Tityustoxin K alpha blocks voltage-gated noninactivating K+ channels and unblocks inactivating K+ channels blocked by alpha-dendrotoxin in synaptosomes.

Two nonhomologous polypeptide toxins, tityustoxinK alpha (TsTX-K alpha) and tityustoxin K beta (TsTX-K beta), purified from thevenom of the Brazilian scorpion Tityus serrulatus, selectively blockvoltage-gated noninactivating K+ channels in synaptosomes (IC50 values of 8 nMand 30 nM, respectively). In contrast, alpha-dendrotoxin (alpha-DTX) andcharybdotoxin (ChTX) block voltage-gated inactivating K+ channels insynaptosomes (IC50 values of 90 nM and 40 nM, respectively). We studiedinteractions among these toxins in 125I-alpha-DTX binding and 86Rb effluxexperiments. Both TsTX-K alpha and ChTX completely displaced specifically bound125I-alpha-DTX from synaptic membranes, but TsTX-K beta had no effect on boundalpha-DTX. TsTX-K alpha and TsTX-K beta blocked the same noninactivatingcomponent of 100 mM K(+)-stimulated 86Rb efflux in synaptosomes. Both alpha-DTXand ChTX blocked the same inactivating component of the K(+)-stimulated 86Rbefflux in synaptosomes. Both the inactivating and the noninactivating componentsof the 100 mM K(+)-stimulated 86Rb efflux were completely blocked when 200 nMTsTX-K beta and either 600 nM alpha-DTX or 200 nM ChTX were present. The effectsof TsTX-K alpha and ChTX on 86Rb efflux were also additive. When TsTX-K alphawas added in the presence of alpha-DTX, however, only the noninactivatingcomponent of the K(+)-stimulated efflux was blocked. The inactivating componentcould then be blocked by ChTX, which is structurally homologous to TsTX-K alpha.We conclude that TsTX-K alpha unblocks the voltage-gated inactivating K+channels in synaptosomes when they are blocked by alpha-DTX, but not when theyare blocked by ChTX. TsTX-K alpha binds to a site on the inactivating K+ channelthat does not occlude the pore; its binding apparently prevents alpha-DTX (7054Da), but not ChTX (4300 Da), from blocking the pore. The effects of TsTX-K alphaon 125I-alpha-DTX binding and 86Rb efflux are mimicked by noxiustoxin, which ishomologous to TsTX-K alpha and ChTX.     

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.     

ATP-sensitive K+ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone.

The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide named from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and 86Rb+ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of ATP-sensitive K+ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamide. Glibenclamide blocks the galanin-activated ATP-sensitive K+ channel. 86Rb+ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced 86Rb+ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K+ channel reported earlier. 86Rb+ efflux studies confirm the electrophysiological demonstration that galanin activates an ATP-dependent K+ channel.     

Angiotensin increases Na+ entry and Na+/K+ pump activity in cultures of smooth muscle from rat aorta.

Angiotensin markedly altered the Na+ permeability of smooth muscle cells cultured from explants of rat aorta. The rate of net Na+ uptake was followed in the presence of ouabain in order to block Na+ efflux via the Na+/K+ pump. Angiotensin II (AII) or angiotensin III (AIII) increased net Na+ uptake by about 3-fold. Maximal stimulation of Na+ uptake was produced by about 10 nM AII. Bradykinin and the angiotensin antagonist [Sar1, Ileu5, Ala8]AII had no significant effect on net Na+ uptake. Angiotensin also enhanced the activity of the Na+/K+ pump, which was assayed by following the rate of ouabain-sensitive 86Rb+ uptake by the cells. AII and AIII nearly doubled ouabain-sensitive 86Rb+ uptake, but bradykinin, norepinephrine, and [Sar1, Ileu5, Ala8]AII had no effect. In the presence of ouabain, 86Rb+ uptake was not significantly affected by AII or AIII, indicating that angiotensin did not alter passive permeability to Rb+. Loading the cells with Na+, either by incubation in K+-free medium or exposure to the Na+-selective ionophore monensin, markedly increased ouabain-sensitive 86RB+ uptake. This result indicates that the activity of the Na+/K+ pump is limited by the low level of Na+ that is normally in the cells. AII had no effect on the activity of the Na+/K+ pump in Na+-loaded cells. These results suggest that AII or AIII stimulates the Na+/K+ pump in cultured aortic muscle cells by increasing its Na+ supply.     

