Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Micropuncture and microperfusion study ofl-glucose secretion in rat kidney

Summary Studies with the free flow micropuncture technique have shown that the ratio of TF/P_l-glucose to TF/P_Inulin in proximal tubular fluid, in distal tubular fluid, and in more than half of the final urine samples measured was greater than one, which suggests thatl-glucose was actively secreted. Studies with the microperfusion technique confirmed this finding and showed thatl-glucose was secreted by the proximal tubules. A maximum rate of secretion was reached at a plasma concentration of 4 mM. The tubular secretion ofl-glucose was augmented by the presence of 16.6 mMd-glucose in tubular lumen and inhibited by 10^–4 M phlorizin. Kinetic analysis showed that theV _max values forl-glucose secretion in the absence and in the presence ofd-glucose are 5.0×10^–10 and 6.3×10^–10 mol×cm^–2×sec^–1 respectively which were very close to the value reported for theV _max ford-glucose reabsorption. However, theK _m forl-glucose secretion was 3.1 mM and was reduced to 1.6 mM whend-glucose was present in the perfusion fluid. TheK _m ford-glucose reabsorption has been reported to be 0.6 mM (8). The results of this investigation were interpreted as being consistant with the hypothesis thatl-glucose secretion andd-glucose reabsorption share the same carrier system.On sabbatical leave from Univ. Louisville, School of Medicine and supported by a NIAMD Special Fellowship (1-F3-AM-32,720-01) and a research grant of USPHS (AMO2217-10).     

Transport of 3-O-methyl-d-glucose into mammalian pancreatic β-cells

Summary The transport and oxidation of 3-O-methyl-d-(U-¹⁴C)glucose was studied in microdissected pancreatic islets of obese-hyperglycemic mice. There was no significant production of¹⁴CO₂ during incubation for 2 h. A comparison with the uptake of sucrose and mannitol indicated that 3-O-methyl-d-glucose was uniformly distributed across the -cell plasma membrane. Externald-glucose inhibited the entry of 3-O-methyl-d-glucose and caused a significant net loss of 3-O-methyl-d-glucose from islets equilibrated with this compound. The transport of 3-O-methyl-D-glucose was also markedly reduced in the presence of phlorizin or phloretin, whereasd-mannoheptulose ord-glucosamine exerted a slight inhibition. The results support our hypothesis that the transport ofd-glucose into the pancreatic -cells is carriermediated, and indicate that 3-O-methyl-d-glucose is a non-metabolizable substrate for this carrier in the pancreatic islets. Since in contrast tod-glucose 3-O-methyl-d-glucose does not stimulte insulin release from the type of islets used, the secretagoric recognition system ford-glucose is probably not identical with the membrane transport system.     

Kinetische Studien derd-Glucoseresorption im proximalen Konvolut der Rattenniere

Zusammenfassung Proximale Konvolute von Rattennieren wurden bei fehlendem Nettofluß von Natriumionen und Wasser kontinuierlich mit Lösungen perfundiert, die eined-Glucosekonzentration zwischen 0,5 und 2,0 mmol/l enthielten. Der Abfall der intraluminalend-Glucosekonzentration entlang eines Konvolutes wurde durch Absaugen der perfundierten Lösung in abnehmender Entfernung von der Perfusionsstelle verfolgt.Die pro innere Tubulusoberfläche und Zeit transportierted-Glucosemenge wird mit Abnahme der intraluminalen Glucosekonzentration kleiner. Dieses Verhalten läßt sich durch eine 2-parametrige Gleichung analog der Michealis-Menten-Kinetik beschreiben. Es errechnet sich eine maximale Transportrate,V _max, von 6 · 10^–10 mol · cm^–2 · sec^–1 und eine Halbsättigungskonzentration,K _m, von 0,6 mmol/l.Die so beschriebene aktive Resorption und die von uns gefundene passive Permeabilität des proximalen Konvolutes fürd-Glucose reichen, nach angestellten Computerberechnungen zu schließen, allein nicht aus, um den Nettoglucosetransport der Gesamtniere unter Freiflußbedingungen quantitativ zu beschreiben.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft und The National Institutes of Health, Grant-No. 1 RO1 AM 10688-01.     

