Cytosensor Microphysiometer Research Articles

Progesterone's role in deep infiltrating endometriosis: Progesterone receptor and estrogen metabolism enzymes expression and physiological changes in primary endometrial stromal cell culture

To study progesterone signaling activation, we measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) using a cytosensor microphysiometer and assessed progesterone receptor (PR) and estrogen metabolism enzymes mRNA expression in cultured endometrial cells from women with deep infiltrating endometriosis and healthy controls using real-time quantitative PCR. This study was conducted at a University hospital and included patients with and without deep infiltrating endometriosis (DIE). Primary endometrial stromal cells (ECs) from women with DIE and controls were treated with 17β-estradiol and progesterone prior to microphysiometer measurements and qPCR evaluations. Decreased progesterone responsiveness and decreased total nuclear PR and HSD17B1 mRNA expression were observed in cultured ECs from women with deep infiltrating endometriosis relative to those from control samples before and after hormone treatment. These cells also showed increased 17β-hydroxysteroid dehydrogenases types 2 (HSD17B2) relative to control group and increased expression of aromatase (CYP19) after exposure to progesterone. These physiological and expression patterns observed in ECs cultures from women with DIE reinforces previous findings in the literature supporting the progesterone resistance hypothesis in the pathogenesis of endometriosis.

Extension of the cytosensor microphysiometer test method toward cell impedance measurement

Extension of the cytosensor microphysiometer test method toward cell impedance measurement

Using the cytosensor microphysiometer to assess ocular toxicity.

Measuring in vitro cytotoxicity is one method currently used to estimate damage to the eye after chemical exposure. The Cytosensor Microphysiometer method evaluates cytotoxicity by measuring the test material-induced reduction in the metabolic rate of L929 cells. Changes in metabolic rate are measured indirectly as a function of changes in the extracellular acidification rate of the cells. During exposure to increasing concentrations of a cytotoxic material, there is a decrease in the release of acid byproducts into the surrounding medium as the cells die. These acidic metabolic byproducts cause a measurable change in the pH of a lightly buffered medium, which can be measured by the Cytosensor Microphysiometer. The change in the pH of the medium over time is then converted into a metabolic rate estimate for the cells. The endpoint measurement from the assay is the metabolic rate decline of 50%, the MRD50 value (in units of mg/ml).

Receptor-independent effects of 2′(3′)-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH

The effect of the relatively potent P2X7 receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pHi) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pHi was measured fluorimetrically using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3-1.5mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5'-triphosphate disodium salt (5mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pHi. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pHi. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca(2+) in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pHi underlie BzATP-TEA-induced Ca(2+) signaling.

A critical comparison of cell-based sensor systems for the detection of Cr(VI) in aquatic environment

The toxicity of chromium ions was investigated using mammalian cell cultures on impedance sensors as well as physiological in vitro sensor systems. The performance of commercially available systems like the 2500 Analyzing System (Bionas), xCELLigence (Roche) and Cytosensor Microphysiometer (Molecular Devices) was compared with a novel CMOS-based impedance-to-frequency converter device. Cell-based sensor systems are shown to be powerful tools to detect Cr(VI) pollutions within several hours in the range of multinational drinking water regulations. The ability to distinguish between toxic Cr(VI) and non-toxic Cr(III) species is one advantage of these integral sensor systems. Impedance only devices are not sufficient for the fast detection of toxic chromium species as rapid cellular changes occur only in the respiration system and the cell physiology.

The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines

BackgroundThe vasoactive peptide bradykinin (BK) acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas.PurposeIn this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells.MethodsThe presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK) activation by Western blotting.ResultsExposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl)-amiloride (MIA) blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity.ConclusionWe conclude that BK exerts mitogenic effects in A498 cells via the BK B2 receptor activation of growth-associated NHE and ERK.

Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos

The transient receptor potential vanilloid type 1 (TRPV1) channel is one of the most well-characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity, as measured by increase in intracellular free Ca(2+). The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels, was used to test formulations containing a variety of surfactants, preservatives, and fragrances. TRPV1-specific Ca(2+) influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, an adult shampoo that contains cocamide monoethanolamine (CMEA), a known stinging ingredient, was the most active sample tested in the NociOcular test. The negative control, a marketed baby shampoo, was negative in the NociOcular and human tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve formulations were classified as nonstinging in the human test, and of those ten were negative in the NociOcular test. There was no correlation between the clinical stinging results for the baby formulations and the data generated from other in vitro eye irritation assays (cytosensor microphysiometer, neutral red uptake, EpiOcular, transepithelial permeability). Our data support that the TRPV1 channel is a principal mediator of eye-stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye-stinging sensation.

