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.

     

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.

     

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.

     

Pharmacological changes in cellular Ca2+ homeostasis parallel initiation of atrial arrhythmogenesis in murine langendorff-perfused hearts

• 1 Intracellular Ca²⁺ overload has been associated with established atrial arrhythmogenesis. The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff‐perfused mouse hearts with changes in Ca²⁺ homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca²⁺ or inhibited entry of extracellular Ca²⁺.
• 2 Caffeine (1 mmol/L) elicited diastolic Ca²⁺ waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca²⁺ depletion.
• 3 Cyclopiazonic acid (CPA; 0.15 µmol/L) produced more gradual reductions in evoked Ca²⁺ transients and abolished diastolic Ca²⁺ events produced by the further addition of caffeine.
• 4 Nifedipine (0.5 µmol/L) produced immediate reductions in evoked Ca²⁺ transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca²⁺ transients, without eliciting diastolic Ca²⁺ events.
• 5 These findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf‐perfused hearts. Thus, caffeine was pro‐arrhythmogenic immediately following but not > 5 min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis.
• 6 Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca²⁺ events in atrial myocytes that, in turn, depend upon a finite SR Ca²⁺ store and diastolic Ca²⁺ release following Ca²⁺‐induced Ca²⁺ release initiated by the entry of extracellular Ca²⁺.

     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

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.     

MECHANISMS FOR REGULATION OF ARTERIAL TONE BY Ca2+- DEPENDENT K+ CHANNELS IN HYPERTENSION

1. The membrane potential and reactivity of arterial smooth muscle cells is regulated by a variety of K⁺ channels, which are highly expressed in vascular smooth muscle meuscle membrances. 2. Of these K⁺ channel types, the high-conductance, Ca²⁺-ependent K⁺ channel appears to be up-regulated in arterial smooth muscle membrances from hypertensive animals. 3. Patch-clamp studies show that whole-cell membrances and membrane patches of arterial smooth muscle obtained from rats with genetic or renal hypertension show an increased macroscopic and single-channel Ca²⁺-activated K⁺ current. Pharmacological block of this K⁺ current profoundly constricts aortic, renal, mesenteric and femoral arteries obtained from the same hypertensive animals, suggesting that Ca²⁺-dependent K⁺ current is a critical determinant of resting membrane potential in arterial muscle exposed to elevated blood pressure. 4. Thus, K⁺ efflux through Ca²⁺-dependent K⁺ channels appears to constitute an important homeostatic mechanism for buffering increases in arterial reactivity in hypertension.     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

ROLE OF ENDOTHELIUM AND K+ CHANNELS IN DOBUTAMINE-INDUCED RELAXATION IN RAT MESENTERIC ARTERY

1. In order to examine the possible involvement of the endothelium and K⁺ channel activation in the relaxation induced by dobutamine, a β-adrenoceptor agonist, in rat isolated mesenteric arteries, the effects of inhibitors of nitric oxide (NO) activity, blockers of K⁺ channels and high extracellular K⁺ were studied by measuring isometric tension in both endothelium-intact and -denuded arteries. 2. Dobutamine inhibited the phenylephrine (PE)-induced sustained tension with a pEC₅₀ of 7.40±0.08 in endothelium-intact arteries. Removal of functional endothelium attenuated the effect of dobutamine. The relaxation induced by dobutamine was inhibited by the β₁-adrenoceptor antagonist CGP20712A (3μ.mol/L) but not by the β₂-adrenoceptor antagonist ICI 118 551 (3 μmol/L) in endothelium-denuded arteries. 3. Pretreatment with N^G-nitro-l -arginine (l -NNA; 100 μmol/L) or methylene blue (3 μmol/L) induced a similar degree of inhibition of the dobutamine-induced relaxation in endothelium-intact arteries, while N^G-nitro-d -arginine (100 (μmol/L) and indomethacin (10 μmol/L) had no effect. In contrast, pretreatment with L -NNA (100 μmol/L) did not affect the relaxation induced by sodium nitroprusside (SNP) or forskolin. Methylene blue (3μmol/L) inhibited the relaxant response to SNP. 4. Charybdotoxin (CTX; 100nmol/L), iberiotoxin (IBX; 100nmol/L) and tetraethylammonium ions (TEA⁺; 3mmol/L) significantly reduced the dobutamine-induced relaxation. Tetrapentylammonium ions (TPA⁺; 5μmol/L) markedly inhibited the relaxant effect of dobutamine. The pECso values for control and in the presence of TPA⁺ in endothelium-intact arteries were 7.35±0.11 and 6.14±0.17, respectively, and 6.35±0.09 and 5.87±0.17 for control and in the presence of TPA⁺ in endothelium-denuded arteries, respectively. In contrast, glibenclamide (3 μ-mol/L) was ineffective. At 5 μmol/L, TPA⁺ also inhibited the relaxation induced by forskolin. 5. The maximal relaxation of PE-contracted arteries induced by 3μmol/L dobutamine was completely abolished in the 60mmol/L K⁺-contracted arteries with and without endothelium, while dobutamine at a concentration greater than 3 μmol/L induced inhibition of the high-K⁺ response. 6. The present results indicate that endothelium, probably NO but not prostacyclin, was involved in the dobutamine-induced relaxation in rat mesenteric arteries. Activation of CTX-, IBX- and TPA⁺-sensitive K⁺ channels contributed towards the observed relaxation. Loss of the ability to relax the 60 mmol/L K⁺-contracted arteries suggests that endothelium-derived vasoactive factors affected by concentrations of dobutamine less than 3 μmol/L may also act through K⁺ channels in our preparations. Higher concentrations of dobutamine may have a direct, endothelium-independent relaxant effect.     

