Na/Ca exchanger and PMCA localization in neurons and astrocytes: functional implications.

Immunocytochemistry reveals that the Na/Ca exchanger (NCX) in neuronal somata and astrocytes is confined to plasma membrane (PM) microdomains that overlie sub-PM (junctional) endoplasmic reticulum (jER). By contrast, the PM Ca(2+) pump (PMCA) is more uniformly distributed in the PM. At presynaptic nerve terminals, the NCX distribution is consistent with that observed in the neuronal somata, but the PMCA is clustered at the active zones. Thus, the PMCA, with high affinity for Ca(2+) (K(d) congruent with 100 nM), may keep active zone Ca(2+) very low and thereby "reprime" the vesicular release mechanism following activity. NCX, with lower affinity for Ca(2+) (K(d) congruent with 1,000 nM), on the other hand, may extrude Ca(2+) that has diffused away from the active zones and been temporarily sequestered in the endoplasmic reticulum. The PL microdomains that contain the NCX also contain Na(+) pump high ouabain affinity alpha2 (astrocytes) or alpha 3 (neurons) subunit isoforms (IC(50) congruent with 5-50 nM ouabain). In contrast, the alpha1 isoform (low ouabain affinity in rodents; IC(50) >10,000 nM), like the PMCA, is more uniformly distributed in these cells. The sub-PM endoplasmic reticulum in neurons (and probably glia and other cell types as well) and the adjacent PM form junctions that resemble cardiac muscle dyads. We suggest that the PM microdomains containing NCX and alpha 2/alpha 3 Na(+) pumps, the underlying jER, and the intervening tiny volume of cytosol (<10(-18) l) form functional units (PLasmERosomes); diffusion of Na(+) and Ca(2+) between these cytosolic compartments and "bulk" cytosol may be markedly restricted. The activity of the Na(+) pumps with alpha 2/alpha 3 subunits may thus regulate NCX activity and jER Ca(2+) content. This view is supported by studies in mice with genetically reduced (by congruent with 50%) alpha 2 Na(+) pumps: evoked Ca(2+) transients were augmented in these cells despite normal cytosolic Na(+) and resting Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)). We conclude that alpha 2/alpha 3 Na(+) pumps control PLasmERosome (local) [Na(+)](CYT). This, in turn, via NCX, modulates local [Ca(2+)](CYT), jER Ca(2+) storage, Ca(2+) signaling, and cell responses.

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.

Structural complexity and functional diversity of endoplasmic reticulum Ca(2+) stores.

Considerable evidence, including recent direct observations, suggest that endoplasmic reticulum (ER) Ca(2+) stores in neurons, glia, and other cell types, consists of spatially-distinct compartments that can be individually loaded and unloaded. In addition, sub-plasmalemmal ('junctional') components of the ER (jER) are functionally coupled to the overlying plasmalemmal (PL) microdomains in PL-jER units named 'PLasmERosomes'. The PL microdomains and the jER contain clusters of specific transport proteins that regulate Na(+) and Ca(2+) concentrations in the tiny cytosolic space between the PL and jER. This organization helps the ER to produce the many types of complex local and global Ca(2+) signals that are responsible for the simultaneous control of numerous neuronal and glial functions.

Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes.

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration.

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation.

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation.

Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E(m)) regulates [Ca(2+)](cyt) by governing Ca(2+) influx through voltage-dependent Ca(2+) channels. Thus intracellular Ca(2+) may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K(+) (Kv) channels regulates resting E(m). In this study, we investigated whether changes of Kv currents [I(K(V))], E(m), and [Ca(2+)](cyt) affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca(2+) abolished PASMC growth. Resting [Ca(2+)](cyt) was significantly higher, and resting E(m) was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell I(K(V)) was significantly attenuated in PASMC during proliferation. Furthermore, E(m) depolarization significantly increased resting [Ca(2+)](cyt) and augmented agonist-mediated rises in [Ca(2+)](cyt) in the absence of extracellular Ca(2+). These results demonstrate that reduced I(K(V)), depolarized E(m), and elevated [Ca(2+)](cyt) may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca(2+)](cyt) in PASMC.

