The effect of bromazepam on psychomotor activity and subjective mood.

The effects of short-term (acute) doses of bromazepam were studied in a double-blind trial with the aid of three dosage groups comprising a total of fifty-five healthy male medical students (who received placebo, and 1.5 mg or 3.0 mg bromazepam, respectively). Subjective well-being was recorded through self-ratings by the volunteers, and the variables of psychomotor function by standard testing instruments. In terms of subjective well-being, fatigue and decreased performance (statistically confirmed throughout) were reported by the probands in all three dosage groups after they were administered either the drug or placebo. None of the dose-effect relationships were statistically significant, although this trend was more pronounced, purely in quantitative terms, in the group that received 3 mg bromazepam than in either the placebo or the 1.5 mg bromazepam group. In the reaction time and in critical flicker-frequency (CFF) testing, the trend mentioned above was confirmed. In the attentiveness and memory span test, learning effects were statistically confirmed in equally uniform fashion. The action of the substance was again not statistically significant. It may be concluded from this that subjective, and also in part objective, fatigue and decreased performance were related to the type of trial design employed, and not, generally speaking, to the action of the substance. However, again independently of the drug's activity, statistical confirmation was obtained of improved performance and/or learning activity in three variables of the alertness testing apparatus. Variables of driving ability were not adversely affected, but--if anything--stabilized. Our investigation studied the single-dose schedules of bromazepam--viz. 1.5 mg and 3 mg--that are most commonly prescribed for patients. The subacute and personality-related effects of the drug will be the subject of a later report.

Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate.

The effect of the extracellular concentration of Pi on the Na+-dependent phosphate transport activity of OK cells was investigated. When incubated with extracellular Pi at concentrations of 200 microM or less, Na+-Pi cotransport increased approximately twofold in OK cells compared with control cells (kept in 0.85 mM Pi), whereas other Na+-dependent transport activities were not affected. After Pi deprivation, Na+-Pi cotransport could be inhibited to a similar extent (80%) by parathyroid hormone (PTH) as in control cells, suggesting that the PTH-sensitive Na+-Pi cotransport activity is also regulated by extracellular Pi. The increase of Na+-Pi cotransport was maximally expressed after 6 h and could be prevented by cycloheximide (70 microM) but not by actinomycin D (0.5-5 g/ml). However, the adaptive response was completely blocked by 3'-deoxyadenosine (cordycepin) at 100 microM. From these data, it is concluded that the upregulation of Na+-Pi cotransport in OK cells due to low extracellular Pi is controlled at a posttranscriptional level.

Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells.

The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (Pi) was investigated in opossum kidney (OK) cells. Up-regulation of Na/Pi cotransport was achieved by incubating OK cells in a medium containing 0.1 mM Pi; down-regulation of Na/Pi cotransport was provoked by refeeding adapted cells with 2 mM Pi. Up-regulation of Na/Pi cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/Pi cotransport similar to that observed after refeeding adapted cells with 2 mM Pi. No effects of the microtubule disrupting drugs were observed on Na/L-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/Pi cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/Pi cotransport systems and that microtubules are not involved in the internalization of Na/Pi cotransport systems.

Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: phosphate "adaptation".

The polarity (apical vs basolateral cell surface) of the up-regulatory response ("adaptation") to low medium phosphate (Pi) concentration on apical and basolateral Pi transport systems was investigated in opossum kidney (OK) cell monolayers grown on permeant supports. Incubation of cultures in low-Pi medium, given either only to the apical or simultanously to the apical and basolateral compartments, increased the rate of transport of both the apical and the basolateral Na/Pi cotransport systems. The basolateral Na-independent, 4,4-diisothiocyanatostilbene-2,2'-disulphonic-acid-sensitive Pi transport system was unaffected by Pi deprivation. Incubation with low-Pi medium from only the basolateral side failed to elicit any "adaptive" response in Pi transport. When cells were Pi-limited either apically or on both sides for short periods of time, adaptation was apparent within 2 h and close to maximal by 6 h, and the alteration in Pi transport was consistant with an increase in Jmax for both the apical and basolateral Na/Pi cotransport systems. These data suggest that apical Na-dependent Pi influx is important in signalling the adaptive response to low extracellular Pi.

Apical and basolateral effects of PTH in OK cells: transport inhibition, messenger production, effects of pertussis toxin, and interaction with a PTH analog.