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine

Inward Na⁺ currents associated with the cloned intestinal Na⁺/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (V _m from +20 to –75 mV) and relatively voltage-independent (V _m from –75 to –150 mV) regions. The apparent I _max for Na⁺ and glucose increased with negative membrane potentials, and the apparent K _0.5 for glucose (K _0.5 ^Glc ) depended on V _m and [Na]_o. Increasing [Na]_o from 7 to 110 mmol/l had the same effect in decreasing K _0.5 ^Glc from 0.44 to 0.03 mmol/l as increasing the V _m from –40 to –150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]_o) were identical for d-glucose, d-galactose, -methyl d-glucopyranoside and 3-O-methyl d-glucoside. The specificity of the cotransporter for sugars was: d-glucose, d-galactose, -methyl d-glucopyranoside > 3-O-methyl d-glucoside d-xylose > d-allose d-mannose. K _i for phlorizin ( 10 mol/l) was independent of V _m at saturating [Na]_o. We conclude that a variety of sugars are transported by the cloned Na⁺/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K _0.5 of the cotransporter for Na⁺ and glucose.     

Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50±12% after 3 min and to 27±6% (n=5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (l)] the substrates are taken up. From the l-side only butyrate (10 mmol · l^–1) sustained the Isc at 95±2% (n=7). All other tested substrates (10 mmol · l^–1): pyruvate, acetate, -OH-butyrate,d-glucose, andl-lactate lead to a marked decline in Isc. From the b-side several substrates (5–10 mmol · l^–1) sustained the Isc:d-glucose,d-mannose, butyrate, -OH-butyrate, acetoacetate,l-lactate, acetate and pyruvate. Other compounds (1–10 mmol · l^–1): citrate, -ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of thed-glucose,l-lactate, acetate, pyruvate or -OH-butyrate concentration with apparentK _m's between 0.05–1.0 mmol · l^–1. Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mol · l^–1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition ocurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol · l^–1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. -Cyano-m-OH-cinnamate (10 mmol · l^–1) inhibited the Isc specifically whenl-lactate was the bath substrate. Probenecid (1 mmol · l^–1) had a similar yet less marked inhibitory effect. Thed-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 · 10^–6 mol · l^–1. We conclude that the cTAL segment of the rabbit utilizesd-glucose and/or small anions such as pyruvate orl-lactate or acetate to energize salt reabsorption. The link between substrate availability and salt reabsorption is extremely close in this nephron segment. Substrate uptake occurs from the blood side. Sugar uptake can be inhibited by cytochalasin B andl-lactate uptake by probenecid and -cyano-m-OH-cinnamat. These data suggest that substrate uptake at the basolateral cell side occurs probably via carrier systems.Parts of this study have been presented at the 57th and 58th Tagung Deutsche Physiologische Gesellschaft, 17th Meeting Europ. Soc. Clin. Investigation, and XXIX Int. Congress Physiol. Sciences. This study was supported by Deutsche Forschungsgemeinschaft Gr 480/5-7     

Differenzierung zwischen aktiver und passiver Komponente desd-Glucosetransports am proximalen Konvolut der Rattenniere

Zusammenfassung Der Nettotransport und der unidirektionale Transport vond-Glucose wurden in Mikroperfusionsversuchen am proximalen Konvolut der Rattenniere unter der Bedingung des fehlenden Nettoflusses von Natriumchlorid und Wasser gemessen. Nach vollständiger Hemmung des aktiven Transportanteils durch Phlorrhizin (10^–4 mol/l in der Perfusionslösung) läßt sich eine passive Komponente desd-Glucosetransports nachweisen. Diese ist als additives Glied zum aktiven Transportanteil auch im nicht phlorrhizin-vergifteten Zustand zu beobachten. Der aktive Transportanteil beträgt imV _max Bereich 6·10^–10 mol·cm^–2·sec^–1.Die passive Transportkomponente ist derd-Glucosekonzentrationsdifferenz zwischen Perfusat und Serum im angewandten Konzentrationsbereich direkt proportional. Zwischen den Ergebnissen der Ein- und Ausstrommessung besteht kein signifikanter Unterschied. Es wird daraus geschlossen, daß der passived-Glucosefluß ein einfacher Diffusionsprozeß ist.Der sich aus drei verschiedenen Versuchsanordnungen ergebende mittlere Permeabilitätskoeffizient der proximalen Tubuluswand für Glucose beträgt 1,7·10^–5 cm ·sec^–1. Es ist experimentell möglich, den aktiven Austransport durch passiven Einwärtstransport vollständig zu kompensieren. Dazu ist allerdings eine Konzentrationsdifferenz von 33 mmol/l nötig.Wird der unidirektionale Austransport mit Hilfe von¹⁴C markierter Glucose gemessen, der Nettotransport jedoch chemisch, so kann man die experimentell gemessene Änderung der spezifischen Aktivität mit den aus der Zweikomponentenhypothese (aktiver Transport + passive Diffusion) voraussagbaren Veränderungen vergleichen. Eine Übereinstimmung von Voraussage und Befund ist bei Benutzung der oben angeführten Werte gegeben.Unter normalen Freiflußbedingungen spielt die passive Transportkomponente keine wesentliche Rolle. Nur bei größeren transtubulären Konzentrationsdifferenzen — wie sie bei niedrigem Glomerulumfiltrat und hoher Serumglucosekonzentration auftreten — wird der passive Glucoseeinstrom für died-Glucoseresorption der Gesamtniere bedeutend.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft und durch The National Institutes of Health Grant-No. 1 RO1 AM 10688-01.     