Unloading and refilling of two classes of spatially resolved endoplasmic reticulum Ca(2+) stores in astrocytes.

Signaling by two classes of endoplasmic reticulum (ER) Ca(2+) stores was studied in primary cultured rat astrocytes. Cytosolic and intra-ER Ca(2+) concentrations ([Ca(2+)](CYT) and [Ca(2+)](ER)) were measured with, respectively, Fura-2 and Furaptra, in separate experiments. The agonists, glutamate and ATP, released Ca(2+) primarily from cyclopiazonic acid (CPA)-sensitive ER Ca(2+) stores (CPA inhibits ER Ca(2+) pumps). Agonist-evoked release was abolished by prior treatment with CPA but was unaffected by prior depletion of caffeine/ryanodine (CAF/RY)-sensitive ER Ca(2+) stores. Conversely, prior depletion of the CPA-sensitive stores did not interfere with Ca(2+) release or reuptake in the CAF/RY-sensitive stores. Unloading of the CPA-sensitive stores, but not the CAF/RY-sensitive stores, promoted Ca(2+) entry through "store-operated channels." Resting [Ca(2+)](ER) averaged 153 microM (based on in situ calibration of Furaptra: K(D) = 76 microM, vs 53 microM in solution). The releasable Ca(2+) in both types of ER Ca(2+) stores was increased by Na(+) pump inhibition with 1 mM ouabain or K(+)-free medium. Using high spatial resolution imaging and image subtraction methods, we observed that some regions of the ER (45-58% of the total ER) unloaded and refilled when CPA was added and removed. Other regions of the ER (24-38%) unloaded and refilled when CAF was added and removed. The overlap between these two classes of ER was only 10-18%. These data indicate that there are two structurally separate, independent components of the ER and that they are responsible for the functional independence of the CPA-sensitive and CAF/RY-sensitive ER Ca(2+) stores.

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration.

Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K(+) channels (Kv) controls membrane potential (E(m)) that regulates cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) by governing the driving force for Ca(2+) influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K(+) channel gene expression, E(m), and [Ca(2+)](cyt) in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, alpha-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca(2+) channels, the depolarized E(m) in DFMO-treated cells decreased [Ca(2+)](cyt) as a result of reduced driving force for Ca(2+) influx through capacitative Ca(2+) entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E(m), and [Ca(2+)](cyt) but also restored cell migration to normal. Removal of extracellular Ca(2+) or blockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca(2+)](cyt) by increasing the driving force (the electrochemical gradient) for Ca(2+) influx and thus stimulates cell migration.

Cell proliferation is associated with enhanced capacitative Ca(2+) entry in human arterial myocytes.

Depletion of Ca(2+) stores in the sarcoplasmic reticulum (SR) activates extracellular Ca(2+) influx via capacitative Ca(2+) entry (CCE). Here, CCE levels in proliferating and growth-arrested human pulmonary artery smooth muscle cells (PASMCs) were compared by digital imaging fluorescence microscopy. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in proliferating PASMCs was twofold higher than that in growth-arrested cells. Cyclopiazonic acid (CPA; 10 microM), which inhibits SR Ca(2+)-ATPase and depletes inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, transiently increased [Ca(2+)](cyt) in the absence of extracellular Ca(2+). The addition of 1.8 mM Ca(2+) to the extracellular solution in the presence of CPA induced large increases in [Ca(2+)](cyt), indicative of CCE. The CPA-induced SR Ca(2+) release in proliferating PASMCs was twofold higher than that in growth-arrested cells, whereas the transient rise of [Ca(2+)](cyt) due to CCE was fivefold greater in proliferating cells. CCE was insensitive to nifedipine but was significantly inhibited by 50 mM K(+), which reduces the driving force for Ca(2+) influx, and by 0.5 mM Ni(2+), a putative blocker of store-operated Ca(2+) channels. These data show that augmented CCE is associated with proliferation of human PASMCs and may be involved in stimulating and maintaining cell growth.

The cellular mechanism of action of cardiotonic steroids: a new hypothesis.