Regulation of ATP-sensitive K+ channels in insulinoma cells: activation by somatostatin and protein kinase C and the role of cAMP.

The actions of somatostatin and of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86Rb+ flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K+ channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86Rb+ efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K+ channels are discussed.     

Endothelin blocks ATP-sensitive K+ channels and depolarizes smooth muscle cells of porcine coronary artery.

ATP-sensitive K+ channels with a conductance of 30 pS in smooth muscle cells of porcine coronary artery were found to be highly active in the intact cell-attached patch configuration when the pipette contained a physiological concentration of Ca2+ (greater than 10(-4) M). In the inside-out configuration, these channels were activated by extracellular Ca2+ and blocked by cytosolic ATP and glibenclamide. Endothelin applied to the pipette specifically blocked these channels in a concentration-dependent manner in the cell-attached configuration (half-maximal inhibition, 1.3 x 10(-9) M). A K+ channel opener, nicorandil, activated these channels even in the presence of 10(-8) M endothelin. In the whole-cell current-clamp method, the cell membrane was depolarized by endothelin and then repolarized by nicorandil. The membrane depolarization is closely related to contraction of smooth muscle cells. These results suggest that the ATP-sensitive K+ channels are important in controlling the vascular tone of the coronary artery and that endothelin can increase vascular tone by blocking these channels.     

The potassium channel openers: a new class of vasorelaxants.

Cromakalim, pinacidil, nicorandil, diazoxide and RP-49356 belong to the class of drugs termed potassium channel openers. In rat portal vein diazoxide, like cromakalim, abolished spontaneous mechanical and electrical activity and in rat aorta caused an increase in 86Rb efflux and inhibited KCl(20 mM)-induced contractions. However, in contrast to cromakalim, diazoxide (greater than 100 microM) also inhibited mechanical responses evoked by 80 mM KCl in rat aorta suggesting that it possesses pharmacological properties in addition to K channel opening. Since glibenclamide can attenuate the effects of cromakalim and diazoxide in vascular tissues, it is possible that a channel resembling the ATP-sensitive K channel found in pancreatic beta-cells may be involved in the vasorelaxant effects of these agents. However, differences exist in the order of potency of cromakalim and diazoxide for producing smooth muscle relaxation and for decreasing insulin secretion in pancreatic beta-cells. Furthermore galanin (which opens ATP-sensitive K channels in beta-cells) increases mechanical activity in rat portal vein. It is anticipated that new chemical developments will produce K channel opening molecules with greater potency and tissue selectivity.     

Effect of extracellular phosphate on Ca2+ and K+ fluxes in pancreatic islets

The idea that a lowering in cytosolic Ca2+ concentration may cause a decrease in K+ conductance in the pancreatic B-cell was tested by investigating the effect of a high extracellular phosphate concentration on 45Ca and 86Rb efflux from prelabelled rat pancreatic islets. Whether in the absence or presence of glucose, 20 mM phosphate tended to decrease 45Ca efflux. This effect was not suppressed in the absence of extracellular Ca2+, at least in glucose-deprived islets, suggesting that it may reflect a fall in cytosolic Ca2+ concentration. The administration of phosphate failed, however, to decrease 86Rb efflux from the islets. In the presence of extracellular Ca2+, 20 mM phosphate also failed to stimulate insulin release from islets perifused at low glucose concentration and inhibited insulin release stimulated by a high glucose concentration. These data indicate that the sequestration of Ca2+ in intracellular organelles and concomitant decrease in cytosolic Ca2+ concentration, as presumably provoked by a rise in extracellular phosphate concentration, is not sufficient to simulate the effect of glucose on K+ conductance.     