Hexose transport by chicken cecum during development

Hexose accumulation during development has been studied in tissue slices from chicken cecum. The age of birds ranged from 0 to 7 weeks after hatch. Ceca were divided into six portions according to their situation either proximal (PC), medial (MC) or distal (DC) to the ileocecal junction. In 0-day-old chicks all segments can accumulate 3-O-methyl-d-glucose (0.5 mmol/l) against a concentration gradient through a phloridzin-sensitive mechanism.Cumulative capacity is lower in DC than in PC and declines with development. Distal segments lose sugar transport ability 1–2 days after hatch whereas the medial region retains some concentrative ability in older birds. In 7-week chickens, PC slices have a similar cumulative ability to that of jejunum (yolk sac region). Kinetic studies showed that in PC the apparentK _m for phloridzin-sensitive transport was half that in 1-day- than in 7-week-old birds; apparentV _m increased by 50% in this time range. The ability to transport sugars by the cecum was further confirmed in isolated enterocytes from 5- to 7-week-old chickens using -methyl-d-glucoside (0.1 mmol/l) as substrate. Cell sugar concentration was greater in PC than in jejunal cells and jejunal greater than MC enterocytes. Sugar present in cells from DC was the same as in phloridzin-treated cells. It is concluded that cecal epithelium may play a significant role in the absorption of sugars during development.     

Glucose-induced excitation in molluscan central neurons producing insulin-related peptides

The light green cells (LGCs) are a group of identified central neurons in the pond snail, Lymnaea stagnalis, that produce a number of insulin-related peptides. Freshly dissociated LGCs are activated by physiological concentrations of extracellular glucose. The response to glucose consists of a slow depolarization, which, at concentrations of 1 mM or more, rapidly induces regular spiking activity. The response persists during prolonged application of glucose but is completely reversed upon washing. The threshold concentration is 0.2 mM; the maximal effect occurs at 5 mM. In LGCs in central nervous system preparations kept in organ culture for 16–24 h, glucose causes a similar depolarization, which may lead to spiking activity. In freshly isolated preparations, which have very inexcitable LGCs, no direct response to glucose was seen. The response is specific to the LGCs: no other central neurons in Lymnaea showed consistent responses. The glucose response is evoked by d-glucose and the non-metabolized analogue 2-deoxy-d-glucose, but not by related hexoses, including l-glucose, nor pentoses or disaccharides. The response is not affected by interfering with the glucose metabolism, nor is the response mimicked by the metabolite d-glyceraldehyde or by injection of glucose. This suggests that glucose metabolites are not involved in the response. The glucose response depends on the presence of extracellular Na⁺ and is blocked by phlorizin, which specifically inhibits Na⁺-coupled glucose transport. This suggests that the response is due to activation of an electrogenic Na⁺-coupled glucose transporter.     

Insulin enhances l-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on l-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 U/ml) increased l-dopa uptake into PT cells by about 50% (705±186 vs.1117±140 pmol l-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity l-dopa transport sites (l-dopa 0.2 M) (0.59±0.05 vs. 0.82±0.09 pmol l-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1–10 M) or colchicine (50–100 M), whereas it was abolished by cytochalasin D or latrunculin B (both 1 M). This suggests that the process is independent of Na⁺,K⁺-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. l-dopa transport by the low-affinity transport sites (l-dopa 5 M) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal l-dopa reabsorption as compared to control animals (81±4 vs. 51±9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal l-dopa reabsorption.     

l-Lactate andd-lactate carriers on the fetal and the maternal side of the trophoblast in the isolated guinea pig placenta

