     

The binding of inorganic and organic cations and H+ to cartilage in vitro

The binding of H⁺, Na⁺, Ca²⁺, Ni²⁺, Ce³⁺, chlorpromazine, chloroquine, hexamethonium, paraquat and gentamicin to decalcified cartilage from bovine nasal septum has been studied in vitro. The results indicated that the chondroitin sulphate is the binding material in the cartilage and that a stoichiometric binding occurs to the carboxyl and ester sulphate groups of the chondroitin sulphate. An analysis of the binding by the method of Scatchard was performed. The H⁺ ions were bound to two groups of sites, one representing the carboxyl, the other representing the ester sulphate groups. It was shown that the Scatchard association constants for H⁺ may be converted to pK_a values. Such transformation showed that the carboxyl groups of the chondroitin sulphate had a pK_a value of 4.57 and the ester sulphate groups a pK_a value of 2.60. The data for Na⁺ indicated a binding to three groups of sites. Two of these may represent the carboxyl and ester sulphate groups. The third, which was a small group with a very strong affinity, may represent a specific localised link with a few groups on the chondroitin sulphate. The experiments with Ca²⁺, Ni²⁺, Ce³⁺ and the organic substances indicated that, in contrast to H⁺ and Na⁺, only one binding class was present, which implies that the binding to the carboxyl and ester sulphate groups for these ligands occurs with a similar strength. The affinity of these cations to the cartilage was related to their positive charge, which is the general characteristic for an electrostatic interaction.