Arterial smooth muscle (ASM) contraction is triggered by agonist-evoked Ca2+ mobilization from sarcoplasmic reticulum (SR). The amount of Ca2+ released, and thus, the magnitude of the contractions, depends directly on SR Ca2+ content. Na+ pump inhibition by cardiotonic steroids (CTS) indirectly increases the Ca2+ content of the SR and, thus, contractility. This sequence of events does not, however, account for the multiple Na+ pump alpha subunit isoforms with different affinities for Na+ and for CTS, nor does it explain the cardiotonic and vasotonic effects of low doses of CTS that do not elevate cytosolic Na+ or Ca2+. We show that the Na+ pump high ouabain affinity (alpha3) isoform and the plasmalemmal (PM) Na/Ca exchanger are confined to PM domains that overlie junctional SR in ASM, while low ouabain affinity alpha1 and the PM Ca2+ pump are uniformly distributed in the PM. Thus, low doses of CTS, including an endogenous ouabain-like compound, influence cytosolic Na+ and (indirectly) Ca2+ concentrations only in the cytoplasmic clefts between the PM and junctional SR (a functional unit we call the "plasmerosome"). In turn, this modulates the Ca2+ content of the junctional SR and cell responsiveness.

Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells.

K(+)-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca(2+)-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. Five KV-channel alpha-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha-subunit (KV9.3), and a KCa-channel alpha-subunit gene (rSlo), as well as three KV-channel beta-subunit genes (KV beta 1.1, KV beta 2, and KV beta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta-subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.

Abnormal calcium homeostasis in astrocytes from the trisomy 16 mouse.

The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 (Ts16) mouse, an animal model for Down syndrome and Alzheimer's disease (AD). The cytoplasmic ionized Ca2+ concentration ([Ca2+]cyt) was determined using digital imaging of fura-2-loaded cells. The relative Ca2+ content of internal endoplasmic reticulum (ER) stores was estimated from the magnitude of the transient increase in [Ca2+]cyt induced by cyclopiazonic acid (CPA), an inhibitor of Ca2+ sequestration into ER stores. At rest, the average [Ca2+]cyt was 140 nM in euploid (normal) astrocytes, but over twice as high, 320 nM, in Ts16 cells. In the absence of extracellular Ca2+, CPA induced a transient increase in [Ca2+]cyt to over 1200 nM in Ts16 astrocytes as compared to only 500 nM in euploid cells, indicating an increased amount of Ca2+ in the Ts16 astrocyte ER. In contrast to euploid astrocytes, both resting [Ca2+]cyt and the amount of Ca2+ in the ER stores varied widely among individual Ts16 astrocytes. These results show that Ts16 produces a dysregulation of Ca2+ homeostasis leading to increased cytoplasmic and stored Ca2+. Since increases in [Ca2+]cyt have been implicated in the etiology of neurodegenerative diseases, including AD, this finding of abnormal Ca2+ homeostasis in a genetic model of human neurological disorders suggests that Ca2+ dysregulation may be a common feature underlying neurodegenerative processes.

Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum.

The organization of calcium (Ca2+) stores in the sarcoplasmic and endoplasmic reticulum (S-ER) is poorly understood. The dynamics of the storage and release of calcium in the S-ER of intact, cultured astrocytes and arterial myocytes were studied with high-resolution imaging methods. The S-ER appeared to be a continuous tubular network; nevertheless, calcium stores in the S-ER were organized into small, spatially distinct compartments that functioned as discrete units. Cyclopiazonic acid (an inhibitor of the calcium pump in the S-ER membrane) and caffeine or ryanodine unloaded different, spatially separate compartments. Heterogeneity of calcium stores was also revealed in cells activated by physiological agonists. These results suggest that cells can generate spatially and temporally distinct calcium signals to control individual calcium-dependent processes.

Modulation of two functionally distinct Ca2+ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger.

Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, induced a transient, four-fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca(2+)-sensitive stores, induced a two-fold increase in [Ca2+]cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3(CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage-dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller, Ca(2+)- (CAF-) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma.

Effects of ryanodine on ouabain-induced spontaneous mechanical and electrical oscillations in guinea-pig heart.