Opening of glibenclamide-sensitive K+ channels in follicular cells promotes Xenopus oocyte maturation.

The vasorelaxing K+ channel opener P1060 (a pinacidil analog), gonadotropins, and cAMP were shown to activate a glibenclamide-sensitive 86Rb+ efflux from fully grown follicle-enclosed Xenopus oocytes. Glibenclamide-sensitive K+ channels are located in follicular cells. Glibenclamide (i) depressed the gonadotropin- but not the progesterone-induced maturation and (ii) did not significantly modify progesterone production in oocytes exposed to Xenopus gonadotropin. In follicle-enclosed oocytes, the opener P1060 very significantly enhanced the oocyte sensitivity to progesterone. This increased sensitivity to the hormone induced by the K+ channel opener was reversed by glibenclamide. Thus these results suggest that the opening of glibenclamide-sensitive K+ channels in follicular cells by gonadotropins (and other activators of this channel) induces a hyperpolarization in the oocyte that greatly facilitates maturation by increasing the oocyte sensitivity to progesterone.     

Differences in the mechanism of metabolic regulation of ATP-sensitive K+ channels containing Kir6.1 and Kir6.2 subunits

AIMS: ATP sensitive K(+) channels (K(ATP)) sense adenine nucleotide concentrations and thus couple the metabolic state of the cell to membrane potential. The hetero-octameric complex of a sulphonylurea receptor (SUR2B) and an inwardly rectifying K(+) channel (Kir6.1) and the corresponding native channel in smooth muscle are relatively insensitive to variations in intracellular ATP. Activation of these channels in blood vessels during hypoxia/ischaemia is thought to be mediated via hormonal regulation such as cellular adenosine release or the release of mediators from the endothelium. In contrast, intracellular ATP prominently inhibits Kir6.2 containing complexes, such as those present in cardiac myocytes. Thus, we investigated differences in the mechanism of metabolic regulation of Kir6.1 and Kir6.2 containing K(ATP) channels. METHODS AND RESULTS: We have heterologously expressed K(ATP) channel subunits in HEK293 and CHO cells and studied their function using (86)Rb efflux and patch clamping. We show that rodent Kir6.1/SUR2B has direct intrinsic metabolic sensitivity independent of any regulation by protein kinase A. In contrast to Kir6.2 containing complexes, this was not endowed by the ATP sensitivity of the pore forming subunit but was instead a property of the SUR2B subunit. Mutagenesis of key residues within the nucleotide-binding domains (NBD) implicated both domains in governing the metabolic sensitivity. CONCLUSION: Kir6.1\SUR2B has intrinsic sensitivity to metabolism endowed by the likely processing of adenine nucleotides at the NBD of SUR2B.     

Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio: possible implication in vascular remodelling

OBJECTIVES : Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. METHODS : DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. RESULTS : Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. CONCLUSIONS : Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.     

Are ionic fluxes of pancreatic beta cells a target for gastric inhibitory polypeptide?

Gastric inhibitory polypeptide (GIP), an incretin candidate, is suggested to amplify the glucose-induced insulin secretion. To evaluate its mode of action we examined whether GIP affects 86Rb+ efflux, 45Ca2+ uptake or efflux, and intracellularly recorded electrical activity of mouse pancreatic islets. GIP (5 nM) neither inhibited 86Rb+ efflux at 3 mM glucose nor modulated 86Rb+ efflux that was inhibited by 5.6 mM glucose or stimulated by the calcium ionophore A23187. 45Ca2+ uptake was increased by GIP in the presence of 16.7 mM which was not observed at 3 or 11 mM glucose. GIP elevated 45Ca2+ efflux from islets, but did not modify 45Ca2+ efflux when a virtually Ca2+ free medium was used. Electrical activity of beta cells induced by 16.7 mM glucose was significantly increased by 5 nM GIP. It is concluded that the amplification of insulin release by GIP is based on the effect of GIP on Ca2+ uptake.     

