Transport ATPases of Cardiac Sarcolemma in 20, 25-Diazacholesterol Induced Myopathy

Abstract. 20, 25-Diazacholesterol, known to induce myotonia in skeletal muscle, also affects cardiac muscle as can be concluded from the development of cardiomegaly. At the same time (Na⁺, K⁺) ATPase of cardiac sarcolemmal membranes of the 20, 25-diazacholesterol treated rats showed an increased activity as compared with control animals (91 % and 46 % stimulation respectively). The Ca⁺⁺ stimulated ATPase showed the same tendency (96 % and 64 % stimulation).
In the plasma of the treated rats creatine phosphokinase activity was found to be elevated whereas the amount of protein-bound iodine was decreased, a finding that is common in myotonic dystrophy.     

A single isoform of the Na+/K+-ATPase α-subunit in Diptera: evidence from characterization of the first extracellular domain

The first extracellular domain of the α-subunit of the Na⁺/K⁺-ATPase (sodium/potassium pump) is functionally important, affecting sensitivity of the enzyme to cardiac glycosides (e.g. ouabain) and being implicated in the transport of K⁺. This domain is also variable among mammalian isoforms of the α-subunit. Using PCR, we have isolated from seven insect species with contrasting physiologies a DNA fragment containing this region, in order to help determine whether tissue-specific expression might be associated with isoforms encoded by a gene family, as it is in mammals. A single sequence (with one ORF) characteristic of Na⁺/K⁺-ATPase was obtained from genomic DNA of each species. Only the fragment from Manduca sexta contained an intron, but at a location different to that found in mammals. For all Diptera so far characterized, the species phylogeny is the same as the α-subunit gene phylogeny (based on the sequences of the first extracellular domain and flanking transmembrane domains). The results strongly indicate a single, ouabain-sensitive isoform of the α-subunit of Na⁺/K⁺-ATPase is present in Diptera.     

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery

Summary— The involvement of large conductance Ca²⁺-activated K⁺ channels (BK) and ATP-sensitive K⁺ (K_ATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca²⁺]_i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca²⁺]_i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca²⁺]_i with force in the presence of serotonin at different extracellular Ca²⁺ concentration ([Ca²⁺]_o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca²⁺-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.     

Erythrocyte Na+–H+ exchanger kinetics and Na+–Li+ countertransport activity in essential hypertensive patients

The authors measured Na⁺–H⁺ exchanger kinetics together with Na⁺–Li⁺ countertransport V _max in the erythrocytes of 21 subjects with essential hypertension and 16 normotensive control subjects. Na⁺–H⁺ exchanger V _max appeared to be increased in patients with essential hypertension, while the Na⁺–H⁺ exchanger affinity for intracellular proton sites ( K _50%) proved to be unchanged and the index of cooperativity among intracellular proton binding sites as measured by Hill's coefficient (Hill's n ) decreased as compared with normotensive control subjects. Na⁺–Li⁺ countertransport V _max appeared to be higher in patients with essential hypertension than in control subjects. The authors were unable to find any correlations between Na⁺–H⁺ exchanger kinetic parameters and metabolic variables such as parameters of insulin resistance and plasma lipids. On the basis of the data obtained, erythrocyte Na⁺–H⁺ exchanger activity was found to be abnormal in two kinetic variables in essential hypertensive patients and showed no simple linear correlations with the main variables of glucose metabolism, plasma lipids, renin or aldosterone.     

Relevance of erythrocyte Na+/Li+ countertransport measurement in essential hypertension, hyperlipidaemia and diabetic nephropathy: a critical review

In this review the usefulness of the measurement of erythrocyte Na⁺/Li⁺ countertransport (Na⁺/Li⁺ CT) activity is evaluated. In particular, the association between enhanced erythrocyte Na⁺/Li⁺ CT activity and essential hypertension, hyperlipidaemia and diabetic nephropathy is discussed. The conclusion of this review is that elevated erythrocyte Na⁺/Li⁺ CT activity is associated with essential hypertension and hyperlipidaemia. A relationship between Na⁺/Li⁺ CT activity and diabetic nephropathy is less evident. Despite a significant link of Na⁺/Li⁺ CT activity with hypertension and hyperlipidaemia, the diagnostic significance of Na⁺/Li⁺ CT activity is low. This is due to the large overlap between the results of control subjects and patients. The factors that contribute to this broad range are discussed in detail.     

