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Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 +/- 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa. Funding Agencies|European TRANS-INT Consortium; European Union [281035]; TRANS-INT; Swedish Research Council [2822, 2020-01586]; FRS-FNRS (Fonds de la Recherche Scientifique) , Belgium; Competitive Reference Groups, Conselleria de Educacion e Ordenacion Universitaria, Xunta de Galicia [ED431C 2021/17]; European Commission, Education, Audiovisual and Culture Executive Agency (EACEA) [20120028]; Swedish Government, Sweden

The oral delivery of bioactive peptides and proteins is prevented by the intestinal epithelial barrier, in which intercellular tight junction complexes block the uptake of macromolecules. Here, we show that anionic nanoparticles induce tight junction relaxation, increasing intestinal permeability and enabling the oral delivery of proteins. This permeation-enhancing effect is a function of nanoparticle size and charge, with smaller (≤ 200 nm) and more negative particles (such as silica) conferring enhanced permeability. In healthy mice, silica nanoparticles enabled the oral delivery of insulin and exenatide, with a 10 U/kg of orally delivered insulin sustaining hypoglycaemia for a few hours longer than a 1 U/kg dose of subcutaneously injected insulin. In healthy, hyperglycaemic and diabetic mice, the oral delivery of 10 U/kg of insulin led to a dose-adjusted bioactivity of, respectively, 35%, 29%, and 23% compared to 1 U/kg subcutaneously injected insulin. The permeation-enhancing effect of the nanoparticles was reversible, non-toxic, and attributable to the binding to integrins on the surface of epithelial cells.

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Negatively charged nanoparticles of about 50 nm in size permit the oral delivery of insulin and other peptide drugs by temporally enhancing the permeability of the intestinal wall.

Genes and proteins show variable expression patterns throughout the human body. However, it is not clear whether relative differences in mRNA concentrations are retained on the protein level. Furthermore, inter-individual protein concentration variability within single tissue types has not been comprehensively explored. Here, we used the Gini index for in-depth concentration variability analysis of publicly available transcriptomics and proteomics data, and of an in-house proteomics dataset of human liver and jejunum from 38 donors. We found that the transfer of concentration variability from mRNA to protein is limited, that established ‘reference genes’ for data normalization vary markedly at the protein level, that protein concentrations cover a wide variability spectrum within single tissue types, and that concentration variability analysis can be a convenient starting point for identifying disease-associated proteins and novel biomarkers. Our results emphasize the importance of considering individual concentration levels, as opposed to population averages, for personalized systems biology analysis.

Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation-including brush-border hydrolases, integrins, and adherens and tight junctions-gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (

Nanoparticulate based drug delivery systems have been extensively studied to efficiently encapsulate and deliver peptides orally. However, most of the existing data mainly focus on the nanoparticles as a drug carrier, but the ability of nanoparticles having a biological effect has not been exploited. Herein, we hypothesize that nanostructured lipid carriers (NLCs) could activate the endogenous glucagon-like peptide-1 (GLP-1) secretion and also act as oral delivery systems for GLP-1 analogs (exenatide and liraglutide). NLCs effectively encapsulated the peptides, the majority of which were only released under the intestinal conditions. NLCs, with and without peptide encapsulation, showed effective induction of GLP-1 secretion in vitro from the enteroendocrinal L-cells (GLUTag). NLCs also showed a 2.9-fold increase in the permeability of exenatide across the intestinal cell monolayer. The intestinal administration of the exenatide and liraglutide loaded NLCs did not demonstrate any glucose lowering effect on normal mice. Further, ex vivo studies depicted that the NLCs mainly adhered to the mucus layer. In conclusion, this study demonstrates that NLCs need further optimization to overcome the mucosal barrier in the intestine; nonetheless, this study also presents a promising strategy to use a dual-action drug delivery nanosystem which synergizes its own biological effect and that of the encapsulated drug molecule.

Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na(+)-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved hepatocytes.

Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM β-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.

