Effect of melittin on ion transport across cell membranes.
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Extensive work with melittin has shown that the venom has multiple effects, probably, as a result of its interaction with negatively changed phospholipids. It inhibits well known transport pumps such as the Na(+)-K(+)-ATPase and the H(+)-K(+)-ATPase. Melittin increases the permeability of cell membranes to ions, particularly Na+ and indirectly Ca2+, because of the Na(+)-Ca(2+)-exchange. This effect results in marked morphological and functional changes, particularly in excitable tissues such as cardiac myocytes. In some other tissues, e.g., cornea, not only Na+ but Cl- permeability is also increased by melittin. Similar effects to melittin on H(+)-K(+)-ATPase have been found with the synthetic amphipathic polypeptide Trp-3. (+info)
Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.
(18/5144)
Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension. (+info)
Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.
(19/5144)
We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells. (+info)
Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis.
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It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion. (+info)
Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors.
(21/5144)
Hyperalgesic and nociceptor sensitizing effects mediated by the beta-adrenergic receptor were evaluated in the rat. Intradermal injection of epinephrine, the major endogenous ligand for the beta-adrenergic receptor, into the dorsum of the hindpaw of the rat produced a dose-dependent mechanical hyperalgesia, quantified by the Randall-Selitto paw-withdrawal test. Epinephrine-induced hyperalgesia was attenuated significantly by intradermal pretreatment with propranolol, a beta-adrenergic receptor antagonist, but not by phentolamine, an alpha-adrenergic receptor antagonist. Epinephrine-induced hyperalgesia developed rapidly; it was statistically significant by 2 min after injection, reached a maximum effect within 5 min, and lasted 2 h. Injection of a more beta-adrenergic receptor-selective agonist, isoproterenol, also produced dose-dependent hyperalgesia, which was attenuated by propranolol but not phentolamine. Epinephrine-induced hyperalgesia was not affected by indomethacin, an inhibitor of cyclo-oxygenase, or by surgical sympathectomy. It was attenuated significantly by inhibitors of the adenosine 3',5'-cyclic monophosphate signaling pathway (the adenylyl cyclase inhibitor, SQ 22536, and the protein kinase A inhibitors, Rp-adenosine 3',5'-cyclic monophosphate and WIPTIDE), inhibitors of the protein kinase C signaling pathway (chelerythrine and bisindolylmaleimide) and a mu-opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Consistent with the hypothesis that epinephrine produces hyperalgesia by a direct action on primary afferent nociceptors, it was found to sensitize small-diameter dorsal root ganglion neurons in culture, i. e., to produce an increase in number of spikes and a decrease in latency to firing during a ramped depolarizing stimulus. These effects were blocked by propranolol. Furthermore epinephrine, like several other direct-acting hyperalgesic agents, caused a potentiation of tetrodotoxin-resistant sodium current, an effect that was abolished by Rp-adenosine 3',5'-cyclic monophosphate and significantly attenuated by bisindolylmaleimide. Isoproterenol also potentiated tetrodotoxin-resistant sodium current. In conclusion, epinephrine produces cutaneous mechanical hyperalgesia and sensitizes cultured dorsal root ganglion neurons in the absence of nerve injury via an action at a beta-adrenergic receptor. These effects of epinephrine are mediated by both the protein kinase A and protein kinase C second-messenger pathways. (+info)
Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons.
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Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons. (+info)
Intrinsic theta-frequency membrane potential oscillations in hippocampal CA1 interneurons of stratum lacunosum-moleculare.
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The ionic conductances underlying membrane potential oscillations of hippocampal CA1 interneurons located near the border between stratum lacunosum-moleculare and stratum radiatum (LM) were investigated using whole cell current-clamp recordings in rat hippocampal slices. At 22 degrees C, when LM cells were depolarized near spike threshold by current injection, 91% of cells displayed 2-5 Hz oscillations in membrane potential, which caused rhythmic firing. At 32 degrees C, mean oscillation frequency increased to 7.1 Hz. Oscillations were voltage dependent and were eliminated by hyperpolarizing cells 6-10 mV below spike threshold. Blockade of ionotropic glutamate and GABA synaptic transmission did not affect oscillations, indicating that they were not synaptically driven. Oscillations were eliminated by tetrodotoxin, suggesting that Na+ currents generate the depolarizing phase of oscillations. Oscillations were not affected by blocking Ca2+ currents with Cd2+ or Ca2+-free ACSF or by blocking the hyperpolarization-activated current (Ih) with Cs+. Both Ba2+ and a low concentration of 4-aminopyridine (4-AP) reduced oscillations but TEA did not. Theta-frequency oscillations were much less common in interneurons located in stratum oriens. Intrinsic membrane potential oscillations in LM cells of the CA1 region thus involve an interplay between inward Na+ currents and outward K+ currents sensitive to Ba2+ and 4-AP. These oscillations may participate in rhythmic inhibition and synchronization of pyramidal neurons during theta activity in vivo. (+info)
Chronic lithium treatment inhibits amiloride-sensitive sodium transport in the rat distal nephron.
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Chronic treatment of rats with lithium leads to Na+ loss and a reduced antinatriuretic response to aldosterone, suggesting that lithium reduces conductive Na+ transport in the distal nephron. This was investigated in the present study by measuring the renal response to aldosterone infusion followed by amiloride in chronically instrumented conscious rats given lithium for 3 to 4 weeks to achieve plasma Li+ concentrations of approximately 0.5 mM. A servo-controlled infusion system was used to maintain sodium and water homeostasis, thereby preventing misinterpretation of the findings as a consequence of drug-induced changes in Na+ balance. In a control group of rats, Na+ excretion decreased in response to aldosterone (p <.01) and subsequent amiloride administration led to a marked increase in Na+ excretion (p <.001). In contrast, in the lithium-treated group, there was no significant response to either aldosterone or amiloride. It is concluded that long-term treatment with lithium, even when plasma Li+ concentrations are below 1 mM, reduces aldosterone-stimulated Na+ transport through the amiloride-sensitive Na+ channels in the principal cells of the distal nephron. (+info)