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.     

EFFECTS OF INTRACELLULAR Ca2+ CHELATING ON NORADRENALINE RELEASE IN NORMOXIC AND ANOXIC HEARTS

1. Ischaemia and anoxia induce excessive noradrenaline (NA) release in the heart by a mechanism independent of both nerve activity and extracellular Ca²⁺. The present study was designed to examine the potential role of intracellular Ca²⁺ mobilization in anoxic NA release in the heart by chelating intracellular free Ca²⁺. 2. In normoxic hearts, preloading with an intracellular fre. Ca²⁺ chelator (BAPTA) reduced neuronal NA release by 65%, confirming the effectiveness of the loading protocol. Release of NA independent of nerve activity occurred in hearts subjected to a 40 min period of anoxic, substrate-free and nominal Ca²⁺-free perfusion. Loading hearts with BAPTA prior to anoxia failed to reduce NA overflow (1561 ± 147 vs 1496 ± 206 pmol/g over 40 min). Infusion with BAPTA (20 μmol/L) during the first 25 min of the anoxic period reduced the quantity of anoxic NA release by approximately 25% from 2013 ± 124 to 1476 ± 207 pmol/g (P < 0.05). 3. Our results confirm that anoxic NA release is predominantly. Ca²⁺-independent process with Ca²⁺ mobilization from endogenous storage playing only a minor contributing role.     

MOLECULAR AND FUNCTIONAL DIVERSITY OF K+ CHANNELS

1. Amino acid sequences encoding K⁺ channels belong to several subfamilies of the voltage-gated ion channel superfamily which includes Na²⁺-, Ca²⁺-, and cyclic nucleotide gated channels. The Kv family is the largest group, and encodes delayed rectifier, A-type, and large conductance Ca²⁺ activated K⁺ channels. 2. The α-subunits of Kv channels form as tetramers of four independent subunits. Each subunit has six membrane spanning regions and a pore forming ‘P’ region. Subunits belong to subfamilies (Kv1–4, BK, Eag) comprising multiple members, each of which has distinct properties resembling each of the major types of native Kv channel when expressed as homomultimers in heterologous systems. 3. Enormous diversity of Kv channel function arises from the multiplicity of subunits, the formation of heteromultimers within subfamilies and from association with intracellular β-subunit proteins. 4. In the absence of direct structural information, mutational analyses have provided considerable insights into the structure of the voltage-sensor, pore-forming region and the sites of action of drugs, toxins and associated proteins. 5. Another subfamily, the inwardly rectifying, or K_IR, family, appears to have arisen from a deletion of the first four membrane spanning regions of ancient Kv channels, changing gating properties from outward to inward rectification. These include the G-protein gated inward rectifiers and K_atp channels.     

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.

     

Inhibitory effect of 1,8-cineole on guinea-pig airway challenged with ovalbumin involves a preferential action on electromechanical coupling

• 1 1,8‐Cineole is a terpenoid constituent of essential oils with anti‐inflammatory properties. It reduces the neural excitability, functions as an antinociceptive agent and has myorelaxant actions in guinea‐pig airways. The aim of the present study was to investigate the mechanism underlying the myorelaxant effects of 1,8‐cineole in guinea‐pig isolated trachea from either naïve guinea‐pigs or ovalbumin (OVA)‐sensitized animals subjected to antigenic challenge.
• 2 Isometric recordings were made of the tone of isolated tracheal rings. Rings with an intact epithelium relaxed beyond basal tone in the presence of 1,8‐cineole (6.5 × 10^?6 to 2 × 10^?2 mol/L) in a concentration‐dependent manner (P < 0.001, anova ) with a pD₂ value of 2.23 (95% confidence interval 2.10–2.37). Removal of the epithelium or pretreatment of intact tissue for 15 min with 50 µmol/L N^G‐nitro‐l ‐arginine methyl ester, 5 mmol/L tetraethylammonium, 0.5 µmol/L tetrodotoxin or 5 µmol/L propranolol did not alter the potency (pD₂) or the maximal myorelaxant effect (E_max) of 1,8‐cineole.
• 3 1,8‐Cineole also significantly decreased the Schultz‐Dale contraction induced by OVA, mainly in preparations from OVA‐sensitized animals submitted to antigen challenge. 1,8‐Cineole decreased tracheal hyperresponsiveness to KCl and carbachol caused by antigen challenge and almost abolished the concentration–response curves to KCl, whereas it had little effect on the concentration–response curves to carbachol. Under Ca²⁺‐free conditions and in the presence of 10^?4 mol/L acetylcholine, neither 1,8‐cineole (6.5 × 10^?3 mol/L) nor verapamil (1 × 10^?5 mol/L) affected Ca²⁺‐induced contractions, but they almost abolished Ba²⁺‐induced contractions.
• 4 In conclusion, the findings of the present study show that 1,8‐cineole is a tracheal myorelaxant that acts preferentially on contractile responses elicited electromechanically.