Effect of endotoxic shock on basal and insulin-mediated Na+/K(+)-pump activity in rat soleus muscle.

This study evaluated basal and insulin-mediated Na+/K+ transport in skeletal muscle during endotoxic shock. Fasted male Holtzman rats (80-100 g) were killed 5 hr after the i.v. injection of saline (control) or 20 mg/kg Salmonella enteritidis endotoxin. 86Rb+ uptake into the cellular compartment of soleus muscle was determined in the presence and absence of 100 mU/ml insulin. The rate coefficient and rate of 22Na+ efflux and muscle intracellular 22Na+ content (in percentage of total muscle 22Na+) were determined in the presence and absence of 1 mM ouabain. The rate of basal cellular 86Rb+ uptake (cpm x 10(3).g wet weight-1.min-1) was not significantly different between control (77.7 +/- 2.9) and endotoxic soleus muscles (76.1 +/- 3.9). Insulin increased the rate of cellular 86Rb+ uptake by 19% in control (92.4 +/- 3.7) and 23% in endotoxic soleus muscles (93.8 +/- 4.1). Cellular 22Na+ content was 40% greater in endotoxic muscle (16.8 +/- 1.4) as compared to control muscles (12.0 +/- 0.9). The rate coefficient for ouabain-sensitive 22Na+ efflux in endotoxic muscles (0.028 min-1) was 1.7-fold greater than that in control muscles (0.017 min-1). The rate of ouabain-sensitive 22Na+ efflux was more than doubled in endotoxic muscles (0.470%/min) compared with control muscles (0.204%/min). Endotoxic shock did not alter insulin's ability to stimulate Na+/K+ transport in muscle. An increase in the electrogenicity of the Na+/K+ pump is consistent with an increased rate coefficient for 22Na+ efflux, with no change in the rate of 86Rb+ uptake in endotoxic soleus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased (Na+K+Cl) cotransport in rat arterial smooth muscle in deoxycorticosterone (DOCA)/salt-induced hypertension

The inhibition by loop diuretics of K efflux (tracer (86)Rb) from the rat femoral arterial smooth muscle was measured in normotension and in DOCA-salt hypertension. The sensitivity sequence (bumetanide > piretanide > furosemide) was the characteristic pharmacological profile of (Na+K+Cl) cotransport. In hypertension, cotransport activity was 46% greater than in normotension and the sensitivity to loop diuretics was threefold less. Intracellular ?K and the Na, K and Cl permeability ratios and electrogenic Na pump activity were assessed electrophysiologically in normotension and hypertension. ?K(i) was lower in hypertension (173 mM) than normotension (198 mM) but the other parameters (P(Na/Cl) = 0.14, P(Cl)/P(K) = 0.19 and electrogenic pump = -8.3 mV in normotension) were not significantly different. Ionic permeabilities to Na, K and Cl were significantly lower in hypertension than normotension. Plasma ?Na, but not ?K, was higher in hypertension than normotension. The conclusion is that increased activation of (Na+K+Cl) cotransport in hypertension plays a major role in the elevation of ?Cl(i) and depolarisation of the membrane potential in vascular smooth muscle in DOCA-salt hypertension. The role of (Na+K+Cl) cotransport in vascular smooth muscle in this model of hypertension is discussed in relation to ?Cl(i), depolarisation of the membrane potential and contraction and in relation to cell growth.     

