Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison.

INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

An overview of the safety pharmacology society strategic plan.

Safety Pharmacology studies are conducted to characterize the confidence by which biologically active new chemical entities (NCE) may be anticipated as safe. Non-clinical safety pharmacology studies aim to detect and characterize potentially undesirable pharmacodynamic activities using an array of in silico, in vitro and in vivo animal models. While a broad spectrum of methodological innovation and advancement of the science occurs within the Safety Pharmacology Society, the society also focuses on partnerships with health authorities and technology providers and facilitates interaction with organizations of common interest such as pharmacology, physiology, neuroscience, cardiology and toxicology. Education remains a primary emphasis for the society through content derived from regional and annual meetings, webinars and publication of its works it seeks to inform the general scientific and regulatory community. In considering the future of safety pharmacology the society has developed a strategy to successfully navigate forward and not be mired in stagnation of the discipline. Strategy can be defined in numerous ways but generally involves establishing and setting goals, determining what actions are needed to achieve those goals, and mobilizing resources within the society to accomplish the actions. The discipline remains in rapid evolution and its coverage is certain to expand to provide better guidance for more systems in the next few years. This overview from the Safety Pharmacology Society will outline the strategic plan from 2016 to 2018 and beyond and provide insight into the future of the discipline which builds upon a previous strategic plan established in 2009.

The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate.

BACKGROUND AND PURPOSE: BAF312 is a next-generation sphingosine 1-phosphate (S1P) receptor modulator, selective for S1P(1) and S1P(5 ) receptors. S1P(1) receptors are essential for lymphocyte egress from lymph nodes and a drug target in immune-mediated diseases. Here, we have characterized the immunomodulatory potential of BAF312 and the S1P receptor-mediated effects on heart rate using preclinical and human data. EXPERIMENTAL APPROACH: BAF312 was tested in a rat experimental autoimmune encephalomyelitis (EAE) model. Electrophysiological recordings of G-protein-coupled inwardly rectifying potassium (GIRK) channels were carried out in human atrial myocytes. A Phase I multiple-dose trial studied the pharmacokinetics, pharmacodynamics and safety of BAF312 in 48 healthy subjects. KEY RESULTS: BAF312 effectively suppressed EAE in rats by internalizing S1P(1) receptors, rendering them insensitive to the egress signal from lymph nodes. In healthy volunteers, BAF312 caused preferential decreases in CD4(+) T cells, T(naïve) , T(central memory) and B cells within 4-6 h. Cell counts returned to normal ranges within a week after stopping treatment, in line with the elimination half-life of BAF312. Despite sparing S1P(3) receptors (associated with bradycardia in mice), BAF312 induced rapid, transient (day 1 only) bradycardia in humans. BAF312-mediated activation of GIRK channels in human atrial myocytes can fully explain the bradycardia. CONCLUSION AND IMPLICATIONS: This study illustrates species-specific differences in S1P receptor specificity for first-dose cardiac effects. Based on its profound but rapidly reversible inhibition of lymphocyte trafficking, BAF312 may have potential as a treatment for immune-mediated diseases.

Oscillations of cardiac wave length and proarrhythmia.

Drug-induced action potential duration (APD) prolongation was first proposed to be antiarrhythmic, but is now widely presumed to be torsadogenic. To elucidate this paradox, we tested the effect of APD upon liability for torsade de pointes. In addition, torsadogenicity is commonly associated with disturbances of repolarization, but at least in theory, it could also result from disturbances of conduction. These possibilities were tested in female rabbit hearts. Dofetilide, ATX II, and sodium channel blockers that did not prolong the action potential duration were used to modulate the APD and induce disturbances of conduction and disturbances of repolarization. Torsadogenicity could be induced by dofetilide and ATX II starting at normal APD (210 ms), reaching a peak incidence around a doubling of APD (400 to 450 ms), to then sharply decline with further APD prolongation, until torsade de pointes disappeared above 725 ms. Early afterdepolarizations (EAD) were regular triggers for torsade de pointes; while most of the EADs occurred in the plateau range, their incidence declined with repolarization but their potential for torsadogenicity increased. Sodium channel blockers that shorten the APD, even when devoid of hERG blocking properties, can yield torsade de pointes. Torsade de pointes can occur at normal, prolonged, and shortened APD, so that QT prolongation is an incomplete predictor of torsadogenicity. Torsade de pointes can result not only from disturbances of repolarization but also from disturbances of conduction.