The cellular distribution (apical vs. basolateral) of parathyroid hormone (PTH) signal transduction systems in opossum kidney (OK) cells was evaluated by measuring the action of PTH on apically located transport processes (Na/Pi cotransport and Na/H exchange) and on the generation of intracellular messengers (cAMP and IP3). PTH application led to immediate inhibition of Na/H-exchange without a difference in dose/response relationships between apical and basolateral cell-surface hormone addition (half-maximal inhibition at approximately 5 x 10(-12) M). PTH required 2-3 hr for maximal inhibition of Na/Pi cotransport with a half-maximal inhibition occurring at approximately 5 x 10(-10) M PTH for basolateral application and approximately 5 x 10(-12) M for apical application. PTH addition to either side of the monolayer produced a dose-dependent production of both cAMP and IP3. Half-maximal activation of IP3 was at about 7 x 10(-12) M PTH and displayed no differences between apical and basolateral hormone addition, while cAMP was produced with a half maximal concentration of 7 x 10(-9) M for apical PTH application and 10(-9) M for basolateral administration. The PTH analog [nle8.18,tyr34]PTH(3-34), (nlePTH), produced partial inhibition of Na/Pi cotransport (agonism) with no difference between apical and basolateral application. When applied as a PTH antagonist, nlePTH displayed dose-dependent antagonism of PTH inhibition of Na/Pi cotransport on the apical surface, failing to have an effect on the basolateral surface. Independent of addition to the apical or basolateral cell surface, nlePTH had only weak stimulatory effect on production of cAMP, whereas high levels of IP3 could be measured after addition of this PTH analog to either cell surface. Also an antagonistic action of nlePTH on PTH-dependent generation of the internal messengers, cAMP and IP3, was observed; at the apical and basolateral cell surface nelPTH reduced PTH-dependent generation of cAMP, while PTH-dependent generation of IP3 was only reduced by nlePTH at the apical surface. Pertussis toxin (PT) preincubation produced an attenuation of both PTH-dependent inhibition of Na/Pi cotransport and 1P3 generation while producing an enhancement of PTH-dependent cAMP generation; these effects displayed no cell surface polarity, suggesting that PTH action through either adenylate cyclase or phospholipase C was transduced through similar sets of G-proteins at each cell surface.

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): effect of parathyroid hormone (PTH).

Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10(-8) M; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10(-8) M); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10(-8) M) resulted in a rapid and transient increase in [Ca2+]i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/Pi cotransport.

Functional asymmetry in phosphate transport and its regulation in opossum kidney cells: parathyroid hormone inhibition.

The sidedness (apical vs basolateral) of the inhibitory of phosphate (Pi) transport by parathyroid hormone (PTH) was investigated in opossum kidney (OK)-cell monolayers grown on permeant support. PTH was found to regulate the activity of only the apical Na Pi cotransporter, having no effect on the basolateral transport systems. Transport inhibition was approximately 100-fold more sensitive to apical PTH application (Kd: 5 x 10(-12) M) than to basolateral application (Kd: 5 x 10(-10) M). The time-course of the inhibitory response was identical from the two cell surfaces, with half-maximum inhibition occurring at about 20 min and almost full inhibition by 90 min. Experiments on diffusion and degradation demonstrated that the difference in Kd at the two cell surfaces was not due to differential metabolism or diffusion. Tests of cooperativity between the apical and basolateral regulatory events at intermediate concentrations suggested that the presence of PTH on one side of the monolayer reduced the scope of response from the other side. At maximum doses of PTH (10(-7)-10(-8) M) the transport inhibition from either side was equal and not additive. We conclude that in OK-cell monolayers grown on permeant support only apical Na/Pi co-transport is sensitive to PTH inhibition and that PTH receptor properties may be different on the apical and basolateral surfaces.

Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: phosphate transport.

The polarized distribution of phosphate (Pi) transport systems in a continuous renal cell line derived from opossum kidney (OK) was measured in monolayers grown on permeant filter support. When cultured on collagen-coated nitrocellulose filters, OK cells formed tight, functionally polarized monolayers. Three Pi transport systems were identified in these monolayers: one apical sodium (Na)-dependent system and two systems on the basolateral surface, one Na-dependent and one Na-independent. The apical system was high-affinity (Km = 0.4 mM Pi), low-capacity (Jmax = 1100 pmol Pi/mg protein per minute) with a Na:Pi stoichiometry greater than 1 (n = 3) and a high interaction coefficient (KNa = 105 mM Na). On the basolateral surface the Na-independent system comprised about 30% of the total Pi transport at this surface. Both basolateral systems were of low affinity (Km: Na-independent, 2.6 mM; Na-dependent, 5.2 mM) and high capacity (Jmax: Na-independent, 2100; Na-dependent, 2400 pmol/mg protein per minute). The basolateral Na-dependent system had a Nai stoichiometry of 1 and a relatively low interaction coefficient (KNa = 25 mM Na). Only the basolateral Na-independent system was inhibitable by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These results are compatible with a net vectorial transcellular transport of Pi from the apical through the basolateral cell surfaces. The presence of a basolateral Na-dependent system may reflect additional metabolic requirements that cannot be met only by apical influx. Taken together, these results demonstrate the ability to grow cell monolayers successfully, displaying polarized transport activities similar to in situ.