The transfer of¹⁴C-labelledd- orl-lactate (test substance) relative to³H-l-glucose (control substance, extracellular marker) into the trophoblast of the isolated guinea pig placenta was determined during an open loop perfusion on both sides. Using a single passage, paired tracer dilution technique, the maximal lactate uptake (U _max) was derived from the venous concentration ratio of lactate tol-glucose.A significant metabolism ofl-lactate was not observed. The lactate uptake, which occurred in all placentas studied, was not significantly different on the fetal and maternal side. Within one placenta thel-lactate uptake was always higher than thed-lactate uptake. The uptake of bothl- andd-lactate could be inhibited by phloretin. The lactate uptake was inversely correlated with the pH of the perfusate fluid within the range from 6.2–8. A first order saturation kinetic (Hofstee-plot) was used to approximate the relationship between thel-lactate uptake and the chemicall-lactate concentration. We conclude that similar lactate carriers exist in the membranes on both the maternal and the fetal side of the trophoblast.Supported by the Deutsche Forschungsgemeinschaft     

Effect of rabbit duodenal mRNA on phosphate transport in Xenopus laevis oocytes: dependence on 1,25-dihydroxy-vitamin-D3

Expression of Na⁺-dependent transport of phosphate (P_i) was analysed in Xenopus laevis oocytes after injection of poly(A)-rich RNA isolated from the duodenal mucosa of rabbits with increased levels of 1,25-(OH)₂-VitD₃ (injection of vitamin D₃ or low-P_i diet) or from control animals. In parallel, the effect of elevated levels of vitamin D₃ was studied in isolated duodenal brush-border membrane vesicles. In brush-border membrane vesicles, the rate of Na⁺-P_i cotransport was found to be doubled after 1,25-(OH)₂-VitD₃ injections while Na⁺ — d-glucose cotransport (measured as a control) was not altered. In X. laevis oocytes, Na⁺-dependent P_i uptake was increased after injection of poly(A)-rich RNA isolated from duodenal mucosa of animals with increased levels of 1,25-(OH)₂-VitD₃ but not after injection of poly(A)-rich RNA isolated from control animals; between the two groups of mRNA no difference in the expression of the Na⁺ — d-glucose transport system was observed. Sucrose density gradient fractionation suggests that mRNA species related to the increased Na⁺-dependent P_i uptake are of average chain lengths between 2×10³ and 3×10³ bases (2–3 kb). It is concluded that in duodenal enterocytes 1,25-(OH)₂-VitD₃ increases the content of mRNA species of 2–3 kb that might be involved either directly in Na-P_i cotransport or at least in controlling its activity.     

d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

Coupling of metabolic CO2 production to ion transport in isolated rat thick ascending limbs and collecting tubules

Metabolic CO₂ production from appropriate [U-¹⁴C]-labelled substrates (eitherl-lactate ord-glucose) was measured in single pieces of tubule as previously described (Le Bouffant et al. 1984). Changing the incubate osmotic pressure by mannitol addition resulted in an increase in oxidative metabolism which was more marked in outermedullary segments (MAL and MCT) than in cortical segments (CAL and CCT). Availability of metabolic substrate was not rate limiting under these conditions because FCCP addition (1 mol·l^–1) produced a marked rise in CO₂ production in these structures.Ouabain (1 mmol·l^–1) decreased by more than 50% the CO₂ production by CAL, MAL, CCT and MCT samples, indicating that the larger part of oxidative metabolism was coupled to active Na transport. Furosemide addition (10^–5 mol·l^–1) to CAL and MAL samples, or amiloride addition (10^–4 mol·l^–1) to CCT and MCT samples reduced the rate of CO₂ production to an extent almost similar to that obtained with ouabain, an observation suggesting that apical entry of Na⁺ was present in these non-perfused tubules.Finally, the effects of changing the concentration of either K⁺ or Cl^– was tested in CAL samples. K⁺ suppression greatly depressed the rate of CO₂ production. Replacement of chloride with sulfate also decreased this rate to an extent similar to that observed with furosemide. The CO₂ production increased in a sigmoid way (apparentK _a=41 mmol·l^–1, Hill coefficient=2.12) as a function of [Cl^–] in the incubate, suggesting that oxidative metabolism was coupled to bath chloride via the Cl^–-requiring Na entry along the 1 Na⁺–1K⁺–2Cl^– luminal contrasport system.     

