Intracellular mechanisms and receptor types for endothelin-1-induced positive and negative inotropy in mouse ventricular myocardium

We examined the intracellular mechanisms for endothelin-1-induced positive and negative inotropic components that coexist in the mouse ventricular myocardium using isolated ventricular tissue and myocytes from 4-week-old mice. In the presence of SEA0400, a specific inhibitor of the Na⁺–Ca²⁺ exchanger, endothelin-1 produced positive inotropy. Endothelin-1, when applied to cardiomyocytes in the presence of SEA0400, did not change the peak amplitude of the Ca²⁺ transient but increased intracellular pH and Ca²⁺ sensitivity of contractile proteins. On the other hand, in the presence of dimethylamiloride (DMA), a specific inhibitor of the Na⁺–H⁺ exchanger, endothelin-1 produced negative inotropy. In cardiomyocytes, in the presence of DMA, endothelin-1 produced a decrease in peak amplitude of the Ca²⁺ transient. In the presence of both DMA and SEA0400, endothelin-1 produced neither positive nor negative inotropy. Positive inotropy was blocked by BQ-123 and negative inotropy by BQ-788. These results suggested that endothelin-1-induced positive inotropy is mediated by ET_A receptors, activation of the Na⁺–H⁺ exchanger and an increase in intracellular pH and Ca²⁺ sensitivity and that the negative inotropy is mediated by ET_B receptors, activation of the Na⁺–Ca²⁺ exchanger and decrease in Ca²⁺ transient amplitude.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

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。     

Angiotensin II-induced increase in slowly exchanging 45Ca2+ in relation to contractile responses of rat and guinea-pig aorta

Summary To gain more information about sources of activator Ca²⁺ involved in the contraction of rat and guinea-pig aorta evoked by angiotensin II and their sensitivity to Ca ²⁺ entry blockers, measurement of slowly exchanging ⁴⁵Ca²⁺ was established. A more physiological procedure was used, replacing La³⁺- and EGTA- containing solutions by a normal Ca²⁺-containing buffer. It was demonstrated that the angiotensin 11-induced increase in slowly exchanging ⁴⁵Ca²⁺ in rat aorta was incompletely (by approximately 60%–70%) inhibited by the organic Ca²⁺ entry blockers nifedipine, verapamil and diltiazem and by other Ca⁺ entry blocking compounds like CoCl₂ and chlorpromazine. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) was able to inhibit the angiotensin II-induced increase in ⁴⁵Ca²⁺ content completely, but this may be an intracellular storage effects. By contrast, the organic Ca²⁺ entry blockers completely inhibited that part of the angiotensin II-induced contraction of rat aorta which was dependent upon extracellular Ca²⁺.In guinea-pig aorta, the increase in ⁴⁵Ca²⁺ content elicited by angiotensin 11 could be completely suppressed by all compounds under study. The results of these experiments correlated well with data from the functional experiments in guinea-pig aorta. In both preparations the release of Ca ²⁺ from a rapidly as well as a slowly exchanging intracellular pool appears to contribute to the contractile response elicited by angiotensin 11. Offprint requests to P. N. M. van Heiningen at the above address     

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.     

Large- and small-conductance Ca2+-activated K+ channels: their role in the nicotinic receptor-mediated catecholamine secretion in bovine adrenal medulla

In cultured bovine adrenal chromaffln cells, charybdotoxin and iberiotoxin (inhibitors of the large-conductance Ca²⁺-activated K⁺ channel) as well as apamin (an inhibitor of the small-conductance Ca²⁺-activated K⁺ channel), at 1–100 nM, suppressed carbachol-induced ⁸⁶Rb⁺ efflux, augmented carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion and had no effect on carbachol-induced ²²Na⁺ influx via nicotinic receptors, a prerequisite for Ca²⁺ channel activation by carbachol. ⁴⁵Ca²⁺ influx caused by high K⁺ (a direct activation of voltage-dependent Ca²⁺ channels) was also enhanced by these K⁺ channel inhibitors, with the concentration-response curves being similar to those for carbachol-induced ⁴⁵Ca²⁺ influx. Dendrotoxin and mast cell degranulating peptide (inhibitors of voltage-dependent K⁺ channels), on the other hand, did not alter carbachol-induced ⁸⁶Rb⁺ efflux or ⁴⁵Ca²⁺ influx.These results suggest that the stimulation of nicotinic receptors eventually opens large- and small-conductance Ca²⁺-activated K⁺ channels, and that the blockade of these Ca²⁺-activated K⁺ channels results in gating of voltage-dependent Ca²⁺ channels and thereby augments catecholamine secretion from bovine adrenal chromaffln cells.     