Vascular effects of antiarrhythmic drugs and the roles of K+ channels

Since many of antiarrhythmic drugs can act on variable ion channels of cardiac myocytes, these compounds may also play a role in the activity of similar ion channels expressed on the vascular smooth muscle cells. In contrast, some of these ion channels expressed on vasculature have different subtypes of the channels, indicating that antiarrhythmic drugs may differentially affect ion channels on cardiac myocytes and vascular smooth muscle cells. Therefore, it is crucial to note the effects of antiarrhythmic drugs on the regulation of vascular function. Previous studies using isolated blood vessels as well as cultured vascular smooth muscle cells indicate that antiarrhythmic drugs have some modulator effects on K+ channels expressed on the vascular smooth muscle cells. In addition, this modulation may be modified and dependent on the sort of stimuli, including those of pharmacological and pathophysiological. The K+ channel is one of the most important ion channels modulating vascular function to preserve the organ blood flow including that of the brain as well as the heart, and that several available K+ channel openers are expected to treat cardiovascular disorders, including hypertension, ischemic heart disease. Therefore, these results may provide us a hint to understand the advantage and/or disadvantage of these compounds on the vascular function.     

The ionic, electrical, and secretory effects of protein kinase C activation in mouse pancreatic B-cells: studies with a phorbol ester

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to study the effects of protein kinase C activation on stimulus-secretion coupling in mouse pancreatic B-cells. At a nonstimulatory concentration of glucose (3 mM), 100 nM TPA, but not 10 nM TPA, slightly and slowly increased insulin release and 45Ca2+ efflux and decreased 86Rb+ efflux, but did not affect the membrane potential of B-cells. At a threshold concentration of glucose (7 mM), 100 nM TPA markedly increased insulin release without triggering electrical activity in B-cells. At a stimulatory concentration of glucose (10 mM), TPA caused a dose-dependent irreversible increase in insulin release, 45Ca2+ efflux, and 86Rb+ efflux and slightly augmented islet cAMP levels. Omission of extracellular Ca2+ abolished the effects of 10 nM TPA and partially inhibited those of 100 nM TPA on insulin release and 45Ca2+ efflux. In contrast, their effect on 86Rb+ efflux was paradoxically augmented. Glucose-induced electrical activity in B-cells was only marginally affected by TPA; the duration of the slow waves with spikes was not modified, but a small shortening of the polarized intervals raised their frequency and slightly increased the overall activity. This increase was significant only with 10 nM TPA, whereas only 100 nM TPA brought about a minute increase in 45Ca2+ influx. These results thus show that TPA induces insulin release or potentiates glucose-induced insulin release without mimicking or amplifying the initial ionic and electrical signals triggered by glucose. They suggest that protein kinase C activation affects stimulus-secretion coupling by modulating intracellular and/or nonelectrogenic membrane events.     

Effects of valinomycin on Rb+ fluxes, ATP content and insulin release in pancreatic islets.

Valinomycin, 0.5-500 nM, was tested for its effects on pancreatic islets microdissected fron non-inbred ob/ob-mice. Valinomycin decreased the islet accumulation Rb+ and the content of ATP in a dose-dependent manner; efflux of Rb+ from pre-loaded islets was not noticeably changed. Rb+ accumulation and ATP content correlated markedly; on the model of linear regression, less than 10% of the change Rb+ accumulation in valinomycin-treated islets was statistically attributable to factors other than ATP. Valinomycin did not cause a prompt inhibition of glucose-stimulated insulin release that could reflect hyperpolarization due to increased K+ permeability. The following conclusions are drawn: 1) The plasma membranes of beta-cells resemble those of neurons in having such a high ion permeability as to be relatively little influenced by valinomycin; 2) Islet accumulation of Rb+ is due to a vectorial catalyst in theplasma membrane rather than to uptake by mitochondria; 3) Rb+ accumulation in islets is ATP-dependent.     

