Voltage-gated Na+ channels in neuropathic pain

Neuropathic pain occurs as a result of some form of injury to the nervous system. Although the basis of the disease remains to be fully elucidated, numerous studies have suggested a major role for ion channels in the pathogenesis of neuropathic pain. As Na⁺ channels play a fundamental role in not only the generation but also in the conduction of an action potential, they have received considerable attention in the aetiology of pain sensation and have become important pharmacological targets. In this review, the authors discuss the importance of specific Na⁺ channel isoforms in the pathophysiology of neuropathic pain and the present use of Na⁺ channel antagonists in the treatment of neuropathic pain.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

Differential blockade of neuronal voltage-gated Na(+) and K(+) channels by antidepressant drugs

The effects of a range of antidepressants were investigated on neuronal voltage-gated Na(+) and K(+) channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na(+) uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na(+) channel. Imipramine also produced a differential action on macroscopic Na(+) and K(+) channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na(+) channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na(+) channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na(+) channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K(+) currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.     

Effects of carbamazepine and amitriptyline on tetrodotoxin-resistant Na<Superscript>+</Superscript> channels in immature rat trigeminal ganglion neurons

Although anticonvulsant drugs that block voltage-dependent Na⁺ channels have been widely used for neuropathic pain, including peripheral nerve injury-induced pain, much less is known about the actions of these drugs on immature trigeminal ganglion (TG) neurons. Here we report the effects of carbamazepine (CBZ) and amitriptyline (ATL) on tetrodotoxin-resistant (TTX-R) Na⁺ channels expressed on immature rat TG neurons. TTX-R Na⁺ currents (I_Na) were recorded in the presence of 300 nM TTX by use of a conventional whole-cell patch clamp method. Both CBZ and ATL inhibited TTX-R I_Na in a concentration-dependent manner, but ATL was more potent. While CBZ and ATL did not affect the overall voltage-activation relationship of TTX-R Na⁺ channels, both drugs shifted the voltage-activation relationship to the left, indicating that they inhibited TTX-R Na⁺ channels more efficiently at depolarized membrane potentials. ATL showed a profound use-dependent blockade of TTX-R I_Na, but CBZ had little effect. The present results suggest that both CBZ and ATL, common drugs used for treating neuropathic pain, efficiently inhibit TTX-R Na⁺ channels expressed on immature TG neurons, and that these drugs might be useful for the treatment of trigeminal nerve injury-induced neuropathic pain, as well as the inhibition of ongoing central sensitization, even during immature periods.     

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K and Na channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K⁺ and Na⁺ currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K⁺ and Na⁺ currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K⁺ than Na⁺ channels. P-CTX-4B shifted the voltage-dependence of Na⁺ conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca²⁺ in the external solution. A negative shift of about 16 mV in the steady-state Na⁺ inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na⁺ currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K⁺ channels, whereas it is about 50 times less efficient to affect Na⁺ channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.     

The Na+/H+ exchanger modulates long-term potentiation in rat hippocampal slices

Although present in great variety in the brain, the role of Na⁺/H⁺ exchangers (NHEs) in hippocampal plasticity is still unknown and the effect of NHE inhibition on long-term potentiation (LTP) has not been studied yet. As it is conceivable that NHE inhibitors may severely affect mechanisms that are considered to underlie learning and memory we investigated whether the broad-spectrum NHE inhibitor 5′-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 μM) influences LTP induced by different stimuli based on a theta burst in interface hippocampus slices from 7–8-week-old Wistar and 30-month-old Fischer 344/Brown–Norway F1 hybrid (F344/BN) rats. EIPA did not affect basal synaptic transmission, paired pulse inhibition, or LTP induced by a weak stimulus, but improved the maintenance of the LTP of the population spike induced by a strong tetanus. Our data suggest that NHE activity serves as a negative feedback mechanism to control neuronal excitability and plasticity in both young and senescent animals.     

Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels

The effect of the Na+/Ca(2+)-exchange inhibitor KB-R7943 was investigated in spinal cord dorsal column ischemia in vitro. Oxygen/glucose deprivation at 37 degrees C for 1 h causes severe injury even in the absence of external Ca2+. KB-R7943 was very protective in the presence and absence of external Ca2+ implicating mechanisms in addition to extracellular Ca2+ influx through Na+/Ca(2+)-exchange, such as activation of ryanodine receptors by L-type Ca2+ channels. Indeed, blockade of L-type Ca2+ by nimodipine confers a certain degree of protection of dorsal column against ischemia; combined application of nimodipine and KB-R7943 was not additive suggesting that KB-R7943 may also act on Ca2+ channels. KB-R7943 reduced inward Ba2+ current with IC50 = 7 microM in tsA-201 cells expressing Ca(v)1.2. Moreover, nifedipine and KB-R7943 both reduced depolarization-induced [Ca2+]i increases in forebrain neurons and effects were not additive. Nimodipine or KB-R7943 also reduced ischemic axoplasmic Ca2+ increase, which persisted in 0Ca2+/EGTA perfusate in dorsal column during ischemia. While KB-R7943 cannot be considered to be a specific Na+/Ca2+ exchange inhibitor, its profile makes it a very useful neuroprotectant in dorsal columns by: reducing Ca2+ import through reverse Na+/Ca2+ exchange; reducing influx through L-type Ca2+ channels, and indirectly inhibiting Ca2+ release from the ER through activation of ryanodine receptors.     

Cholinergic regulation of Na+-K+-ATPase activity in rat parotid gland: changes after castration

In this study, we investigated the different signalling pathways involved in muscarinic acetylcholine M(3) receptor-dependent modulation of Na(+)-K(+)-ATPase in parotid glands from normal and castrated rats. Carbachol inhibited the enzyme activity in parotid glands from control rats while it stimulated the enzyme activity in castrated rats. The inhibition of Ca(2+) calmodulin by trifluoperazine abolished the inhibitory effect of carbachol in control rats, while the inhibition of protein kinase C by staurosporine stimulated Na(+)-K(+)-ATPase. In castrated rats, trifluoperazine inhibited the carbachol-stimulant effect while staurosporine had no effect. Results indicate that in control glands the activation of a phospholipid-Ca(2+) calmodulin-dependent protein kinase C is responsible for the inhibitory effect of carbachol on Na(+)-K(+)-ATPase activity. In castrated rats, the activation of the enzyme by carbachol is regulated by its Ca(2+) calmodulin-stimulating action, and not by activation of protein kinase C. The activation of the Na(+)-K(+)-ATPase observed in castrated rats resulted in a decrease in carbachol-induced net K(+) efflux and thereby could decrease salivary fluid production.     

Inhibition of Na+ channel currents in rat myoblasts by 4-aminopyridine

Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na+ currents (I(Na)) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive I(Na) recorded from cultured rat myoblasts. 4-AP inhibited I(Na) amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K+ channel blocker 4-AP inhibits both neuron and myoblast Na+ channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research.     

Characteristics and possible mechanisms of low - Na+ induced contractions in rat aorta

The influence of reducing external Na⁺ concentration ([Na⁺]^ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na⁺]_ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na⁺]_ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10^–6 M. Elimination of the endogenous catecholamine participation using either phentolamine 10^–5 M or guanethidine 3.10^–6 M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10^–5 M, an antagonist of the voltage operated Ca²⁺ channels (25–30% residual tension for all the substitutes). Large reductions in [Na⁺]_ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65–75 %, sucrose replacement) and insensitive to D600 (all the substitutes). The Na⁺/K⁺ ATPase inhibitor ouabain (10^–4 M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10^–6 M control.Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na⁺]_ex dependence. Phenylephrine 10^–6 M failed to further contract the ouabain 10^–4 M precontracted rings.We conclude that relatively small reductions in [Na⁺]_ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na⁺/Ca²⁺ exchange. Larger reductions in [Na⁺]_ex appear to cause contraction through muscular Na⁺/Ca²⁺ exchange.     