Role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> content in Ca<Superscript>2+</Superscript> entry of bovine airway smooth muscle cells

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2⁺ channels with methoxyverapamil or by the sustained K⁺-induced depolarisation. The slow Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Role of sodium ions in angiotensin tachyphylaxis in the guinea-pig ileum and taenia coli

Summary We have examined the responsiveness of the guinea-pig ileum and taenia coli to angiotensin (ANG) analogues that were previously shown to be either able (|1-sarcosine|-ANG, Sar¹-ANG) or unable (|2-lysine|-ANG, Lys²-ANG) to induce tachyphylaxis in the ileum. The taenia coli, in which tachyphylaxis had not been previously shown to occur, was strongly tachyphylactic to Sar¹-ANG, but not to Lys²-ANG. The contractile responses of the ileum, as well as the contractile and electrical (sucrose-gap) events in the taenia coli, in response to the two ANG analogues, were used to investigate the tachyphylactic phenomenon and the role of Na⁺ in the manifestation. Relaxation of the ileum and repolarizations of the taenia coli were faster after treatment with Lys²-ANG than after Sar¹-ANG. In the tachyphylactic state, relaxation and repolarization after Sar¹-ANG became as fast as after Lys²-ANG. In low-Na⁺ (80 mmol/l) medium, as well as in ouabain-treated preparations, the responses of the ileum to ANG analogues were similar to those of tissues in the tachyphylactic state. Addition of Ca²⁺ to taenia coli preparations previously treated with the two ANG analogues in Ca⁺-free medium, caused contractile and electrical responses only in the case of Sar¹-ANG. It is proposed that ANG tachyphylaxis is due to changes at the level of the receptor causing increased Na⁺ permeability which leads to a decreased Na⁺ gradient across the cell membrane. Send offprint requests to T. B. Paiva at the above address     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Carbachol-induced nonspecific desensitization in guinea-pig ileum

Summary The effects of repeated stimulation by carbachol on force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. Carbachol was applied at 20°C for 5 min. Each application was followed by a 25-min washout period and the desensitization was expressed by the decline of the maximal force development. Three hours after the first carbachol-induced contraction the peak amplitude was about 40% of the initial value. Increasing the frequency of application, thereby decreasing the washout time, enhanced the desensitization, while the presence of the competitive blocker atropine reduced the phenomenon. At 35°C no desensitization could be observed. Blocking the Na⁺/K⁺ pump by ouabain or by K⁺-free solution reduced the force development to less than 20%. Increasing [K⁺]₀ in the washout solution at 20°C reduced the desensitization phenomenon, while decreasing [K⁺]₀ resulted in an enhanced desensitization as expressed by a decline of the force development.The total cellular Na⁺ content after various stimulation sequences was determined at 20° and 35°C from the ²²Na⁺ effluxes. At 35°C the cellular Na⁺ content did not change significantly during stimulation for 10 min with 10^–4 mol/l carbachol. At 20°C the resting Na⁺ content was significantly increased, and it doubled during carbachol stimulation for 10 min. Furthermore, the recovery of the cellular Na⁺ content after washout proceeded extremely slowly at that temperature. The appearance of desensitization was increased by 10 mol/l ryanodine, while it was reduced by adding the Ca²⁺ agonist Bay K 8644. Also the presence of pertussis toxin reduced the desensitization. The desensitizing effects of other agonists as histamine and substance P were less pronounced but also frequency dependent. The nonspecific nature of the carbachol desensitization was confirmed by the reduced response to histamine after stimulations with carbachol.We conclude that nonspecific desensitization is an agonist-, time- and temperature-dependent phenomenon. This desensitization could be the result of a G-protein-mediated inactivation of voltage-gated Ca²⁺ currents, which would thereby reduce the amount of [Ca²⁺]_i mobilized during repeated stimulation. Send offprint requests to: B. Himpens at the above address     