Imaging of Ca2+-induced cytoplasmic Ca2+ responses in normal and pathological parathyroid cells

Ca²⁺-induced changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) were studied in bovine and normal and pathological human parathyroid cells using digital image analysis of fura-2-loaded cells. When raising external Ca²⁺ from 0.5 to 3.0 mmol L⁻¹, about 95% of all cells reacted rapidly and simultaneously with sustained elevation of [Ca²⁺]_i. In approximately two out of three bovine parathyroid cells, normal human cells and cells from most patients with hyperparathyroidism (HPT) the sustained phase was preceded by an overshooting [Ca²⁺]_i transient. The proportion of cells with such a transient was decreased in cells from severe cases of uraemic parathyroid hyperplasia only. However, pathological human cells from adenomas and normal-sized glands associated with adenomas, as well as cells from primary and uraemic hyperplasias, had lower peak and sustained levels than normal human and bovine cells. The results indicate that both normal and pathological parathyroid cells exhibit heterogeneity in their [Ca²⁺]_i responses to elevation of external Ca²⁺. The Ca²⁺-induced [Ca²⁺]_i transients and the sustained elevations are attenuated in pathological human parathyroid cells. However, the presence of the overshooting transient represents physiological variability rather than being a consequence of the pathophysiology associated with HPT.     

Inhibitory effects of okadaic acid on rat uterine contractile responses to different spasmogens

Summary— In the present study, we examined the effects of okadaic acid, a selective inhibitor of type 1 and 2A protein phosphatases, on the mechanical responses evoked by oxytocin, K⁺- and Na+-modified solutions and ouabain in estrogen-primed rat myometrium. Oxytocin elicited a rapid, phasic contraction followed by rhythmic oscillations. The phasic response was partially resistant to the absence of external Ca²⁺. Okadaic acid (1 μM) and the L-type calcium channel blocker nifedipine (1 μM) abolished the oscillatory component and reduced the initial, phasic response to about 80% of the control response. High K⁺ (60 mM) solution, ouabain (1 mM), K⁺-free medium and low Na⁺ (25 mM) solution induced extracellular Ca²⁺-dependent biphasic responses composed by an early rapid (KCl, ouabain and K⁺-free solution) or slower developed (25 mM Na⁺ solution) phasic contraction followed by a sustained increase in tension. Okadaic acid and nifedipine, alone or in combination, abolished or decreased similarly the contractile response evoked by these stimulants. The okadaic acid- and nifedipine-insensitive responses to ouabain, K⁺-free and low Na⁺ solution were enhanced by increasing the extracellular concentration of Ca²⁺ in the medium and were inhibited in a dose-dependent manner by amiloride (0.05–0.5 mM). These data suggest that, in estrogen-primed rat uterus, dephosphorylating mechanisms by OA-sensitive protein phosphatases play an important role in regulating myometrial contractions elicited by Ca²⁺ entry through voltage-sensitive Ca²⁺ channels.     

CHANGES IN PLATELET FREE Ca2+ CONCENTRATION AFTER CHRONIC DIGOXIN TREATMENT

Summary— Cell Na+ and Ca²⁺ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca²⁺ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca²⁺-countertransport system. Cytosolic free Ca²⁺ concentration is also controlled by the Na+–H+ exchange activity. To analyze the changes in cytosolic Ca²⁺ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca²⁺]_i was increased in platelets from 169±30 to 321±61 nmol/l ( n = 7, P <0.02) and unchanged in erythrocytes (121±6 and 104±7 nmol/l). This increase in platelet [Ca²⁺]_i was not accompanied by a change in serotonin content (5.43±0.67 vs 5.49±0.61 10⁻⁷ mol per 10¹¹ cells) and could not be reproduced by in vitro addition of 10⁻⁴ mol/l ouabain (198±33 vs 186±73 nmol/l). The enhanced [Ca²⁺]_i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca²⁺ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.     