Veratridine-induced intoxication in the isolated left atrium of the rat: Effects of some anti-ischemic compounds

Summary Veratridine-induced Na⁺ and Ca²⁺ uptake was used as a simulation of ischemia-induced Na⁺ and Ca²⁺ uptake. Therefore, electrically driven (1 Hz) isolated left atria of the rat were intoxicated with veratridine and the ⁴⁵Ca²⁺ uptake was determined. Veratridine (10^–4 mol/l) increased the ⁴⁵Ca²⁺ uptake from 575±13 to 2320±86 dpm/mg ww (n=20). The total tissue content of ⁴⁵Ca²⁺ was elevated from 4328±132 to 5136 ±303 dpm/mg ww (n = 13). The veratridine-induced ⁴⁵Ca²⁺ uptake was completely suppressed by tetrodotoxin (10^–7 and 10^–6 mol/l), whereas amiloride (6·10^–6 mol/1) and phentolamine (10^–6 and 10^–5 mol/l) exhibited no effect on the veratridine-induced ⁴⁵Ca²⁺ uptake. Nifedipine (10^–7 and 10^–6 mol/l) was ineffective on veratridine-induced ⁴⁵Ca²⁺ uptake. Verapamil (10^–5 mol/l) suppressed the veratridine-induced ⁴⁵Ca²⁺ uptake, but the ⁴⁵Ca²⁺ uptake in the absence of veratridine was also suppressed by verapamil (10^–6 and 10^–5 mol/l). The novel anti-ischemic compounds R 56865 (10^–8–10^–5 mol/l) and R 59494 (10^–8 -10-5 mol/l) totally abolished veratridine-induced ⁴⁵Ca²⁺ uptake.It is speculated that Ca²⁺ enters the cell via a Na⁺ channel which changes its selectivity upon veratridine treatment. Consequently, R 56865 and R 59494 could display their protective effect by either inhibiting the modified Na⁺ channel or preventing the transition of the normal Na⁺ channel to its altered state. As ischemia- and veratridine-induced Na⁺ and Ca²⁺ uptake share some similarities, it is proposed that veratridine-induced ⁴⁵Ca²⁺ uptake of the isolated left atrium of the rat could be used to study the mechanism of action of novel antiischemic drugs. Send offprint requests to D. Wermelskirchen at the above address     

CompB (J-113397), selectively and competitively antagonizes nociceptin activation of inwardly rectifying K(+) channels in rat periaqueductal gray slices

A novel opioid receptor family, the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors, has been identified to be involved in many physiological functions including pain regulation. CompB (also known as J-113397) is the first non-peptide antagonist of NOP receptors. Using the patch-clamp recording technique in brain slices, we have quantitatively studied the interactions of CompB with N/OFQ at native NOP receptors of ventrolateral neurons of the midbrain periaqueductal gray (PAG), a crucial region for N/OFQ-induced reversal of opioid analgesia. N/OFQ concentration-dependently activated inwardly rectifying K(+) channels in response to hyperpolarization ramps from -60 to -140 mV. CompB attenuated the magnitude but not the reversal potential of the K(+) current activated by N/OFQ in a concentration-dependent manner. The presence of CompB produced a parallel right-shift of the concentration-response curve to N/OFQ. The Schild plot analysis yielded a pA(2) value of 8.37. At concentrations up to 1 microM, CompB affected neither the membrane current per se nor the inwardly rectifying K(+) current activated by [D-Ala(2), N-Me-Phe(4),Gly-ol(5)]-enkephalin or baclofen, a mu-opioid and GABA(B) receptor agonist, respectively. It appears that CompB, at nanomolar concentrations, is a pure, selective and competitive antagonist of postsynaptic NOP receptors that mediate inwardly rectifying K(+) channel activation in ventrolateral PAG neurons.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Involvement of Na+/H+ exchanger in hypoxia/re-oxygenation-induced neonatal rat cardiomyocyte apoptosis

