gramicidin-a and ethylisopropylamiloride

The Na+/H+-exchange inhibitor aethylisopropylamyloride was found to enhance the membrane resistance, the AP duration, and the duration of the guinea pig ureter's smooth muscle contraction. Tetraethylammonium and incubation in the Ca-free EG-TA-containing solution eliminated the activating effect of this inhibitor. Gramycidin A shortens the AP plateau. The Na+/H+-exchange mechanism seems to act under normal conditions. Alterations occurring in electrical and contractile activities of smooth muscle under the effect of the Na+/H+-exchange seem to result from the changes in the membrane Ca-dependent potassium conductivity.

Topics: Action Potentials; Amiloride; Animals; Calcium Chloride; Cell Membrane; Gramicidin; Hydrogen-Ion Concentration; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Potassium; Rabbits; Sodium-Hydrogen Exchangers; Taenia; Ureter

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na(+) [via the Na(+)-H(+) exchanger (NHE)]. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na(+) removal. Na(+)-gradient reversal induced an efflux of (3)H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na(+)-gradient reversal, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]MPP(+) efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.

Topics: 1-Methyl-4-phenylpyridinium; Amiloride; Animals; Biological Transport, Active; Desipramine; Drug Interactions; Gramicidin; Humans; Imidazoline Receptors; LLC-PK1 Cells; Mazindol; Models, Biological; Myocardial Ischemia; Nigericin; Norepinephrine; Receptors, Drug; Sodium; Swine; Time Factors

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.

Topics: Adult; Amiloride; Cheek; Electrolytes; Gramicidin; Humans; In Vitro Techniques; Ionophores; Kinetics; Mouth Mucosa; Nigericin; Potassium; Saliva; Sodium; Sodium-Hydrogen Exchangers; Valinomycin

Sodium orthovanadate caused a 2-fold stimulation of system A transport activity in soleus muscle, as assessed by the uptake of the nonmetabolizable analog 2-(methylamino)isobutyric acid (MeAIB). The effect of vanadate on system A was rapid, concentration-dependent and was characterized by an increased Vmax without modification of Km for MeAIB. Under these conditions, vanadate also activated 3-O-methylglucose uptake and lactate production. The effects of vanadate on muscle metabolism showed a complex interaction with the effects of insulin. Thus, the stimulatory effects of vanadate and insulin on MeAIB and 3-O-methylglucose uptake were not additive; however, the effects of insulin and vanadate on lactate production were additive. In spite of the lack of additivity, insulin- and vanadate-induced stimulation of system A differed in their sensitivity to gramicidin D, being the vanadate effect more susceptible to inhibition by gramicidin D than the insulin effect. System A transport activity shows a dependence on pH, and recent results suggest the presence of critical histidine residues on the A carrier that may be responsible for its pH dependence (Bertran, J., Roca, A., Pola, E., Testar, X., Zorzano, A. & Palacín, M. (1991) J. Biol. Chem. 266, 798-802). In this regard, a rise in extracellular pH led to a substantial activation of system A. Furthermore, lowering of muscle intracellular pH induced by ethylisopropylamiloride (EIPA), a specific inhibitor of sodium/proton exchange activity, led to inhibition of system A. This suggests that critical histidine residues are present in an intracellular localization on the A carrier. Furthermore, the rate of muscle glycolysis was also altered in response to a rise in extracellular pH or to EIPA treatment. Regarding the mechanisms involved in vanadate action, vanadate treatment in the incubated soleus muscle did not cause any significant stimulation of tyrosine kinase activity after partial purification of muscle insulin receptors. On the other hand, vanadate but not insulin caused a substantial increase in muscle intracellular pH as assessed by 5,5'-dimethyloxazolidine-2,4-dione equilibrium. This effect of vanadate on intracellular pH was not due to activation of the sodium/proton exchanger, since it was not blocked by EIPA. Based on these findings, we suggest that alkalinization of muscle intracellular pH might mediate the effects of vanadate on system A and on glycolysis.

Topics: Amiloride; Amino Acids; Animals; beta-Alanine; Biological Transport, Active; Cell Membrane; Glucose; Gramicidin; Hydrogen-Ion Concentration; Insulin; Kinetics; Lactates; Male; Muscles; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Receptor, Insulin; Vanadates