ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Cardiac Purkinje fibres

Summary The influence of intracellular calcium concentration [Ca²⁺] _i on the steady state membrane currentsi was studied in a range of clamp potentials between –20 and –100 mV. Injection of CaCl₂ or Ca-EGTA (pCa 6) increasedi whereas injection of K-EGTA diminished it. The changes i were attributed to a change in steady state potassium conductance, g_K, by four arguments: i was restricted to potentials negative to –20 mV and depended on clamp potential in an inward rectifying manner. i displayed a reversal potential, E_rev, which followed log [K⁺]₀ with 60 mV for a tenfold change. Since E_rev obtained during Ca injection agreed with E_rev observed during EGTA injection the potassium driving force had to be constant. Wheng _K was blocked by superfusion with 20 mM Cesium neither CaCl₂ nor K-EGTA injection modifiedi .Supported by SFB 38, Membranforschung, project G2     

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (_mc = 12 mV) followed by a slower repolarization (_mc = –7 mV). The intracellular sodium activity, a_iNa⁺, rises from 13.2±2.4 meq/l by 6.7±0.5 meq/l within 5 min, a_iCl^– rises about 3 meq/l above the control value of 37.7±2.2 meq/l, while a_iK is constant (97.7 ±7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (a_iK⁺) is 5.2±0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1±0.1 meq/l).In chloride-free media a_sK⁺=4.2±0.1 meq/l and _mc hyperpolarizes by –13 mV. The depolarization due to glucose absorption increases (_mc = 14.1 ± 1.4 mV) and the repolarization ( _mc ^repol ) disappears. In addition, a_iNa⁺ rises from 16.3±2.4 meq/l by 9.9±1.5 meq/l within 5 min, a_iK⁺ remains constant and equal to the value in chloride containing solutions (88.5±2.8 meq/l); a_sK⁺ increases transiently (1.1±0.1 meq/l).Serosal Ba²⁺ (5 mM) depolarizes _mc (+14.2±1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes _mc and abolishes the repolarization.These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca²⁺, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.     

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Transport ofl-lysine by rat intestinal brush border membrane vesicles

Brush border membranes were isolated from rat jejunum by a divalent cation precipitation method.³H-l-Lysine uptake was measured by a rapid filtration technique. Uptake after prolongued incubation periods was osmotically insensitive and represented almost exclusively binding to the vesicles. Extrapolating initial linear uptake to a zero incubation time indicated no binding of the amino acid to the external membrane surface.Sodium did not significantly alter the initial uptake rate.l-Lysine transport respresents a carrier mediated uptake in the presence and absence of sodium as indicated by the transstimulation experiments. The transport mechanism operates stereospecifically and is inhibited by other basic amino acids andl-leucine andl-phenylalanine. Saturation experiments result in aK _m of 0.26 mmoles/l and aV _max of 272 pmoles/mg protein/10 s.Inside negative anion diffusion potentials and inside negative potassium diffusion potentials (valinomycin) were unable to increase the transport rate. Transmembrane pH-gradients were also unable to alter transport.Abbreviations HEPES N-2-hydroxethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycolbis-(-aminoethyl-ether)-N,N tetraacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic β-cells

We have examined the temporal relationship between intracellular Ca²⁺ concentration ([Ca²⁺]_i) and secretion in single intact pancreatic -cells. Secretion was detected as the release of 5-hydroxytryptamine from pre-loaded -cells, using amperometry, and changes in [Ca²⁺]_i were monitored by microfluorimetry. Stimulation of -cells by elevation of the extracellular K⁺ concentration ([K⁺]_o), acetylcholine or glucose increased [Ca²⁺]_i and, after a delay of 2–7 s, evoked amperometric currents. In the presence of glucose, we observed oscillations in [Ca²⁺]_i which were associated with oscillations in the amplitude and frequency of amperometric currents: however, the temporal correlation was not exact, suggesting that there is a significant latency between the increase in average [Ca²⁺]_i and exocytosis. Both the amplitude and frequency of the amperometric currents elicited by 50 mM KCl declined with successive stimulation, but were restored by agents which elevate intracellular adenosine 3'5:cyclic monophosphate (cAMP). This suggests that -cells may possess a readily releasable pool of granules which is replenished by cAMP. The variable amplitude of the amperometric currents is discussed in terms of a model in which several secretory granules fuse simultaneously with the plasma membrane.     