Vectorial (transcellular) transport of potassium (86Rb+) by cultured Sertoli cells

Sertoli cells from rats aged 25 days were grown on Millipore filters (pore diameter 0.5 micron) for 7 days and were then used for determination of transport of 86Rb+ through the cells (base to apex); this procedure is referred to as measuring transcellular or vectorial transport. Sertoli cells were also used to measure apical efflux of 86Rb+ by loading the cells with the isotope to steady state and then incubating cells so that the apical surfaces were in contact with medium not containing 86Rb+, from which samples were taken. Basal efflux was measured in the same way except that the opposite surface of the cells was in contact with the medium. Cells grown on filters treated with collagen IV plus fibronectin showed transcellular transport of 86Rb+; t1/2 for equilibration across the cells was 9-12 min. The rate of transport was accelerated by addition of (Bu)2cAMP, forskolin, or FSH to the incubation medium. Half-maximal responses were seen with (Bu)2cAMP at 0.2 mM and with forskolin at 20 microM. Apical efflux (t1/2 9.8 +/- 2.1 min) was not influenced by the presence or absence of K+ in the medium nor by azide or (Bu)2cAMP. Basal efflux showed similar values for t1/2 in the presence of K+ (9.7 +/- 1.9 min) and values of 21.4 +/- 4.2 min in the absence of K+. Vectorial transport of 86Rb+ by these cells may account for the K+ gradient seen in the seminiferous tubule and appears to result from a basolateral potassium pump together with an apical membrane that is permeable to K+.     

Membrane permeability to K+ and the control of aldosterone synthesis: effects of valinomycin and cromakalim in bovine adrenocortical cells.

Stimulation of aldosterone synthesis by angiotensin II (AII) is associated with depolarization of the cell membrane. Since the potential difference of adrenocortical cells is dependent on membrane permeability to potassium ions, the effects of agents which hyperpolarize the cell (by increasing permeability to K+) on the control of aldosterone synthesis were investigated further. Basal and AII-stimulated aldosterone synthesis was increased by 20-70% in cells incubated with 1 or 10 nM of the potassium ionophore valinomycin; higher concentrations markedly inhibited AII-stimulated synthesis. Cromakalim, a potential antihypertensive drug which facilitates the opening of K+ channels in smooth muscle cells, stimulated basal aldosterone synthesis at 2 microM but had no effect at 40 microM. AII-stimulated aldosterone synthesis was not affected by cromakalim except at 40 microM, which was inhibitory. The inhibitory effects of cromakalim, unlike those of valinomycin, were not reversible. Aldosterone synthesis from added hydroxycholesterol and pregnenolone (but not from deoxycorticosterone and corticosterone) was significantly inhibited by 40 microM cromakalim. Potassium efflux from cells preloaded with 43K was unaffected by low concentrations of valinomycin, but was markedly increased by concentrations which inhibited AII-stimulated aldosterone production. Small decreases and increases in 43K efflux, caused by 1 and 40 microM cromakalim respectively, corresponded with increases and decreases in basal aldosterone production; cromakalim did not affect 43K efflux from AII-stimulated cells. We suggest that increasing adrenocortical cell membrane permeability to K+ reduces steroidogenesis, but that valinomycin and cromakalim have other actions which complicate the relationship between 43K efflux and aldosterone production. Cromakalim appears to inhibit 21-hydroxylase activity in the biosynthetic pathway and may also affect 3 beta-hydroxysteroid dehydrogenase activity.     

Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Two ionophores (monensin and gramicidin) that carry Na+ into 3T3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing 86Rb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 86Rb+ uptake and cell Na+ and K+ over a range of monensin concentrations show that the activity of the Na+,K+ pump in 3T3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum rapidly enhances the rate of 22Na+ uptake and net Na+ entry when Na+ exit is inhibited by ouabain. At 0.3 microgram/ml, monensin increases the rate of net Na+ entry and activates the Na+,K+ pump by the same degree as serum. The stimulation of 86Rb+ uptake by serum or the ionophores has an absolute requirement for external Na+. Thus, serum appears to stimulate the Na+,K+ pump in quiescent 3T3 cells by increasing its supply of Na+.