The effects of ryanodine on ventricular arrhythmias in guinea-pigs in vivo, on delayed after potentials and after contractions, and on spontaneous oscillations of the membrane potential (SOP) and of resting tension (SOT) of guinea-pig papillary muscle under ouabain intoxication were studied. After addition of ouabain (1 microM) the after potentials, after contractions, and SOP and SOT amplitude were significantly increased. The power spectra of SOT and SOP under these conditions had a resonance harmonic with the frequency of about 5 Hz. Three to 5 mins after the addition of ryanodine (0.1-0.5 microM), the after potentials, after contractions, and SOP and SOT were abolished, suggesting a close relationship between these oscillations and the oscillatory activity of sarcoplasmic reticulum. In in vivo experiments, ouabain-induced (75-115 micrograms/kg) ventricular arrhythmias were terminated 4 to 5 min after intravenous injection of ryanodine (15 micrograms/kg); within 8-10 min, sinus rhythm was completely restored. We attribute the antiarrhythmic effect of ryanodine to a cellular effect and alteration of SR function, rather than to effects that are secondary to this.

[Effects of ethmozine on ventricular fibrillation threshold in acute occlusion of the coronary artery in dogs]. / Deistvie étmozina na porog fibrilliatsii zheludochkov pri ostroi okkliuzii koronarnoi arterii u sobak.

In experiments on dogs with acute left descending coronary artery occlusion, ethmozine (3 mg/kg) was tested for effects on the threshold of ventricular fibrillation occurring as a result of high-frequency electric stimulation. Two hours after occlusion, the fibrillation threshold became significantly lower than the control values. Ethmozine used in this period enhanced the ventricular fibrillation threshold in some experiments and diminished it in the others. Four hours following the occlusion, the fibrillation threshold did not differ from the control ones. Ethmozine given in this period caused a significant increase in the ventricular fibrillation threshold. It was concluded that 4 hours after the onset of experimental myocardial infarction are the minimal time period following which administration of ethmozine failed to decrease electric stability of the heart.

Analysis of antiarrhythmic effect of ryanodine in guinea-pigs.

The effects of ryanodine on (1) ventricular arrhythmias in guinea-pigs in vivo, (2) delayed afterpotentials and aftercontractions and (3) spontaneous oscillations of the membrane potential (SOP) and of resting tension (SOT) of guinea-pig papillary muscle under ouabain intoxication have been studied. After addition of ouabain (1 microM), the afterpotentials, aftercontractions and the amplitude of SOP and SOT were significantly increased. The power spectra of SOT and SOP under these conditions had a resonance harmonic with a frequency of about 5 Hz. The afterpotentials, aftercontractions, SOP and SOT were abolished 3 to 5 min after ryanodine addition (0.1 to 0.5 microM), suggesting a close relationship between these oscillations and the oscillatory activity of sarcoplasmic reticulum. During in vivo experiments, ouabain-induced (75 to 115 micrograms/kg) ventricular arrhythmias were terminated 4 to 5 min after the intravenous injection of ryanodine (15 micrograms/kg) and within 8 to 10 min, the sinus rhythm was completely restored. We conclude that the antiarrhythmic effect of ryanodine is related to the inhibition of the diastolic fluctuations of the membrane potential.

[Anti-arrhythmic effect of ryanodine in the guinea pig with glycoside poisoning]. / Antiaritmicheskii éffekt rianodina u morskoi svinki pri glikozidnoi intoksikatsii.

The effect of ryanodine on membrane potential oscillations in vitro (in isolated guinea-pig papillary muscle) and on ventricular arrhythmias in vivo (in glycoside intoxication) were studied. 3-5 minutes after ryanodine (0.5 microM) addition the membrane potential oscillations induced by ouabain (1 microM) were abolished. 4-5 minutes after intravenous ryanodine infusion (15 micrograms/kg) ventricular arrhythmias induced by ouabain intoxication (75-115 micrograms/kg) disappeared and 8-10 minutes later sinus rhythm was restored. It is suggested that antiarrhythmic effect of ryanodine is a result of the inhibition of diastolic membrane potential oscillations.