Effects of K+ channel inhibitors and antagonists on NS-004 evoked relaxations in guinea-pig isolated trachea

Summary— The smooth muscle relaxant responses to NS-004, an activator of charybdotoxin-sensitive, large conductance Ca²⁺-dependent K⁺ channels (BKCa) were studied on the basal spontaneous tone in guinea-pig trachea in vitro. The sensitivity of these responses to a range of K⁺ channel inhibitors and antagonists were also evaluated. NS-004 (0.1–30 μM) evoked concentration-related relaxations (pIC₅₀ 5.48 ± 0.13) on the spontaneous tone in guinea-pig tracheal rings, suspended in Krebs bicarbonate solution, with a maximum response not different to that to aminophylline (1 μM). Charybdotoxin (0.03 and 0.1 μM) or iberiotoxin (0.1 μM) significantly displaced the NS-004 concentration-response curve to the right of control with no change in maximum response. In contrast, glibenclamide (1.0 μM), apamin (0.1 μM) and dofetilide (1.0 μM) each failed to modify the responses to NS-004 on spontaneous tone in guinea-pig trachea. These results suggest that relaxations in guinea-pig tracheal smooth muscle to the substituted benzimidazolone, NS-004, involve the activation of BK_Ca channels.     

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells

Summary— The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K⁺, and on voltage-dependent Na⁺ and Ca²⁺ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 μM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 μM) did not. In dispersed cells, both tacrine (1 μM-1 mM) and physostigmine (1 μM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K⁺ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1–100 μM) or physostigmine (1 μM-1 mM). In fura-2 loaded cells, tacrine (100 μM) almost abolished [Ca²⁺]i rise induced by veratridine, but only slightly reduced that evoked by high K⁺. In voltage-clamped cells, tacrine (300 μM) depressed the voltage-dependent Na⁺ and Ca²⁺ currents by about 93% and 69%, and physostigmine (300 μM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na⁺ channels rather than the Ca²⁺ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.     

The cytoplasmic Ca2+ response to glucose as an indicator of impairment of the pancreatic β-cell function

Abstract. The effects of glucose on the cytoplasmic Ca²⁺ concentration (Ca²⁺_i) regulating insulin release were investigated using pancreatic β-cells representative for the normal and diabetic situations. Increase of the glucose concentration resulted in a slight lowering of Ca²⁺_i followed by a rise, often manifested as high amplitude oscillations. The Ca²⁺_i-lowering component in the glucose action associated with suppression of insulin release became particularly prominent when the β-cells were already depolarized by tolbutamide. Glucose-induced inhibition of insulin release was observed also in experiments with rats made diabetic with streptozotocin or alloxan. Other studies indicated lowering of plasma insulin after intravenous glucose administration in patients with insulin- and noninsu-lin-dependent diabetes mellitus. Brief exposure of β-cells to 2–2 mmol 1^-1 streptozotocin resulted in impairment of the response to glucose, manifested as disappearance of the cyclic variation of Ca²⁺_i. The results indicate that glucose-induced depolarisation is a vulnerable process, the disturbance of which may contribute to insulin secretory defects in diabetes mellitus.     

Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L.)

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V-ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3-day-old feeding adult Amblyomma americanum (L.) female ticks. The 5' end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V-ATPase decrease the rate of dopamine-stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na⁺, K⁺ ATPase, indicating that a V-ATPase may participate in the mechanism of salivary fluid secretion in A. americanum , but the volume of saliva secreted is more dependent on an active Na⁺, K⁺ ATPase.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Tonic block of the Na+ current in single atrial and ventricular guineapig myocytes, by a new antiarrhythmic drug, Ro 22-9194

Summary— Ro 22-9194 reduced the Na⁺ current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22-9194 had a higher affinity to the inactivated state Na⁺ channels (Kd₁ = 3.3 μM in atrial myocytes, Kd₁ = 10.3 μM in ventricular myocytes) than to those in the rested state (Kd_R = 91 μM in atrial myocytes, Kd_R = 180 μM in ventricular myocytes), which indicated that Ro 22-9194 had a higher affinity to the Na⁺ channels in atrial myocytes than in ventricular myocytes. Ro 22-9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22-9194 more strongly inhibited the Na⁺ channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na⁺ channels in ventricular myocytes.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

Activation of the respiratory burst in eosinophil leucocytes—a transduction sequence decoupled from cytosolic Ca2+ rise

Abstract. The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5 nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca²⁺]_i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca²⁺]_i, it restored the burst response to agonists in Ca²⁺-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca²⁺-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G_i-proteins, but that in the presence of PMA portions of the agonist response could be recovered.     