Selective block of IKs plays a significant role in MAP triangulation induced by IKr block in isolated rabbit heart.

INTRODUCTION: The role of IKr (rapidly-activating delayed rectifier K(+) current) block in triangulation of monophasic action potentials (MAP) and in development of torsade de pointes (TdP) arrhythmia is known. Combined IKr and IKs (slowly-activating delayed rectifier K(+) current) block has been demonstrated to promote TdP. The aim of this study was to describe a possible implication of IKs block in MAP triangulation. METHODS: Four contact electrodes were placed on the epicardium of the left ventricle of Langendorff-perfused rabbit hearts to record monophasic action potentials (MAP), with an IKr blocker d,l-sotalol (3 to 100 microM, n=6) or a non-selective IKr blocker, quinidine (1 to 30 microM, n=6). Their effects were assessed with or without a specific IKs blocker chromanol 293B (20 microM, n=6), on MAP duration at 30, 60 and 90% of repolarization (APD30, 60 and 90, respectively) and MAP triangulation (APD90-APD30) at 1 and 0.2 Hz. RESULTS: D,L-sotalol increased significantly APD90 and triangulation with reverse use-dependency for concentrations > or =10 microM. Quinidine markedly prolonged APD90 and triangulation with reverse use-dependency at concentrations > or =3 microM. Chromanol 293B alone had no effects on APD, but when combined with D,L-sotalol or quinidine (i) increased APD prolonging effects, (ii) lowered values of pro-arrhythmic concentrations, (iii) increased incidence and length of D,L-sotalol- or quinidine-induced Early Afterdepolarizations (EADs) and TdP. All these events were primarily due to an important slowing of final repolarization, i.e. a marked increased triangulation. CONCLUSION: IKs, even of low amplitude in rabbits, plays a key role in ventricular repolarization. IKs is involved in prolonged MAP duration mainly by triangulation and subsequent increased drug arrhythmogenicity. Therefore drug affinity for IKs must be evaluated with IKr studies as part of preclinical drug cardiac safety assessment.

Relevance of in vitro SCREENIT results for drug-induced QT interval prolongation in vivo: a database review and analysis.

INTRODUCTION: The use of an isolated rabbit heart model (SCREENIT) to predict drug-induced QTc prolongation in animals was assessed using hERG and telemetry data. PURPOSE: We compiled data from (i) hERG assay (IC50s), (ii) SCREENIT assay (APD60) and (iii) in vivo non-rodent telemetry studies (QTc interval) and evaluated the reliability of APD60 to fit with IC50s and QTc prolongation using the ratio to free plasma level (FPL). Eighty-two compounds were separated into three classes based on hERG IC50s (class I: IC50s< or =1 microM, n=7; class II: IC50s>1 microM to < or =10 microM, n=15; class III: IC50s>10 microM, n=60). RESULTS: Three class I compounds did not prolong QTc at the FPL equivalent to their IC50s (43% hERG false positives). There were no false positives in SCREENIT. Six class II compounds prolonged the QTc interval. Results showed 40% hERG false negatives and no SCREENIT false negatives. Nine compounds had no effect on QTc, and two prolonged APD60 at an equivalent concentration/FPL (13% false positives). Three class III compounds prolonged QTc at an FPL lower than maximum SCREENIT concentrations (5% false negatives). Four other compounds generated SCREENIT false positive results (7%). CONCLUSION: SCREENIT increased the predictability of preclinical results for QTc prolongation without generating any false positive results in class I (13% in class II). Making decisions without isolated heart data increases the risk for eliminating efficient drugs displaying hERG inhibition.

Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus.

Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS-receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob-R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob-R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty-seven percent of all Fos-positive cells in the ventromedial arcuate nucleus were also positive for Ob-R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in-vitro slice preparation. Ghrelin stimulated the electrical activity dose-dependently in 80% of all cells tested (n=49) with a threshold concentration of 10(-11) M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10(-7) M) was weakly antagonized by the peptidic GHS-receptor antagonist (D-Lys3)-GHRP-6 (10(-4) M), which also showed a much weaker affinity (IC(50), 0.9 x 10(-6) M) to the GHS-receptor than ghrelin (IC(50), 0.3 x 10(-9) M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.

Regulation of the renal type IIa Na/Pi cotransporter by cGMP.

Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.

An apical membrane Na+/H+ exchanger isoform, NHE-3, is present in the rat epididymal epithelium.

An acidic milieu is required for sperm maturation and for keeping sperm quiescent during storage in the cauda epididymidis. Previous studies have implicated a Na+/H+ exchanger (NHE) in epididymal acidification together with carbonic anhydrase (CA) and vacuolar proton adenosine triphosphatase (H(+)-ATPase). The present studies were undertaken to discover whether the NHE isoform involved is NHE-3, which is known to mediate Na+ and HCO3- absorption in renal tubules. Using the reverse transcription polymerase chain reaction technique (RT-PCR), Northern blot analysis and in situ hybridization, NHE-3 mRNA was detected mainly in the cauda epididymis and to a lesser extent in other regions of the epididymis. Immunohistochemical studies showed that NHE-3 was present in the apical membranes of the epithelial principal cells and confirmed that its expression is strongest in the cauda region, decreasing towards the more proximal regions. Immunoblotting showed a similar expression pattern. These results demonstrate that NHE-3 is expressed in the rat epididymal duct with strongest expression in its cauda region. These findings are thus consistent with the possibility that NHE-3 in the epididymal duct is involved in luminal Na+ and/or HCO3- absorption, as in the renal proximal tubule, and thereby in the regulation of sperm motility and maturation.

Investigating the surface expression of the renal type IIa Na+/Pi-cotransporter in Xenopus laevis oocytes.

We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/Pi cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubation with methanethiosulfonate reagents, leads to complete suppression of cotransport function. This allowed surface labeling of the S460C using MTSEA-Biotin and confirmation by electrophysiology on the same cell. Protein was analyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin treatment resulted in a complete inhibition of S460C-mediated Na+/Pi-cotransport activity, which indicated that all transporters at the surface were biotinylated. After biotinylation, only a small fraction of total S460C protein was precipitated by streptavidin compared with the total amount of S460C protein detected in the lysate. Light- and electron-microscopy analysis of oocytes showed a large amount of WT and S460C transporter protein beneath the oocyte membrane. These data indicate that the apparent weak labeling efficiencies of surface-biotinylation-based assays of membrane proteins heterologously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma.

Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.

The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.

Cleavage of disulfide bonds leads to inactivation and degradation of the type IIa, but not type IIb sodium phosphate cotransporter expressed in Xenopus laevis oocytes.

Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P(i)-cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0 degrees C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/P(i)-cotransporters.

Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-P(i) cotransporter.

Parathyroid hormone (PTH) inhibits proximal tubular reabsorption of P(i) by retrieval of type IIa Na-P(i) cotransporters (NaPi-IIa) from the brush-border membrane (BBM). We analyzed by immunohistochemistry whether PTH analogs, signaling through either protein kinase A (PKA) and C (PKC; 1-34 PTH) or only PKC (3-34 PTH), elicit in rat kidney in vivo or in the perfused murine proximal tubule in vitro a retrieval of NaPi-IIa and whether pharmacological agonists or inhibitors of these kinases are able to either mimic or interfere with these PTH effects. Treatment with either 1-34 or 3-34 PTH downregulated NaPi-IIa in rat kidney. In isolated murine proximal tubules 1-34 PTH was effective when added to either the apical or basolateral perfusate, whereas 3-34 PTH acted only via the luminal perfusate. These effects were mimicked by an activation of PKA with 8-bromoadenosine 3',5'-cyclic monophosphate or PKC with 1, 2-dioctanoylglycerol. The luminal action of both PTH peptides was blocked by inhibition of the PKC pathway (calphostin C), whereas the basolateral effect of 1-34 PTH was completely abolished by inhibiting both pathways (H-89 and calphostin C). These results suggest that 1) NaPi-IIa can be internalized by cAMP-dependent and -independent signaling mechanisms; 2) functional PTH receptors are located in both membrane domains; and 3) apical PTH receptors may preferentially initiate the effect through a PKC-dependent mechanism.

