Selective tonic inhibition of G-6-Pase catalytic subunit, but not G-6-P transporter, gene expression by insulin in vivo

The regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6-P) transporter gene expression by insulin in conscious dogs in vivo and in tissue culture cells in situ were compared. In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6-P transporter mRNA was unchanged. In contrast, a 5-h period of hyperinsulinemia resulted in a suppression of both G-6-Pase catalytic subunit and G-6-P transporter gene expression. Similarly, insulin suppressed G-6-Pase catalytic subunit and G-6-P transporter gene expression in H4IIE hepatoma cells. However, the magnitude of the insulin effect was much greater on G-6-Pase catalytic subunit gene expression and was manifested more rapidly. Furthermore, cAMP stimulated G-6-Pase catalytic subunit expression in H4IIE cells and in primary hepatocytes but had no effect on G-6-P transporter expression. These results suggest that the relative control strengths of the G-6-Pase catalytic subunit and G-6-P transporter in the G-6-Pase reaction are likely to vary depending on the in vivo environment.     

Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats

High-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates. The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling. Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days. The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs. STD (0.40 +/- 0.10). G-6-Pase activity was significantly higher in HSD and HFD vs. STD in both intact and disrupted liver microsomes. HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only. Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups. Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase. Tracer-estimated glucose phosphorylation and glucose cycling (glucose <--> glucose 6-phosphate) were significantly higher in cells isolated from HSD only. After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol. mg protein(-1). min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs. HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9). Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit. Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.     

Membrane phosphatase activities in isolated and cultured adult rat hepatocytes

Membrane bound phosphohydrolysing enzymes, such as Na-K-ATPase, Mg-ATPase, ALPase and G-6-Pase were assayed in intact liver, in freshly isolated cells and in cultured hepatocytes to evaluate the effects of the isolation procedure and culture on these enzyme activities. Na-K-ATPase and Mg-ATPase are significantly reduced following cell dispersion while ALPase and G-6-Pase are nearly unaffected. During culture, Na-K-ATPase is restored to the "in vivo" level within the first two days, but rapidly declines in the following days. The Na dependent, energy requiring AIB uptake shows a similar pattern; Mg-ATPase is practically unmodified. A significant increase in ALPase activity and a net decrease of G-6-Pase activity, as a function of the culture time has been observed.     

Properties and regulation of organic cation transport in freshly isolated human proximal tubules.

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.     

Biochemical, functional, and morphological characterization of a primary culture of rabbit proximal tubule cells.

Primary cultures of renal rabbit proximal tubule cells were initiated from a pure suspension of proximal tubule fragments. Proximal tubule cells were grown in a hormone-supplemented, serum-free medium containing low concentrations of antibiotics. Confluent monolayers exhibited multicellular dome formation, indicating the presence of transepithelial solute and water transport. Ultrastructural examination revealed a monolayer of polarized epithelial cells with tight junctions and sparse membraneous microvilli facing the culture medium. Time course biochemical characterization was performed using a palette of 12 enzymes, representative of important metabolic functions or pathways. Brush-border-associated enzymes (gamma-glutamyl transpeptidase and alanine aminopeptidase) were moderately reduced throughout the culture whereas alkaline phosphatase was markedly decreased at confluency. Mitochondrial and lysosomal marker enzymes were well preserved over the culture period. Glutathione-S-transferase activity remained stable during the 16-day culture period investigated. Glycolysis enzyme activities (lactate dehydrogenase and hexokinase) were enhanced, as a function of culture age. Na(+)-K(+)-ATPase activity rise was concomitant with the increase of glycolysis marker enzymes. In contrast, the gluconeogenesis marker enzyme, glucose-6-phosphatase, fell dramatically to reach a low level equivalent to 4% of the activity measured in isolated proximal tubules. Primary cultures exhibited several differentiated functions of the proximal tubule cell: (a) PTH alone was able to induce a significant stimulation of adenylate cyclase activity, unlike isoproterenol, thyrocalcitonin, and arginine vasopressin, and (b) sodium-dependent alpha-methylglucoside (AMG) transport was detected. This AMG uptake was selectively inhibited by phlorizin (5 X 10(-3) M), which is a competitive inhibitor of glucose uptake at the apical membrane. Complete characterization made it possible to investigate hitherto unexplored aspects of in vitro cultured proximal tubule cells. This primary culture model could provide a useful and reliable tool to investigate in vitro renal proximal tubule function, under normal conditions or after a drug-induced toxicity.     