Inhibition by carbamazepine of various ion channels-mediated catecholamine secretion in cultured bovine adrenal medullary cells

The effects of carbamazepine (CBZ) on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, catecholamine secretion and cyclic GMP production were examined in cultured bovine adrenal medullary cells. 1 CBZ (40–120 mol/l) inhibited ²²Na⁺ influx evoked by carbachol in a concentration-dependent manner. CBZ inhibited carbachol-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion at concentrations similar to those which suppressed ²²Na⁺ influx. 2 CBZ (4–120 mol/l) inhibited veratridine-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion. 3 CBZ (12 or 40–120 mol/l) suppressed 56 mmol/1 K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, respectively. 4 Combination of CBZ with nitrendipine or -agatoxin-IVA produced further inhibition of 56 mmol/l K⁺ - evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, compared to the effect of CBZ alone, whereas CBZ plus -conotoxin-GVIA did not produce any further inhibition. 5 CBZ (40 mol/1) attenuated the production of cyclic GMP caused by muscarine. These results suggest that CBZ at therapeutic concentrations (16–48 mol/l: 4–12 g/ml) inhibits catecholamine secretion by interfering with nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and N-type voltage-dependent Ca²⁺ channels, and may have an antimuscarinic effect in adrenal medullary cells.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Methanol-Induced Contraction of Canine Cerebral Artery and Its Possible Mechanism of Action

In the present report, we investigated the effects of methanol on canine basilar cerebral arterial rings. Our data indicate that acute methanol exposure (5–675 mM) induces potent contractile responses of cerebral arteries in a concentration-dependent manner. Pharmacological antagonists, such as propranolol, phentolamine, haloperidol, methysergide, naloxone, diphenhydramine, and cimetidine, did not exert any effects on these methanol-induced contractions. Likewise, a potent antagonist of cyclo-oxygenase, and subsequent synthesis of prostanoids (i.e., indomethacin), failed to exert any effect on methanol-induced contractions. No differences in responsiveness to methanol in canine cerebral arteries were found in vessel segments with or without endothelial cells. Removal of extracellular Ca²⁺([Ca²⁺]_o) partially attenuated methanol-induced contractions, while withdrawal of extracellular Mg²⁺([Mg²⁺]_o) potentiated the contractions. In the complete absence of [Ca²⁺]_o, 10 mM caffeine and 400 mM methanol induced similar, transient contractions followed by relaxation in K⁺-depolarized cerebral vascular tissues. Methanol-induced contractions were, however, completely abolished by pretreatment of tissue with 10 mM caffeine. Our results indicate that (1) methanol causes contractile responses of cerebral arterial smooth muscle (independent of amine, prostanoid, or opioid mediation; (2) in addition to a need for [Ca²⁺]_o, an intracellular release of Ca²⁺is required for methanol-induced contractions; and (3) Mg deficiency potentiates the contractile responses of methanol on these brain vessels. The data presented in the study suggest that methanol-induced contractions occur via an sarcoplasmic reticulum-releasable store of [Ca²⁺]_i; via mediation of either ryanodine–caffeine type receptors or a caffeine-releasable intracellular store of Ca²⁺.     

Effects of calcium entry blocking agents on 5-hydroxytryptamine- and noradrenaline-induced contractions of rat isolated jugular vein and aorta