We recently published an analysis in Trends in Pharmacological Sciences, assessing the impact of transporters on cellular drug permeability based on quantitative data for drug transport kinetics and transporter expression levels [1,2]. Our aim was to explore what properties transporters would need to have if – as hypothesized in a series of publications over the past few years – drug diffusion across the lipid parts of cell membranes is negligible, and transporter-mediated processes explain all experimental observations of cellular drug flux.

In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro–in vivo extrapolation are reviewed.

During calcification of bone, large amounts of phosphate (Pi) must be transported from the circulation to the osteoid. Likely candidates for osteoblast Pi transport are the type

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

Aim The present study was set up to develop a protocol for detection of Enterococcus faecalis and Enterococcus faecium from the root canal. Methodology A collection of type strains and clinical isolates of E. faecalis and E. faecium was used. Specific polymerase chain reaction (PCR) primers targeted against the 16S/23S rDNA intergenic region were used and PCR reactions were set up. PCR products were run on TBE-agarose gel and analysed. The sensitivity of the PCR system was studied using serial dilutions of (i) bacterial DNA and (ii) bacterial cells from E. faecalis. The specificity of the identification was tested against closely related species. Results All strains of E. faecalis and E. faecium produced identical amplicon profiles composed of two major bands corresponding to sizes of 320 and 420 bp. When amplifying DNA of higher purity, a third band of 600 bp became evident as well. Closely related species demonstrated single bands of various sizes and were easily distinguished from enterococci. The detection level of DNA from serial dilutions of DNA was 10−13 g. The DNA extraction protocol from bacterial cell suspensions resulted in a detection level of 10 bacterial cells per sample. Conclusions The present study demonstrated a good potential for using PCR technology in the detection of E. faecalis and E. faecium from root canal samples. With a high specificity the methodology was able to detect 10 cells of E. faecalis.

Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.

Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.

We recently published an analysis of the limits of carrier-mediated drug transport and whether transporters alone can explain experimental observations that are typically rationalized by drug diffusion through the lipid portions of cell membranes [1]. We found that carrier-mediated transport rates can be high enough to explain observed rates for high-permeability drugs across monolayers of Caco-2 cells, but only if the drugs are substrates of transporters with extreme capacity or if transport is mediated by several medium-to-high-capacity transporters working in parallel.