Although increased Na(+)/H(+) exchanger type-1 (NHE-1) activity has been implicated in the pathogenesis of myocardial infarction, the role of NHE-1 in induction of apoptosis, and the potential mechanisms involved have not been fully characterized. This study tested the hypothesis that NHE-1 activity is involved in hypoxia (H)/re-oxygenation (Re)-induced cardiomyocyte apoptosis by increasing mitochondrial Ca(2+) ([Ca(2+)]m). Primary cultured neonatal rat cardiomyocytes were subjected to 4.5 h of H followed by 12 h of Re. Relative to H alone, the level of X-rhod-1 acetoxymethyl (AM)-labeled [Ca(2+)]m was increased, and the frequency of cell death (propidium iodide (PI) staining) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling [TUNEL]), confirmed by Annexin-V, were augmented at the end of Re, along with appearance of cytosolic cytochrome c, activation of caspase-3, and increased ratio of Bax and Bcl-2. Addition of cariporide (20 micromol/l), a well-known NHE-1 inhibitor, to cultured cells before H significantly reduced [Ca(2+)]m, the number of PI and TUNEL positive cells relative to the levels at end of Re, but did not completely eliminate these changes compared to Sham control. There was a strong trend for attenuation in increased levels of [Ca(2+)]m, and the number of PI and TUNEL positive cells when same dose of cariporide was added only at Re, but the difference in these variables did not reach significance. In contrast, the levels of [Ca(2+)]m and the number of PI and TUNEL positive cells were significantly reduced to a level comparable to Sham control when cariporide (20 micromol/l) was administered before H and during Re, respectively, associated with a reduction in cytosolic cytochrome c, caspase-3 activity and ratio of Bax and Bcl-2. In conclusion, these data suggest that NHE-1 is involved in induction of cardiomyocyte apoptosis during both H and Re through a [Ca(2+)]m-dependent manner, thereby resulting in activation of cytochrome c-caspase-3 signaling pathways.     

Mechanisms of Na+ and Ca2+ influx into respiratory neurons during hypoxia

Changes in intracellular Na+ and Ca2+ in inspiratory neurons of neonatal mice were examined by using ion-selective fluorescent indicator dyes SBFI and fura-2, respectively. Both [Na+]i and [Ca2+]i signals showed rhythmic elevations, correlating with the inspiratory motor output. Brief (2-3 min) hypoxia, induced initial potentiation of rhythmic transients followed by their depression. During hypoxia, the basal [Na+]i and [Ca2+]i levels slowly increased, reflecting development of an inward current (Im). By antagonizing specific mechanisms of Na+ and Ca2+ transport we found that increases in [Na+]i, [Ca2+]i and Im due to hypoxia are suppressed by CNQX, nifedipine, riluzole and flufenamic acid, indicating contribution of AMPA/kainate receptors, persistent Na+ channels, L-type Ca2+ channels and Ca2+-sensitive non-selective cationic channels, respectively. The blockers decreased also the amplitude of the inspiratory bursts. Modification of mitochondrial properties with FCCP and cyclosporine A decreased [Ca2+]i elevations due to hypoxia by about 25%. After depletion of internal Ca2+ stores with thapsigargin, the blockade of NMDA receptors, Na+/K+ pump, Na+/H+ and Na+/Ca2+ exchange, the hypoxic response was not changed. We conclude that slow [Na+]i and [Ca2+]i increases in inspiratory neurons during hypoxia are caused by Na+ and Ca2+ entry due to combined activation of persistent Na+ and L-type Ca2+ channels and AMPA/kainate receptors.     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

A common "hot spot" confers hERG blockade activity to alpha-scorpion toxins affecting K+ channels

While alpha-KTx peptides are generally known for their modulation of the Shaker-type and the Ca(2+)-activated potassium channels, gamma-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily alpha-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative "hot spot" formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K(+) and HERG currents. Biochem J 2004;378:745-52]. In this work, we investigated if the "hot spot" is a commonality in subfamily alpha-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the "hot spot" in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active alpha-KTx. Additionally, we could extend our results to other alpha-KTx subfamily members belonging to alpha-KTx1, 4 and 6, therefore, the "hot spot" represents a common pharmacophore serving as a predictive tool for yet to be discovered alpha-KTxs.     

Insulin induced translocation of Na^＋/K^＋-ATPase is decreased in the heart of streptozotocin diabetic rats

Aim:

To investigate the effect of acute insulin administration on the subcellular localization of Na⁺/K⁺-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.

Methods:

Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na⁺/K⁺-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.

Results:

In control rat heart muscle α1 isoform of Na⁺/K⁺ ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P<0.05) in plasma membrane and α2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2- but not α1-subunits was detected. α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and β1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.

Conclusion:

This study demonstrate that insulin influences the plasma membrane localization of Na⁺/K⁺-ATPase isoforms in the heart. α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na⁺/K⁺-ATPase α1- and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.     

Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.