Bile acids regulate hepatic gluconeogenic genes and farnesoid X receptor via G��i-protein-coupled receptors and the AKT pathway

Bile acids are important regulatory molecules that can activate specific nuclear receptors and cell signaling pathways in the liver and gastrointestinal tract. In the current study, the chronic bile fistula (CBF) rat model and primary rat hepatocytes (PRH) were used to study the regulation of gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) and the gene encoding short heterodimeric partner (SHP) by taurocholate (TCA). The intestinal infusion of TCA into the CBF rat rapidly (1 h) activated the AKT (∼9-fold) and ERK1/2 (3- to 5-fold) signaling pathways, downregulated (∼50%, 30 min) the mRNA levels of PEPCK and G-6-Pase, and induced (14-fold in 3 h) SHP mRNA. TCA rapidly (∼50%, 1–2 h) downregulated PEPCK and G-6-Pase mRNA levels in PRH. The downregulation of these genes by TCA was blocked by pretreatment of PRH with pertussis toxin (PTX). In PRH, TCA plus insulin showed a significantly stronger inhibition of glucose secretion/synthesis from lactate and pyruvate than either alone. The induction of SHP mRNA in PRH was strongly blocked by inhibition of PI3 kinase or PKCζ by specific chemical inhibitors or knockdown of PKCζ by siRNA encoded by a recombinant lentivirus. Activation of the insulin signaling pathway appears to be linked to the upregulation of farnesoid X receptor functional activity and SHP induction.     

Glucose-6-phosphatase as a cytochemical marker of endoplasmic reticulum in human leukocytes and platelets

Leukocytes and platelets, freshly isolated from normal human blood, were tested cytochemically for glucose-6-phosphatase (G-6-Pase) by a modified Wachstein-Meisel method. The enzyme was present in the endoplasmic reticulum (ER) and perinuclear cisternae of all five types of leukocytes and in the ER of platelets. The reaction product from the cytochemical test distinguished the ER from other intracellular membrane-limited cisternae (i.e., the smooth pinocytic tubules of monocytes and the surface-connected canalicular system of platelets) and thus is a valuable marker of the ER. The cytochemical test also showed that the ER of polymorphonuclear leukocytes (PMN), usually obscured by abundant granules in cells prepared for morphological examination, is more extensive than formerly appreciated. This is the first demonstration of G-6-Pase in human leukocytes. Its precise role in leukocyte metabolism can now be investigated.     

A high-sucrose diet increases gluconeogenic capacity in isolated periportal and perivenous rat hepatocytes

A high-sucrose (SU) diet increases gluconeogenesis (GNG) in the liver. The present study was conducted to determine the contribution of periportal (PP) and perivenous (PV) cell populations to this SU-induced increase in GNG. Male Sprague-Dawley rats were fed an SU (68% sucrose) or starch (ST, 68% starch) diet for 1 wk, and hepatocytes were isolated from the PP or PV region of the liver acinus. Hepatocytes were incubated for 1 h in the presence of various gluconeogenic substrates, and glucose release into the medium was used to estimate GNG. When incubated in the presence of 5 mM lactate, which enters GNG at the level of pyruvate, glucose release (nmol x h(-1) x mg(-1)) was significantly increased by the SU diet in both PP (84.8 +/- 3.4 vs. 70.4 +/- 2.6) and PV (64.3 +/- 2.5 vs. 38.2 +/- 2.1) cells. Addition of palmitate (0.5 mM) increased glucose release from lactate in PP cells by 11.6 +/- 0.5 and 20.6 +/- 1.5% and in PV cells by 11.0 +/- 4.4 and 51.1 +/- 9.1% in SU and ST, respectively. When cells were incubated with 5 mM dihydroxyacetone (DHA), which enters GNG at the triosephosphate level, glucose release was significantly increased by the SU diet in both cell types. In contrast, glucose release from fructose (0.5 mM) was significantly increased by the SU diet in PV cells only. These changes in glucose release were accompanied by significant increases in the maximal specific activities of glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in both PP and PV cells. These data suggest that the SU diet influences GNG in both PP and PV cell populations. It appears that SU feeding produces changes in GNG via alterations in at least two critical enzymes, G-6-Pase and PEPCK.     

大鼠活体注射台盼蓝后肝和肾的组织学与组织化学变化的观察

大鼠连续4天腹腔注射1%台盼蓝后,观察隔天、隔周、隔二周后肝和肾的组织结构及PAS、AlP、AcP、G-6-Pase、Mg~(2+)-ATPase和SDH的活性变化。结果发现:肝细胞和肾小管的上皮细胞中有台盼蓝颗粒;肝PAS反应阴性;隔天后肝AlP、AcP、G-6-Pase和SDH活性增强,Mg~(2+)-ATPase活性减弱;肾的上述组化反应活性都减弱;隔二周后肝和肾的上述组化反应接近对照。实验结果提示:活体注射台盼蓝对肝和肾未构成实质性损伤,隔天后的组化变化可能是一种生理适应性反应。     

Ultrastructure and osmoregulatory function of the kidney in larvae of the Persian sturgeon Acipenser persicus