During calcification of bone, large amounts of phosphate (P(i)) must be transported from the circulation to the osteoid. Likely candidates for osteoblast P(i) transport are the type II sodium-phosphate cotransporters NaPi-IIa and NaPi-IIb that facilitate transcellular P(i) flux in kidney and intestine, respectively. We have therefore determined the 'cotransporters' expression in osteoblast-like cells. We have also studied the 'cotransporters' regulation by P(i) and during mineralization in vitro. Phosphate uptake and cotransporter protein expression was investigated at early, late and mineralizing culture stages of mouse (MC3T3-E1) and rat (UMR-106) osteoblast-like cells. Both NaPi-IIa and NaPi-IIb were expressed by both osteoblast-like cell lines. NaPi-IIa was upregulated in both cell lines one week after confluency. After 7 days in 3mM P(i) NaPi-IIa was strongly upregulated in both cell lines. NaPi-IIb expression was unaffected by both culture stage and P(i) supplementation. The expression of both cotransporters was unaffected by P(i) deprivation. In vitro mineralization at 1.5mM P(i) was preceded by a three-fold increase in osteoblast sodium-dependent P(i) uptake and a corresponding upregulation of both NaPi-IIa and NaPi-IIb. Their expression thus seem regulated by phosphate in a manner consistent with their playing a role in transcellular P(i) flux during mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this thesis was, therefore, to study the expression of components of the transcellular ion transport system, Na+/Ca2+ exchangers and Na(+)-Pi contransporters, in odontoblastic and osteoblastic cells. Their activity was assayed in osteoblast-like cells and in the recently developed MRPC-1 odontoblast-like cell line. To assess the relationship between ion transport and mineralization, Ca2+ and Pi uptake activities were determined in mineralizing cultures of MRPC-1 cells. Osteoblastic and odontoblastic cells showed an identical expression pattern of Na+/Ca2+ exchanger splice-variants, NCX1.3, NCX1.7 and NCX1.10, derived from the NCX1 gene, while NCX2 was not expressed. The cells showed a high sodium-dependent calcium extrusion activity. Regarding Na(+)-Pi cotransporter expression, Glvr-1, Ram-1 and the two high capacity cotransporters Npt-2a and Npt-2b were found to be expressed in odontoblasts and MRPC-1 cells. Osteoblast-like cells differed from this in expressing the Npt-1 but not the Ram-1 gene but were otherwise identical to the odontoblastic cells. Odontoblast-like cells exhibited almost twice the sodium-dependent Pi uptake activity of osteoblast-like cells. The presence of NaPi-2a and NaPi-2b, gene products of Npt-2a and Npt-2b, was verified in vivo by immunohistochemistry on mouse teeth. Both cotransporters could be detected in fully differentiated, polarized odontoblasts but not in preodontoblasts prior to dentin formation. Both cotransporters were detected in adjacent bone and in ameloblasts. Studying ion uptake in mineralizing MRPC-1 cultures, large changes were detected concomitant with the onset of mineral formation, when phosphate uptake increased by 400% while calcium uptake started to decline. The increase in Pi uptake was found to be due to activation of the NaPi-2a cotransporter. MRPC-1 cells expressed an odontoblast-like phenotype already at the onset of culture, but in order to form mineral a differentiation involving their ion transporters seems necessary. Calculating the theoretical rate of ion transport needed for dentin formation and comparing with data from the studies in this thesis showed that transcellular ion transport is both possible and sufficient to meet the phosphate and calcium demands of dentinogenesis.

The nptA gene of Vibrio cholerae has significant protein sequence homology with type II sodium-dependent phosphate (P i ) cotransporters found in animals but not previously identified in prokaryotes. The phylogeny of known type II cotransporter sequences indicates that nptA may be either an ancestral gene or a gene acquired from a higher eukaryotic source. The gene was cloned into an expression vector under the control of an inducible promoter and expressed in Escherichia coli . The results demonstrate that nptA encodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent P i uptake with comparable affinities for both sodium and phosphate ions. Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon. P i uptake activity, a component of which appeared to be sodium dependent, was increased in V. cholerae by phosphate starvation. However, it appears from the use of a reporter gene expressed from the nptA promoter that none of this activity is attributable to the induction of expression from nptA . It is thus proposed that the physiological function of NptA protein may be the rapid uptake of P i in preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.

In odontoblasts as well as osteoblasts, a number of mechanisms for the inflow and extrusion of Ca2+ have been demonstrated. The entrance of Ca2+ ions into odontoblasts occurs mainly through voltage-gated calcium channels. Extrusion of Ca2+ is found to be an ATP-dependent process and, in addition, Na+/Ca2+-antiports exist, which are provoked by extracellular Na+. The aim of this study was to identify the Na+/Ca2+-antiport isoforms expressed in dentinogenically active rat incisor odontoblasts and to make a comparison with different osteoblastic cells. Using RT-PCR and RNAse protection assay, we demonstrated the expression of three different isoforms, NaCa 3, 7, and 10, of the NCX1-encoded antiport in odontoblasts and osteoblastic cells. When incubated in the presence of Na+, dissected rat incisor odontoblasts as well as the osteoblastic cells extruded Ca2+ ions, as detected by chlorotetracycline and Fura-2 fluorometry, thus supporting a physiological role for the detected isoform expression. Odontoblasts and rat calvarial osteoblasts, as well as osteoblast-like cell lines UMR-106.01 and Saos-2, were shown to exhibit identical phenotypes of Na+/Ca2+-antiport isoform expression, different from the expression patterns of other tissues. The significance of this specific expression pattern is unknown, but there is a possibility that it is in some way related to the unique demands on these cell types to produce mineralized connective tissue.