Characteristics and regulation of Na/Pi cotransport in a SV-40-transformed rabbit proximal tubular cell line.

An SV-40-transformed cell line of rabbit S2 proximal tubular origin (RKPC-2 cells) was used to characterize Na/P(i) cotransport. P(i) saturation experiments showed simple Michaelis-Menten behaviour and an apparent Km of 106 microM; Hill analysis of Na+ concentration dependence results in an apparent Km for Na+ of about 130 mM and suggests a stoichiometry exceeding unity. Exposure of confluent monolayers to low P(i) medium induced an increase in Na/P(i) cotransport. Incubation with 10(-9) M parathyroid hormone produced a 'paradoxical' stimulation of Na/P(i) cotransport, mimicked by pharmacological activation of protein kinase A or protein kinase C. The above regulatory events, observed on Na/P(i) cotransport, were not observed for Na(+)-dependent amino acid transport (L-proline and/or L-glutamic acid).

Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2).

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition.

We have examined the rapid effect of 1,25-dihydroxyvitamin-D3 [1,25(OH)2D3] on apical Na+/H+ exchange activity in opossum kidney (OK) cells and in MCT cells (a culture of simian-virus-40-immortalized mouse cortical tubule cells) grown on filter support. Addition of 1,25(OH)2D3 (10 nM) for 1 min increased apical Na+/H+ exchange activity [recovery from an acid load; measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] in OK cells (by 56%) and in MCT cells (by 36%). The cellular mechanisms involved in 1,25(OH)2D3-dependent stimulation of Na+/H+ exchange were analysed in OK cells; stimulation of Na+/H+ exchange by 1,25(OH)2D3 was not prevented by actinomycin D. Applying parathyroid hormone (PTH) reduced Na+/H+ exchange activity in OK cells (by 34% at 10 nM, 5 min); 1,25(OH)2D3 "reversed" PTH-induced inhibition, either when PTH was added prior to 1,25(OH)2D3 or when the two agonists were applied together. 1,25(OH)2D3 had no effect on basal OK cell cAMP content or on [Ca2+]i (fura-2). 1,25(OH)2D3 attenuated PTH-induced cAMP accumulation and had no effect on the PTH-dependent increase in [Ca2+]i. These data suggest a regulatory control (stimulation) of proximal tubular brush-border Na+/H+ exchange by 1,25(OH)2D3. This effect is non-genomic and might in part be explained by a release from cAMP-dependent control of transport activity.

Expression of parathyroid hormone receptors in MDCK and LLC-PK1 cells.

Parathyroid hormone (PTH) inhibits renal proximal tubular phosphate (Pi) and bicarbonate reabsorption by regulating the activity of apical Na/Pi cotransport and Na/H exchange. Two renal epithelial cell lines ["proximal tubular", LLC-PK1; "distal tubular", Madin-Darby canine kidney, (MDCK) cells] were stably transfected with complementary deoxyribonucleic acids (cDNAs) encoding a cloned PTH receptor in order to examine the polarity of transfected receptor function and whether or not intrinsic Pi transport is regulated by the transfected PTH receptor. The receptors are functionally coupled to the stimulation of adenosine 3':5' cyclic monophosphate (cAMP) production at both cell surfaces in LLC-PK1 cells, whereas this response is primarily limited to the basolateral surface in MDCK cells. Immunocytochemistry suggests an apical and basolateral localization of the transfected PTH receptor in LLC-PK1 cells and only a basolateral localization in MDCK cells. PTH activation of the transfected receptors is not coupled to the regulation of intrinsic Pi transport in either LLC-PK1 or MDCK cells.

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability.

Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/Pi) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/Pi-cotransport, we measured type II Na/Pi-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10(-8) M) and/or vanadate (10(-3) M) for 3 h led to a non-additive 1.4-fold increase in Na/Pi-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/Pi-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/Pi-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/Pi-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/Pi-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/Pi-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/Pi-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules.

Cloning of a Na/Pi cotransporter from opossum kidney cells.

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Stimulation of Na+/H+ exchange activity by endothelin in opossum kidney cells.