The localization of Na(+) , K(+) -ATPase (NKA) and the ultrastructural features of kidney were examined in larvae of the Persian sturgeon Acipenser persicus (L 31-41 mm total length and 182·3-417·3 mg). Investigations were conducted through light and electron microscopy and through immunofluorescence for NKA detection. The kidney nephrons consisted of a large glomerulus and tubules (neck, proximal, distal and collecting), which connected to the ureters. Posteriorly, ureters extended and joined together into a thin-walled ureter terminal sac. Ultrastructurally, the glomerular cells (podocytes) possessed distinctive pedicels that extended to the basal membrane. The proximal tubule (PT) showed two different cells. The cells lining the anterior part of PT possessed apical tall microvilli (c. 2·7 μm), a sub-apical tubular system, a basal nucleus and dense granules. Posteriorly in the cells, the sub-apical tubular system and granules were absent and round mitochondria associated with basolateral infoldings were found; the apical microvilli were reduced. Distal and collecting tubular cells showed the typical features of osmoregulatory cells, i.e. well-developed basolateral infoldings associated with numerous mitochondria. No immunofluorescence of NKA was detected in the glomeruli. A weak immunostaining was observed at the basolateral side of the cells lining the neck and PT. A strong immunostaining of NKA was observed in the entire cells of the distal tubules, collecting tubules and in some isolated cells of the ureters. In all immunostained cells, the basolateral region showed a much higher fluorescence and nuclei were immunonegative. In conclusion, the epithelial cells of kidney tubules had morphological and enzymatic features of ionocytes, particularly in the distal and collecting tubules. Thus, the kidney of A. persicus larvae possesses active ion exchange capabilities and, beside its implication in excretion, participates in osmoregulation.     

Antigenic expression of aminopeptidase M,dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Summary Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Organ culture of adult mouse intestine IV. Stimulation of glucose-6-phosphatase in vitro.

Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase in measured during the first 24 h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.     

Carnitine metabolism in isolated rat kidney cortex tubules

Renal carnitine metabolism was studied in isolated kidney cortex tubules from fed rats. The tubular distribution of free carnitine (C), acid-soluble short chain acylcarnitine (AcC), and total acid-soluble carnitine was measured. The content of the last-mentioned in rat cortical tubule suspensions was 2.85 +/- 0.15 nmol/mg protein, 46% representing AcC. In the absence of metabolic substrates the AcC/C ratio declined from 0.84 to 0.48 during incubation. The administration of 2mM acetoacetate or 2mM 3-hydroxybutyrate caused an increase in AcC by 45% and 51%, respectively. The rise in AcC was paralleled by a decrease in C, resulting in an increase of the tubular AcC/C ratio to 1.69 and 1.85, respectively. In the presence of 1 mM exogenous L-carnitine 35 +/- 6 nmol AcC/(mg protein X h) was formed. The addition of acetoacetate and 3-hydroxybutyrate led to a 3.5 to 3.8-fold rise in AcC formation. Other substrates which are likewise metabolized by proximal tubules were less effective. More than 90% of the formed AcC was recovered in the extracellular fluid. The results suggest that proximal renal tubule cells are the intrarenal site of carnitine acylation and may be involved in the regulation of blood and/or urinary carnitine acylation state.     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Activation of liver G-6-Pase in response to insulin-induced hypoglycemia or epinephrine infusion in the rat

This study was conducted to test the hypothesis of the activation of glucose-6-phosphatase (G-6-Pase) in situations where the liver is supposed to sustain high glucose supply, such as during the counterregulatory response to hypoglycemia. Hypoglycemia was induced by insulin infusion in anesthetized rats. Despite hyperinsulinemia, endogenous glucose production (EGP), assessed by [3-(3)H]glucose tracer dilution, was paradoxically not suppressed in hypoglycemic rats. G-6-Pase activity, assayed in a freeze-clamped liver lobe, was increased by 30% in hypoglycemia (P < 0.01 vs. saline-infused controls). Infusion of epinephrine (1 microg x kg(-1) x min(-1)) in normal rats induced a dramatic 80% increase in EGP and a 60% increase in G-6-Pase activity. In contrast, infusion of dexamethasone had no effect on these parameters. Similar insulin-induced hypoglycemia experiments performed in adrenalectomized rats did not induce any stimulation of G-6-Pase. Infusion of epinephrine in adrenalectomized rats restored a stimulation of G-6-Pase similar to that triggered by hypoglycemia in normal rats. These results strongly suggest that specific activatory mechanisms of G-6-Pase take place and contribute to EGP in situations where the latter is supposed to be sustained.     

Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.     

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine

Human proximal tubular (PT) epithelial cells were isolated from urine and monoclonally cultured as monolayers for 1 wk, after which they were subcultured between two layers of collagen gel, designated a "collagen gel sandwich." Under these culture conditions, PT cells formed three-dimensional tubular structures exhibiting distinct polarized cell morphology. Scanning and transmission electron microscopic studies showed that they bore numerous microvilli at the apical surface and that they closely contacted the collagen gel at the basal surface. These studies indicate that PT cells exfoliated in urine still exhibit the potential to proliferate and form organized structures mimicking in vivo tubules. Because of the current lack of useful culture systems for human tubular epithelial cells originating from kidney tissue, we suggest that this unique culture system using voided PT cells in urine could open up new avenues to study not only the mechanisms of morphogenesis but also the physiology of human PT cells.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)