Summary We calculated the contribution of the intracellular releasable calcium pool to the contractile responses induced by 5-hydroxytryptamine (5-HT) and noradrenaline (NA) by constructing time-response curves to the agonists in Ca²⁺-deficient medium in the isolated rat jugular vein and aorta. Biexponential curves were obtained compatible with a two compartment model. In the aorta the intracellular calcium pools are likely to be different for both 5-HT and NA. Moreover, we investigated the effect of maximally effective concentrations of calcium entry blocking agents (CEB's) on K⁺, 5-HT- and NA-induced contractions in Ca²⁺-containing medium. Only a moderate inhibiting effect of nifedipine, diltiazem, flunarizine and gallopamil on 5-HT and NA-induced Ca²⁺ influx could be observed; in contrast, K⁺-induced Ca ²⁺ influx could be antagonized completely. The calculated contribution of intracellular Ca ²⁺ to 5-HT-and NA-induced contractions, obtained from the experiments in Ca²⁺-free medium was much lower than that obtained after pretreatment with CEB's, leading to the conclusion that after CEB-pretreatment a Ca²⁺ influx component persists. This hypothesis was supported by the observation that contractions in Ca²⁺-free medium consist of a monophasic, fast response only, whereas after CEB-pretreatment a response similar to the control, including a slow, sustained component, was obtained. The Ca²⁺ influx component not affected by maximally effective concentrations of CEB's seems to represent an inflow of extracellular Ca²⁺ directly into the cytosol and not into an intracellular calcium store. Send offprint requests to M. A. M. Gouw at the above address     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.     

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca homeostasis

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR₉₀), fura-fluorescence intensity (FFI) and intracellular Ca²⁺ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR₉₀ and intracellular Ca²⁺ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na⁺-Ca²⁺ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca²⁺ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca²⁺ regulation.     

Calcium mobilization induced by endothelins and sarafotoxin in cultured canine tracheal smooth muscle cells

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca²⁺ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca²⁺]_i, with half-maximal effect (EC₅₀) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ET_A receptor antagonist, had a high affinity to block the rise in [Ca2⁺]_i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca²⁺ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca²⁺]_i to the resting level. In the absence of external Ca²⁺, only an initial transient peak of [Ca²⁺]_i was seen, the sustained elevation of [Ca²⁺]. could then be evoked by addition of 1.8 mM Ca²⁺. Ca²⁺ influx was required for the changes of [Ca²⁺]_i, since the Ca²⁺-channel blockers, diltiazem, verapamil, and Ni²⁺, decreased both the initial and sustained elevation of [Ca²⁺]_i response to these peptides. ETs exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization of the Ca²⁺ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca²⁺ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2⁺]_i stimulated by these peptides is due to the activation of ET_A receptors and subsequently the release of Ca²⁺ from internal stores, whereas the contribution of external Ca²⁺ follows and partially involves a diltiazem- and verapamil-sensitive process. There is a cross-regulation among ETs and other receptor-coupling signal transduction pathways through PI hydrolysis in canine tracheal smooth muscle cells. Correspondence to: C. Mao Yang at the above address     

Effects of hypoxia on stimulus-release coupling mechanisms in cultured bovine adrenal chromaffin cells

Summary To clarify the effects of hypoxia on stimulus-release coupling, we have examined the effects of hypoxia on nicotine-induced catecholamine (noradrenaline and adrenaline) release from, and ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in, cultured bovine adrenal chromaffin cells. Experiments were carried out in media pre-equilibrated with 21% O₂/79% N₂ (control) or with 0% O₂/100% N₂ (hypoxia). Cells were stimulated with either nicotine (activating nicotinic acetylcholine (ACh) receptors) or a high K⁺ concentration (55 mmol/1 KCI; directly activating voltage-dependent Ca²⁺ channels). Hypoxia reduced both nicotine- and high K⁺-induced catecholamine releases from the cells, but the reduction of the former (to about 30% of the control value) was more pronounced than that of the latter (to about 40% of the control value). Nicotine-induced ²²Na⁺ influx, which is considered to reflect the function of nicotinic ACh receptors, was inhibited by hypoxia. Both nicotine- and high K⁺-induced ⁴⁵Ca²⁺ influx into the cells were reduced by hypoxia, but the reduction of the former was more pronounced than that of the latter. Nicotine- and high K⁺-induced increases in [Ca²⁺]_i were reduced by hypoxia to about 30% and 40% of the control values, respectively. These results suggest that hypoxia reduces cation influxes (Na⁺ and Ca²⁺) through both the ligand-gated cation channels of the nicotinic ACh receptor and the voltage-dependent Ca²⁺ channels. Correspondence to K. Lee at the present address