Atrial Membranes Contain Nucleoside Diphosphate Kinase (NDPK) Activity: Its Role in Regulation of Muscarinic K+ Channels

Guinea pig or rabbit atrial muscarinic K⁺ channels in cell-free inside-out patches ran be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg²⁺,^1,2 This ATP-dependent activation is compatible with the existence of a membrane-bound nudeos de diphosphate kinase (NDPK), which directly phosphorylates G_K-bound GDP. We show that this ATP-dependent activation is also possible in frog atria] cells, and that atrail membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphogphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation o/muscarinic K⁺ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.     

Deficiency In Serum Ionized Magnesium But Not Total Magnesium In Patients With Migraines. Possible Role Of Ica2+/IMg2+ Ratio

SYNOPSIS
It has been suggested that magnesium (Mg) may play a role in the pathogenesis of headaches. Serum and intracellular measurements of Mg in headache patients have produced inconsistent results. The recent development of an ion-selective electrode for Mg²⁺ allowed precise measurement of serum ionized magnesium (IMg²⁺) in patients with various headache syndromes. Low serum Img²⁺ and a high ICa²⁺/IMg²⁺ ratio were found in 42% of patients having an attack of migraine, but only in 23% of patients with e severe continuous headache. Total serum Mg was normal in both groups of patients. However, in patients with low serum IMg²⁺ total serum Mg was lower than in patients with normal serum IMg²⁺. These results are compatible with the serotonin and vascular concepts of migraine pathogenesis. Low IMg²⁺ and a high ICa²⁺/Img²⁺ would result in cerebral vasospasm and reduced blood flow in the brain. The activity of serotonin receptors can also be affected by changes in IMg²⁺ levels. The finding of a difference in IMg²⁺ levels in two different headache types suggests a possible novel classification of headaches and that migraine patients with a low serum IMg²⁺ or a high ICa²⁺/IMg²⁺ ratio may benefit from Mg supplementation.     

Comparative Absorption of Ferri-Haemoglobin-59Fe/Ferro-Haemoglobin-59Fe and 59Fe3+/59Fe2+ in Humans with Normal and Depleted Iron Stores*

Abstract. The intestinal ⁵⁹Fe absorption from ferri- and ferro-haemogIobin-⁵⁹Fe and ⁵⁹Fe³⁺ and ⁵⁹Fe⁺ was calculated from whole body-⁵⁹Fe-retention measurements in subjects with normal and depleted iron stores. A ferri-haemoglobin-⁵⁹Fe/ferro-haemoglobin-⁵⁹Fe absorption ratio of 1.03 ±0.11 was observed for the absorption of ferri-haemoglobin-⁵⁹Fe (8.6± 0.77%) and ferro-haemogIobin-⁵⁹Fe (8.7±0.94%) in persons with normal iron stores. Depletion of iron stores caused a slight but significant higher rise of ferri-haemoglobin-⁵⁹Fe absorption (22 ± 1.7%) than the increase of ferro-haemoglobin-⁵⁹Fe absorption (18 ±0.9%) so that the absorption ratio was 1.24±0.073.—This remarkable iron valence independence of haemoglobin iron absorption is in considerable contrast to the well-established valence dependence of inorganic iron absorption which favours ferrous iron absorption especially with rising iron doses. The ⁵⁹Fe³⁺/⁵⁹Fe²⁺ absorption ratio for a diagnostic 0.56 mg Fe dose increased from 0.43 in subjects with normal iron stores to 0.74 in persons with depleted iron stores, whereas this absorption ratio was augmented only from 0.21 to 0.28 for the therapeutic 50 mg Fe-dose.—The different influence of iron valence on iron absorption from inorganic and haemoglobin iron supports other evidence for the existence of two separate mechanisms for ferrous iron and haem iron absorption in humans.