     

Gossypol stimulates opening of a Ca2+‐ and Na+‐permeable but Ni2+‐ and Co2+‐impermeable pore in bEND.3 endothelial cells

Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti‐cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca²⁺]_i was raised concentration‐dependently by 1‐10 µmol/L gossypol. We then explored the Ca²⁺ signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca²⁺ signal was composed largely of Ca²⁺ influx and to a small extent, intracellular Ca²⁺ release. Such Ca²⁺ influx was much larger than store‐operated Ca²⁺ influx triggered by maximal Ca²⁺ pool depletion. The Ca²⁺ influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn²⁺ and Na⁺, but not Ni²⁺ and Co²⁺. Gossypol‐triggered Ca²⁺ signal was inhibited only by 14% and 37% by 100 µmol/L La³⁺ and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni²⁺. Gossypol‐triggered Ca²⁺ signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non‐selective cation pore, possibly a member of the TRPV family.     

FORCE AND INTRACELLULAR Ca2+ DURING NANC-MEDIATED RELAXATION OF RAT ANOCOCCYGEUS MUSCLE AND THE EFFECTS OF CYCLOPIAZONIC ACID

1. Simultaneous recordings of tension and [Ca²⁺]_i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca²⁺]_i. 3. The nitric oxide synthase inhibitor, N^G-nitro-L-Arginine (NOLA, 100 μmol/L) completely abolished the decreases in the [Ca²⁺]_i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca²⁺]_i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 μmol/ L), partially inhibited the force and [Ca²⁺]_i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, cyclopiazonic acid, (10 μmol/ L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca²⁺ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca²⁺]_i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca²⁺ sequestration into the sarcoplasmic reticulum. However, other Ca²⁺ lowering mechanisms and possible Ca²⁺ independent mechanisms may also contribute to the NANC relaxation response.     

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。     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: PHYSIOLOGICAL ROLE OF LARGE,Ca2+-ACTIVATED K+ CHANNELS IN HUMAN GLOMERULAR MESANGIAL CELLS

• 1 Contraction assays and patch clamp methods were used to determine the role of K⁺ channels in the regulation of contractile tone of human mesangial cells (MC) in culture.
• 2 MC contraction was induced by vasoconstrictor agents, such as angiotensin II (AngII; 100nmol/L) and glybenclamide (Glyb), but not by iberiotoxin (IbTX), a blocker of large Ca²⁺-activated K⁺ channels (BK(Ca)). These results suggest that Glyb-sensitive K⁺ channels, but not BK(Ca) channels, were active at rest.
• 3 In the presence of 100nmol/L IbTX, contraction by Angll was slightly, but not significantly, enhanced, indicating that BK(Ca) has a minimal role as a negative feedback regulator of contraction. Nitroprusside (NP; 100μmol/L), a nitric oxide (NO) donor, atrial natriuretic peptide (ANP; 1.0 μmol/L) and db-cGMP (10μmol/L) attenuated AngII-induced contraction in the absence, but not in the presence, of IbTX, suggesting that BK(Ca) channels were activated by cGMP.
• 4 In patch clamp experiments, three distinct K⁺-selective channels of 9, 65 and 150 pS (outward currents) were found in excised, inside-out patches. The 150 pS channel was completely inhibited by 100nmol/L IbTX and displayed ***voltage- and calcium-dependent gating qualitatively similar to BK(Ca) in other cell types.
• 5 In cell attached (CA) patches, the response of BK(Ca) to bath AngII (100nmol/L) was relatively minor in control solutions, but was considerably greater in the presence of db-cGMP.
• 6 In excised patches, Mg-ATP (1 mmol/L) plus db-cGMP (1 μmol/L) activated BK(Ca) in the absence, but not the presence, of the non-specific kinase inhibitor, staurosporine.
• 7 Separate experiments showed that BK(Ca) were also activated by arachidonic acid and high ambient glucose concentrations.
• 8 These results indicate that: (i) resting MC tone is sensitive to glybenclamide and apamin; and (ii) the role of BK(Ca) as a negative feedback regulator of contraction is minimal under normal conditions but is markedly enhanced by cGMP-stimulating relaxants and arachidonic acid.