Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.     

Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes

Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium.     

ATP-sensitive K(+) channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K(+) (K(ATP)) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated K(ATP) channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 microM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 microM) increased K(ATP) channel activity in cell-attached patches. PKG (5 U/microl) added together with ATP and cGMP (100 microM each) directly to the intracellular surface increased the channel activity. Activation of K(ATP) channels was abolished by the replacement of ATP with ATPgammaS. Rp-pCPT-cGMP (100 microM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the K(ATP) channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated K(ATP) channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of K(ATP) channels in rabbit ventricular myocytes.     

Antagonistic effect of cAMP and cGMP on the kinetic behaviour of phosphofructokinase from rat erythrocytes and reticulocytes

Fructose-6-phosphate (F6P)-saturation curves (up to 5 mM F6P) for phosphofructokinase (PFK) have been studied at physiological pH (7.1) and inhibitory (1.5 mM) or non-inhibitory (0.25 mM) ATP levels, in rat erythrocytes and reticulocytes. The addition of 300 microM cAMP to control samples activates the enzyme and displaces F6P-saturation curve towards the left, while the addition of cGMP inhibits the enzyme and shifts the curve to the right. The cAMP positive allosteric effect is more evident at inhibitory ATP levels, while the inhibitory effect of cGMP is very similar at both ATP levels. This antagonistic effect is exerted at the same regulatory site, since cAMP also activates the enzyme when cGMP is previously present in the reaction mixture. The physiological significance of this antagonism is not yet clear.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells.

The mechanism of 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) action was evaluated in isolated adrenal glomerulosa cells. TMB-8 inhibits both angiotensin II- and K+-stimulated aldosterone secretion in a dose-dependent manner. The ID50 for angiotensin II- and K+-stimulated aldosterone secretion is 46 and 28 microM, respectively. In spite of the fact that 100 microM-TMB-8 inhibits angiotensin II-stimulated aldosterone secretion almost completely, TMB-8 (100 microM) does not inhibit angiotensin II-induced 45Ca2+ efflux from prelabelled cells nor does it affect inositol 1,4,5-trisphosphate-induced calcium release from non-mitochondrial pool(s) in saponin-permeabilized cells. TMB-8 has no inhibitory effect on A23187-induced aldosterone secretion, but 12-O-tetradecanoylphorbol 13-acetate-induced aldosterone secretion is completely abolished. TMB-8 effectively inhibits both angiotensin II- and K+-induced increases in calcium influx but has no effect on A23187-induced calcium influx. TMB-8 inhibits the activity of protein kinase C dose-dependently. These results indicate that TMB-8 inhibits aldosterone secretion without inhibiting mobilization of calcium from an intracellular pool. The inhibitory effect of TMB-8 is due largely to an inhibition of plasma membrane calcium influx, but this drug also inhibits the activity of protein kinase C directly.     

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival¹, development and, calcium signaling². Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death³.The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase^4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol⁶. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL⁷ interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity⁸ and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Photoreceptor channel activation by nucleotide derivatives

Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal transmission within the P2X2 trimeric receptor

P2X₂ receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X₂ requires binding of at least two ATP molecules. (b) D315A mutation in the β-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X₂ gating in saturating [ATP], making P2X₂ equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the β-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.     

Intracellular ADP activates K+ channels that are inhibited by ATP in an insulin-secreting cell line

The effect of ADP on ATP-sensitive K+ channels in the insulin-secreting RINm5F cell line has been investigated with the help of single-channel current recording from saponin-permeabilized cells. ADP (100-500 microM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP-beta-S cannot mimick this effect. During sustained ATP (500 microM)-evoked inhibition of K+ channel opening, 500 microM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP-activated channels. It is suggested that the physiological control of K+ channel opening in the insulin-secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.     

Effect of cGMP-dependent signaling system in regulation of osmotic permeability in the frog urinary bladder

In frogs' isolated urinary bladders, contribution of cytosolic guanylate cyclase and cGMP-dependent protein kinase to regulation of osmotic permeability was studied. ODQ (25-100 microM), an inhibitor of cytosolic guanylate cyclase induced an increase of vasotocin-activated osmotic permeability but had no effect on the hormone-activated transepithelial urea transport. In isolated mucosal epithelial cells ODQ (50 microM) decreased the concentration of intracellular cGMP. In these cells L-NAME (0.5 nM), an inhibitor of NO synthase, also decreased the level of cGMP whereas cAMP was significantly increased. 8-pCPT-cGMP (25 and 50 microM), a permeable cGMP analogue which selectively activates protein kinase G, inhibited vasotocin-induced increase of water transport along osmotic gradient indicating that protein kinase G is involved in regulation of water reabsorption. The data obtained show that NO/cGMP signalling system in the frog urinary bladder appears to be a negative modulator of vasotocin-activated increase of osmotic permeability.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] </= 0.1 mM or 100 microM ouabain) or when IK-ATP was completely inhibited by 10 mM ATP. In whole-cell configuration, ouabain inhibited up to 60% of inwardly rectifying IK-ATP at 1 mM ATP in the pipette but not at 10 mM ATP and 10 mM phosphocreatine when IK-ATP was always blocked. However, mathematical simulation of giant-patch experiments revealed that only 20% of ATP depletion may be attributed to the ATP concentration gradient in the bulk solution, and the remaining 80% probably occurs in the submembrane space.     