Effects of prostaglandins on Na transport in isolated collecting tubules

Direct tubular effects of prostaglandins (PG's) on Na transport were examined in isolated cortical and medullary collecting tubules of rabbits perfused in vitro. The animals were treated with deoxycorticosterone acetate (DOCA, 1 mg kg^–1 day^–1, i.m.) for 3–6 days before experiments. In the cortical collecting tubules PGE₂ (1.2×10^–7–2.5×10^–5 M), E₁ (1.2×10^–5 M) and F₂(1.2×10^–5 M) added to the bath caused reversible decreases in transtubular potential difference (PD_t). But neither PGE₂ (1.2 ×10^–5 M) added to the perfusate nor PGA₂ (1.2 ×10^–5 M) added to the bath had an effect on PD_t. The net Na absorption was decreased with PGE₂ (1.2×10^–5 M) added to the bath from 8.6±1.36 to 1.5±1.04 pEq cm^–1 s^–1 (P<0.02). In rabbits not pretreated with DOCA, the net Na absorption was reduced from 2.73±0.74 to 1.02±0.74 pEq cm^–1 s^–1 (P<0.01). In the outer medullary collecting tubules PGE₂ (1.2×10^–5 M) added to the bath also caused a reversible decrease in PD_t. It is concluded that PGE₂, F₂ and E₁ inhibit Na absorption in the collecting tubules by acting on the peritubular membrane.     

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe,chlorotetracycline

Uptake of⁴⁵Ca²⁺ was studied in dispersed pancreatic islet cells from non-inbredob/ob-mice. Like whole islets the dispersed cells responded to 20 mMd-glucose with a markedly increased⁴⁵Ca²⁺-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect ofd-glucose could not be reproduced with 3-O-methyl-d-glucose,l-glucose,d-mannose,l-leucine, ord-leucine; however,⁴⁵Ca²⁺ uptake was greater in the presence ofl-leucine as compared withd-leucine.⁴⁵Ca²⁺ uptake by dispersed cells or whole islets was stimulated severalfold by 100 M or more chlorotetracycline. At the concentration of only 10 M, chlorotetracycline had no effect on whole islets and partially inhibited⁴⁵Ca²⁺ uptake by the dispersed cells. The ability ofd-glucose to stimulate⁴⁵Ca²⁺ uptake by islets or dispersed cells remained in the presence of 10 M chlorotetracyline. Islet cell suspensions apparently represent a valid model for studying how Ca²⁺ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca²⁺ probe, attention must be paid to its potential ionophoric activity. At only 10 M, the drug seems to monitor a peripheral pool of Ca²⁺, some of which may reside in normal transport channels.     

Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4Cl-d-Phe6Leu17)VIP

The effect of vasoactive intestinal peptide (VIP) and related peptides [glucagon, secretin, PHI 1-27 (peptide with N-terminal histidine and C-terminal isoleucine)] on renal adenylate cyclase (AC) has been determined in several species. The largest stimulation (4.1±0.5-fold basal) of AC by 1 mol · l^–1 VIP was observed in feline cortical plasma membranes. In rabbit and guinea-pig, VIP increased AC activity 1.5±0.3- and 1.8±0.3-fold respectively but glucagon had no such action. Conversely in the rat glucagon stimulated AC some 3-fold over basal activity whereas VIP had little effect. In dog, cat and mouse both peptides were effective in increasing AC activity. For cat, half-maximal stimulation of cortical plasma membrane AC by VIP was seen at 27.0±9.0 nmol · l^–1 (SEN=9 animals). VIP also increased AC activity in both outer (red) and inner (white) medulla. In feline cortical membranes VIP and PTH (parathyroid hormone) when added in combination were fully additive. However for VIP and glucagon in combination there was no cumulative increase in AC activity, indeed the resultant activity was less than that attained by VIP alone. The VIP analogue (4Cl-d-Phe⁶Leu¹⁷)VIP at 10 mol · l^–1 produced a right shift in the VIP-dose response curve and increased the EC₅₀ from 17.2±5.8 nmol · l^–1 to 132.0±22.2 nmol · l^–1 VIP (SEN=4). There was no reduction in the maximum response elicited by VIP consistent with a competitive type of antagonism by this analogue. PHI-stimulated AC was also reduced by (4Cl-d-Phe⁶-Leu¹⁷)VIP resulting in a similar right shift in the dose response curve. However, this analogue of VIP had no effect on glucagon- or secretin-stimulated AC, indicated by no change in EC₅₀ values.     

Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by -cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na⁺/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.     

The effect of harmaline on intestinal sodium transport and on sodium-dependentd-glucose transport in brush-border membrane vesicles from rabbit jejunum

Harmaline inhibition of sodium uptake and of sodium-dependentd-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, initiald-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrationsd-glucose uptake was stimulated by 10–15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations upt to 2 mM, harmaline did not alter the equilibrium uptake ofd-glucose ord-mannitol. After pre-equlibration with sodium (25 mM),d-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependentd-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane.Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependentd-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.