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Summary The cellular distribution (apicalvs. 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).

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.

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. Examination of the intracellular Ca2+ concentration ([Ca2+]i; using fura-2) and cAMP content (using a cAMP binding protein assay) revealed only PTH-dependent stimulation of adenylate cyclase. These results suggest that PTH (mediated by protein kinase A) inhibits in RKPC-2 cells structurally distinct Na/H-exchange activities (NHE-2 and NHE-1); we assume apical location of NHE-2 (‘amiloride-resistant’) and basolateral location of NHE-1 (‘amiloride-sensitive’) isoforms of Na/H-exchange activities.

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: 5×10-12 M) than to basolateral application (Kd: 5×10-10 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.

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.

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.

Abstract:  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.

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.

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 (10-8m; 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 10-8m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10-8m) 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.

Abstract:  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.

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.

Parathyroid hormone (PTH) inhibits proximal tubular brush border membrane Na+/Picotransport activity; this decrease in the transport activity was found to be associated with a decrease in type II Na+/Picotransporter protein content in rat brush border membranes. In the present study we investigated the PTH-dependent regulation of the type II Na+/Picotransporter in opossum kidney cells, a previously established model to study cellular mechanisms involved in the regulation of proximal tubular Na+/Picotransport. We transfected opossum kidney cells with a cDNA coding for NaPi-2 (rat renal type II Na+/Picotransporter). This allowed the study of PTH-dependent regulation of the transfected NaPi-2 and of the corresponding intrinsic cotransporter (NaPi-4). The results show (i) that the intrinsic and the transfected cotransporters are functionally (transport) and morphologically (immunofluorescence) localized at the apical membrane, (ii) that the intrinsic as well as the transfected Na+/Picotransport activities are inhibited by PTH, (iii) that PTH leads to a retrieval of both cotransporters from the apical membrane, (iv) that both cotransporters are rapidly degraded in response to PTH, and (v) that the reappearance/recovery of type II Na+/Picotransporter protein and function from PTH inhibition requires de novoprotein synthesis. These results document that PTH leads to a removal of type II Na+/Picotransporters from the apical membrane and to their subsequent degradation.