Internalization of proximal tubular type II Na-P(i) cotransporter by PTH: immunogold electron microscopy.

Physiological/pathophysiological alterations in proximal tubular P(i) reabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-P(i) cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in P(i) reabsorption by new synthesis and BBM insertion of type II Na-P(i) cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-P(i) cotransporters (NaPi-2). In kidney of rats on a chronic low-P(i) diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1-34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2.

Dietary phosphate and parathyroid hormone alter the expression of the calcium-sensing receptor (CaR) and the Na+-dependent Pi transporter (NaPi-2) in the rat proximal tubule.

Dietary phosphate (Pi) intake and parathyroid hormone (PTH) are essential regulators of proximal tubular (PT) Pi reabsorption; both factors are associated with adaptive changes in PT apical brush border membrane (BBM) Na/Pi-cotransport activity and specific transporter protein (NaPi-2) content. Urinary Pi excretion is also inversely correlated with luminal Ca2+ concentration ([Ca2+]) both in a PTH-dependent and -independent fashion. A cell-surface, Ca2+(/polyvalent cation)-sensing receptor (CaR) has been localized to the PT BBM with unknown function. To investigate whether PTH and/or dietary Pi intake could affect the distribution or the expression of the CaR, we evaluated their effects on rat kidney CaR and the NaPi-2 expression by Western blot analysis and immunofluorescence microscopy. A chronic high-Pi (1.2%) versus low-Pi (0.1%) diet and acute PTH (1-34) infusion significantly reduced the PT BBM expression of both NaPi-2 and CaR proteins. CaR-specific immunoreactivity in nephron segments other than the PT was not affected by PTH or Pi intake. These results suggest that reduced renal PT CaR expression by a high-Pi diet and by increased circulating PTH levels could contribute to the local control of PT handling of Ca2+ and Pi.

Expression of type II Na-P(i) cotransporter in alveolar type II cells.

Type II Na-P(i) cotransporters (type IIa and type IIb) represent apically located Na-P(i) cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-P(i) cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-P(i) diet. After a chronic adaptation to a low-P(i) diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-P(i) cotransporter in alveolar type II cells.

Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary P(i).

The rate of proximal tubular reabsorption of phosphate (P(i)) is a major determinant of P(i) homeostasis. Deviations of the extracellular concentration of P(i) are corrected by many factors that control the activity of Na-P(i) cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule P(i) reabsorption via one particular Na-P(i) cotransporter (the type IIa cotransporter) by parathyroid hormone (PTH) and dietary phosphate intake. Available data indicate that both factors determine the net amount of type IIa protein residing in the apical membrane. The resulting change in transport capacity is a function of both the rate of cotransporter insertion and internalization. The latter process is most likely regulated by PTH and dietary P(i) and is considered irreversible since internalized type IIa Na-P(i) cotransporters are subsequently routed to the lysosomes for degradation.

Regulation of small intestinal Na-P(i) type IIb cotransporter by dietary phosphate intake.

Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin D(3)) of small intestinal apical Na-P(i) cotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-P(i) diet given for several days leads (in parallel to a stimulation of Na-P(i) cotransport) to an increase of the abundance of the type IIb Na-P(i) cotransporter in the brush-border membrane of mouse enterocytes. Similar results were also obtained by an injection of cholecalciferol. The abundance of the type IIb transcript was investigated by Northern blots. These results indicated that the amount of the type IIb transcript was not changed by either low-P(i) diet or cholecalciferol. It is concluded that stimulation of intestinal Na-P(i) cotransport by low-P(i) diet and vitamin D(3) can be explained by an increased amount of type IIb Na-P(i) cotransporters in the brush-border membrane and that augmentation of type IIb Na-P(i) cotransporters is not related to an increased rate of transcription of the type IIb gene.