Adrenocorticotropic hormone and cAMP inhibit noninactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone (ACTH) at picomolar concentrations. Inhibition of IAC may be a critical step in depolarization-dependent Ca2+ entry leading to cortisol secretion. In whole-cell patch clamp recordings from AZF cells, we have characterized properties of IAC and the signalling pathway by which ACTH inhibits this current. IAC was identified as a voltage-gated, outwardly rectifying, K(+)-selective current whose inhibition by ACTH required activation of a pertussis toxin-insensitive GTP binding protein. IAC was selectively inhibited by the cAMP analogue 8-(4- chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (8-pcpt-cAMP) with an IC50 of 160 microM. The adenylate cyclase activator forskolin (2.5 microM) also reduced IAC by 92 +/- 4.7%. Inhibition of IAC by ACTH, 8-pcpt-cAMP and forskolin was not prevented by the cAMP-dependent protein kinase inhibitors H-89 (5 microM), cAMP-dependent protein kinase inhibitor peptide (PKI[5-24]) (2 microM), (Rp)-cAMPS (500 microM), or by the nonspecific protein kinase inhibitor staurosporine (100 nM) applied externally or intracellularly through the patch pipette. At the same concentrations, these kinase inhibitors abolished 8-pcpt-cAMP-stimulated A-kinase activity in AZF cell extracts. In intact AZF cells, 8-pcpt-cAMP activated A-kinase with an EC50 of 77 nM, a concentration 2,000-fold lower than that inhibiting IAC half maximally. The active catalytic subunit of A-kinase applied intracellularly through the recording pipette failed to alter functional expression of IAC. The inhibition of IAC by ACTH and 8-pcpt- cAMP was eliminated by substituting the nonhydrolyzable ATP analogue AMP-PNP for ATP in the pipette solution. Penfluridol, an antagonist of T-type Ca2+ channels inhibited 8-pcpt-cAMP-induced cortisol secretion with an IC50 of 0.33 microM, a concentration that effectively blocks Ca2+ channel in these cells. These results demonstrate that IAC is a K(+)-selective current whose gating is controlled by an unusual combination of metabolic factors and membrane voltage. IAC may be the first example of an ionic current that is inhibited by cAMP through an A-kinase-independent mechanism. The A-kinase-independent inhibition of IAC by ACTH and cAMP through a mechanism requiring ATP hydrolysis appears to be a unique form of channel modulation. These findings suggest a model for cortisol secretion wherein cAMP combines with two separate effectors to activate parallel steroidogenic signalling pathways. These include the traditional A-kinase-dependent signalling cascade and a novel pathway wherein cAMP binding to IAC K+ channels leads to membrane depolarization and Ca2+ entry. The simultaneous activation of A-kinase- and Ca(2+)-dependent pathways produces the full steroidogenic response.     

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.     

Two active Na+/K+-ATPases of high affinity for ouabain in adult rat brain membranes

The degree of heterogeneity of active Na+/K(+)-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+/K(+)-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 +/- 0.15 nM, 460 +/- 4.0 nM and 320 +/- 4.6 microM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 +/- 0.2 nM (very high) and 80 +/- 1 nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.     

Cobalt activates potassium conductance in the plasma membrane of cultured renal epithelioid (MDCK)-cells

Cobalt has been shown to stimulate sodium transport across the distal nephron of the newt kidney. The mechanism of this action remained elusive. The present study has been performed to test for effects of cobalt on electrical properties of cultured subconfluent kidney (MDCK)-cells: cobalt (10 microM) leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cobalt increases the potassium conductance of the cell membrane. The half-maximal effect is elicited by approx. 1 microM. At extracellular calcium concentration reduced to less than 0.1 microM, cobalt (10 microM) leads to a transient hyperpolarization, which can be elicited only once. Thus, cobalt enhances the potassium conductance in a calcium dependent way. At higher concentrations (100 microM) cobalt hyperpolarizes the cell membrane only transiently even in the presence of extracellular calcium. Furthermore 100 microM cobalt interferes with ATP-induced hyperpolarization, which is known to result from calcium mediated activation of K+ channels. Thus, 100 microM cobalt may inhibit ATP-stimulated calcium entry into the cell.