Endothelin-1 (ET-1) controls multiple aspects of kidney function. In this study we have analysed the effects of ET-1 on apical Na+/H+ exchange activity in opossum kidney (OK) cells. ET-1 (at 10(-10) M and 10(-8) M) activated Na+/H+ exchange activity within 5 min of exposure. ET-1 (10(-8) M) prevented PTH-induced (parathyroid hormone; 10(-8) M) inhibition of Na+/H+ exchange activity; it also abolished transport inhibition in response to 10(-3) M IBMX (isobutyl-methylxanthine) and 3 x 10(-7) M TPA (phorbol 12-myristate 13-acetate), but had no effect on the 8-bromo-cAMP-induced (10(-4) M) decrease of transport rate. Basal cAMP content, IBMX- and PTH-stimulated cAMP production were unaffected by ET-1 (10(-8) M). The stimulatory action of ET-1 (10(-8) M) on Na+/H+ exchange activity was prevented by calphostin C (10(-8) M). These data document that OK cells might serve as a useful in vitro model for analysis of cellular mechanisms involved in endothelin action; proteine kinase C activation seems to participate in the observed endothelin effects.

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability.

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6-16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4-6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Cellular mechanisms involved in the acute adaptation of OK cell Na/Pi-cotransport to high- or low-Pi medium.

Variations in dietary phosphate (Pi) intake in rats lead to alterations of renal Pi reabsorption. These effects are associated with corresponding changes in the abundance of the type II Na/Pi-cotransporter protein in proximal tubular brush-border membranes. In the present study we investigated the regulation of the type II Na/Pi-cotransporter in response to high- and low-Pi medium in opossum kidney (OK) cells, an epithelial cell-line of proximal tubular origin. We show that "acute" (4 h) and "chronic" (24 h) exposures of OK cells to high- or low-Pi medium lead to decreases or increases, respectively, in Na/Pi-cotransport activity which are paralleled by alterations in the total cellular amount of the corresponding type II Na/Pi-cotransporter protein (NaPi-4), but not by changes in the amount of the NaPi-4 mRNA. Also in OK cells transfected with the corresponding rat renal type II Na/Pi-cotransporter (NaPi-2) alterations in the Pi concentration in the medium lead to changes in the amount of NaPi-2 protein but not in the amount of NaPi-2 mRNA. Furthermore we show that lysosomal inhibitors prevent the degradation of the transporter, but do not interfere with its inhibition, in response to "acute" exposure of OK cells to high-Pi medium. Inhibition of lysosomal degradation also leads, in control conditions, to an accumulation of the transporter detectable on Western blot. It is concluded that the lysosomal proteolytic pathway is not only involved in the Pi-induced downregulation of the type II Na/Pi-cotransporter but also in its basic turnover.

Inhibition of phosphatidylinositide 3-kinase in OK-cells reduces Na/Pi-cotransport but does not interfere with its regulation by parathyroid hormone.

The importance of phosphatidylinositide 3- kinase(s) [PI 3-kinase(s)] in membrane trafficking processes led us to examine its/their possible role in parathyroid-hormone- (PTH-) induced endocytosis and lysosomal degradation of the type IIa Na/Pi-cotransporter in opossum kidney cells (OK-cells). We used wortmannin, a potent inhibitor of several mammalian PI 3-kinase isoforms, and measured Na/Pi-cotransporter activity and type IIa Na/Pi-cotransporter protein expression; also the induction of a negative dominant subunit (Deltap85) was used to reduce PI 3-kinase activity. Wortmannin and Deltap85 led to a reduction of Na/Pi-cotransport activity but were unable to prevent its inhibition by PTH. Wortmannin led in a dose- and time-dependent manner to a reduction of Na/Pi-cotransport activity and transporter protein expression, and retarded their recovery from PTH-induced inhibition/degradation. The data suggest that a PI 3-kinase "controlled" mechanism is involved in the synthesis (and/or routing) of the apical type IIa Na/Pi-cotransporter in OK-cells.

cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH.

Parathyroid hormone (PTH) leads to the inhibition of Na-Pi cotransport activity and to the downregulation of the number of type II Na-Pi cotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Pi cotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1-34) and PTH-(3-34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Pi cotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (approximately 50%).

Expression of renal transport systems for inorganic phosphate and sulfate in Xenopus laevis oocytes.

As a first step within an experimental strategy (expression cloning) leading to the structural identification of the two brush-border membrane transport systems for phosphate and sulfate, we have studied the expression of Na(+)-dependent uptake of phosphate and sulfate in Xenopus laevis oocytes injected with rabbit kidney cortex poly(A)+ RNA (mRNA). Na(+)-dependent uptake of phosphate and sulfate was stimulated in a dose- and time-dependent manner up to 20-fold as compared to water-injected controls. After fractionation of the mRNA on a sucrose gradient (or by preparative gel electrophoresis), two neighboring fractions were identified to stimulate Na(+)-dependent phosphate uptake (average size: 3.4 kilobases) and Na(+)-dependent sulfate uptake (average size: 3.7 kilobases). The two transport systems can be discriminated by their inhibition by thiosulfate, which reduced sulfate uptake, but not phosphate uptake. Kinetic characterization of the expressed Na(+)-dependent transport activities results in properties similar to those described for transport activity in renal brush-border membrane vesicles.