Pharmacological characterization of P2X7 receptors in rat peritoneal cells

Objective and design: P2X₇ receptor activation by ATP results in the release of IL-1 and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X₇ receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X₇ receptor (1321rP2X₇-11).Materials and methods: RPC were isolated from rats by lavage. P2X₇ agonist induced pore formation in RPC was measured by EtBr uptake. P2X₇-stimulated pore formation and Ca⁺⁺ influx in 1321rP2X₇-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl –2-4-disulfonic acid (PPADS) on pore formation and Ca⁺⁺ influx were examined in both RPC and 1321rP2X₇-11. P2X₇-mediated IL-1 release in RPC and the effect of PPADS were determined.Results: RPC express functional P2X₇ receptors that were activated by ATP analogs with a rank order of potency of 2- 3-O-(4-Benzoylbenzoyl) adenosine 5-triphosphate (BzATP) > ATP > ,-methylene ATP. Activation of P2X₇ receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X₇-11 cells. Activation of P2X₇ receptors on RPC resulted in IL-1 secretion, which was inhibited by PPADS.Conclusions: RPC express functional P2X₇ receptors that form pores and mediate the release of IL-1.Received 20 July 2004; returned for revision 9 September 2004; accepted by N. Boughton-Smith 5 November 2004     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Modification of the Na+ current conducted by the rat skeletal muscle α subunit by coexpression with a human brain β subunit

Sodium channels are multimeric structures composed of and subunits. Oocytes injected with RNA encoding only the subunit express voltage-gated Na⁺ currents. The kinetics of inactivation, however, are abnormal. Co-injection of rat brain and subunits modifies inactivation of I_Na such that it closely resembles endogenous currents [1]. Here we show that a subunit derived from human brain directs the same functional modification of I_Na expressed by a rat skeletal muscle subunit. This implies that functional domains for the interaction of and subunits are highly conserved across both tissues and species.     

Intracellular potassium activity in epithelial cells of frog fundic gastric mucosa

Microelectrodes were used to measure membrane potential and intracellular potassium activity in surface epithelial cells (SEC) of frog (Rana esculenta) fundic gastric mucosa in vitro. Separate measurements were carried out by applying fine-tipped, single barrelled, KCl filled non-selective electrodes and liquid K⁺-selective electrodes. Membrane potentials with respect to the mucosal and serosal surfaces, measured with non-selective electrodes, were –54.5±1.0 S.E. mV (n=59) and –73.0±1.1 S.E. mV (n=59) respectively. The electrical potential difference referred to the mucosal surface, when measured with K⁺-sensitive electrodes, was +21.2±0.8 S.E. mV (n=35), and intracellular K⁺ activity was 98.5 mmol/l. Assuming that intracellular and extracellular K⁺ activity coefficients are equal (_K=_K), the K⁺ concentration is 135.0 mmol/l. The K⁺ equilibrium potential,E _K, was calculated as –90.0 mV i.e. more negative than both membrane potentials. This result indicates active potassium accumulation in the SEC and provides direct evidence of the presence of an active K⁺ pump in either both or in only one of the cell membranes.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Distribution of anti-F(ab′)2 antibodies and the 16/6 idiotype in systemic lupus erythematosus (SLE) probands and kindreds

Levels of serum anti-F(ab)₂ antibodies and expression of the 16/6 anti-DNA idiotype were studied in 103 sera from first-degree relatives of 17 systemic lupus erythematosus (SLE) kindreds. Among healthy SLE relatives, 35.9% showed anti-F(ab)₂ elevations and 24%, Id 16/6 expression. Forty-three and two-tenths percent of healthy SLE relatives with elevated anti-F(ab)₂ also showed expression of 16/6; when Id 16/6 was positive, 16 of 25 relatives (64%) showed parallel elevations of anti-F(ab)₂. However, within individual families, distribution patterns of elevated anti-F(ab)₂ and Id 16/6 often did not coincide. Affinity-isolated anti-F(ab)₂ from four members of a single SLE kindred showed relative enrichment for Id 16/6 in only two of the four individuals studied. Moreover, none of the isolated anti-F(ab)₂ antibodies within this kindred or another kindred showing 16/6 Id expression reacted directly with 16/6 Id. Our studies suggest that whereas both anti-F(ab)₂ and Id 16/6 are increased within SLE kindreds, expression of the two does not always coincide. Furthermore, anti-F(ab)₂ antibodies do not show direct reactivity with Id 16/6. A number of anti-DNA idiotypic markers may play a role in idiotypic networks among such SLE kindreds.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Kinetics ofl-histidine transport in the proximal convolution of the rat nephron studied using the stationary microperfusion technique

Summary The kinetics ofl-histidine reabsorption by the proximal convolution of the rat nephron have been studied by stationary microperfusion with simultaneous perfusion of peritubular capillaries. Steady-state concentrations (C ) and transepithelial concentration differences (c ) were determined over a wide range of peritubular bistidine concentrations. It was found that c increased hyperbolically with increase in luminal and peritubular histidine concentrations suggesting saturation transport kinetics. Furthermore c declined linearly along the convolution suggesting that nett active transport was not constant throughout the tubule. Using an expression to describe the rate of attainment of steady-state concentration in terms of lummal and peritubular histidine concentrations, histidine permeability coefficient (P), the maximum rate of active histidine transport (J _max) and the half saturation constant of the transport reaction (K _m ), we were able to determine the cause of the tubule inhomogeneity. We find thatP (14.1×10^–5 cm/s) andJ _max (45×10^–10 mol/cm²· s) are constant along the convolution but thatK _m increases markedly from about 5.4 mmol/kg 26% of the way along the convolution to 40 mmol/kg at 86%. These findings suggest that the histidine reabsorptive mechanism would be relatively inefficient with histidinuria occurring at all plasma concentrations but it would have enormous reserve capacity so that saturation would not readily occur. This prediction accords with available data on histidine clearance in the rat.A preliminary report of this work was presented to a Regional Meeting of the International Union of Physiological Sciences, held at the University of Sydney in Auguat 1972 [7]. The project was supported by the National Health and Medical Research Council of Australia, The Australian Kidney Foundation, The Max-Planck-Gesellschaft and by the Post-Graduate Medical Foundation of the University of Sydney.Mrs. Lingard was the recipient of an Australian Commonwealth Post-Graduate Studentship.     

Characterization ofcpd-1 andcpd-2 mutants which affect the activity of orthophosphate regulated cyclic phosphodiesterase inNeurospora

Summary Enzymatic and genetic characterization ofcpd-1 andcpd-2, which exhibit rhythmic conidiation in liquid media and on solid media, were described with band (bd) strain as a reference.Cpd-1 andcpd-2 showed reduced growth in orthophosphate-free cyclic 3,5-AMP media, whilebd showed wild-type level of growth in the media. In low-phosphate media,cpd-1 andcpd-2 produced 19.2% and 9.8% of orthophosphate-regulated cyclic phosphodiesterase (cPDase) in culture media, while bd produced 123%. The intracellular levels of cPDase with K_m of 1 × 10^–5 M in high-phosphate media incpd-1, cpd-2 and bd were about 20%, 15%, and 10% of that in wild-type, respectively. In low-phosphate media, roughly equal levels of cPDase with K_m of 1 × 10^–5 M were produced in all strains, whereas the production of cPDase with K_m of 2 × 10^–3 M was reduced incpd-2, and that of cPDase with K_m of 1 × 10^–2 was reduced incpd-1 andcpd-2. The levels of intracellular cyclic 3,5-AMP incpd-1, cpd-2, andbd in high-phosphate media were 13.1%, 10.1%, and 69.6% of that in wild-type. Adenylate cyclase activity incpd-1, cpd-2, bd, andcr-1 was 69.3%, 34.0%, 63.2%, and 20.3% of that of wild-type (74A). The levels of Mg⁺⁺-stimulated cyclic phosphodiesterase incpd-1, cpd-2, bd, andcr-1 at 0.2M cyclic 3,5-AMP were 199%, 137%, 329%, and 293% of that of wild-type. It was suggested thatcpd-1, cpd-2, andbd are the genes controlling the levels of enzymes for cyclic 3,5-AMP.Cpd-I was mapped on LG IVR 19.4% distal topyr-2 andcpd-2 was mapped on LG IL 5.6% proximal toarg-1.