P2X7-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells

The P2X₇ receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X₇ function exhibit altered bone formation. Moreover, activation of P2X₇ in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X₇ agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X₇ receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X₇, failed to elicit a sustained increase in proton efflux. Specific P2X₇ receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²⁺ was required during P2X₇ receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X₇ receptors on osteoblast-like cells triggers a dramatic, Ca²⁺-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.

Epidermal growth factor‐induced proliferation of renal cells from a mouse model of polycystic kidney disease involves multiple signaling pathways

Epidermal growth factor (EGF) is linked to the pathogenesis of cyst formation in polycystic kidney disease (PKD). We explored the effects of EGF in the orpk cilia (−) collecting duct cell line, derived from a mouse model of PKD, and in a control, orpk cilia (+) cell line to determine signaling pathways activated by EGF. RT‐PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na+/H+ exchangers, NHE‐1, NHE‐2, and NHE‐3 in both cell lines. We measured changes in extracellular pH as a reflection of NHE activity with a Cytosensor microphysiometer, assessed extracellular signal regulated kinase (ERK) activation by Western blotting, and cell proliferation by a bromodeoxyuridine uptake. Exposure to 10 ng/ml of EGF caused ≥20% increase in NHE activity, ≥70% increase in ERK phosphorylation, and ~ 30% increase in proliferation in orpk cilia (+) cells. All effects were more pronounced in orpk cilia (−) cells: ≥30% increase in NHE activity, ≥200% increase in ERK phosphorylation, and ~43% increase in proliferation. The amiloride analog, MIA, blocked NHE activation and inhibited ERK phosphorylation suggesting that NHE is critical for ERK activation by EGF. EGF‐induced proliferation was significantly decreased by MIA and by the ERK inhibitor PD98059, suggesting roles for NHE and ERK in EGF mitogenic activity. These studies are important because of the emerging role of EGF in the pathogenesis of PKD.

Different signaling mechanisms are involved in vasopressin‐induced proliferation of orpk cilia (+) and orpk cilia (−) renal cells derived from a mouse model of polycystic kidney disease

Arginine vasopressin (AVP) is a powerful modulator of cystogenesis in polycystic kidney disease (PKD). We studied signaling pathways activated by AVP in the orpk cilia (−) collecting duct cell line, derived from a mouse model of PKD, and in the control, orpk cilia (+) cell line. We measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) activity with a Cytosensor microphysiometer, assessed extracellular signal regulated kinase (ERK) activation by Western blotting, cAMP production by immunoassay, and cell proliferation by a bromodeoxyuridine uptake. AVP stimulated cAMP production, supporting the expression of functional AVP receptors in both cell lines. However, exposure to AVP caused ≥20% increases in NHE activity in control cells but only minor (~10%) increases in orpk cilia (−) cells. Similarly, AVP stimulated ERK phosphorylation ≥80% only in control cells. Exposure to 1 μM AVP resulted in ≥25% increase in proliferation of control cells, and an ERK inhibitor (PD98059) blocked this effect suggesting a critical role for ERK in AVP mitogenic activity. AVP‐induced cell proliferation in orpk cilia (−) cells was more pronounced (~45% increase), and was not sensitive to ERK inhibition. In summary, AVP induces different pathways to stimulate proliferation in orpk cilia (+) and orpk cilia (−) cells. These studies are important because of the proposed role of AVP in the pathogenesis of PKD.

Bradykinin B2 receptor activates extracellular signal regulated kinase in renal cells from a mouse model of polycystic kidney disease

The vasoactive peptide bradykinin (BK) is a mitogen for normal renal cells. We explored the effects of BK in orpk cilia (+) and orpk cilia (−) collecting duct cell lines, which were derived from a mouse model of polycystic kidney disease (PKD). mRNAs for BK B1 and BK B2 receptors were demonstrated by RT‐PCR in both cell lines. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, a Cytosensor microphysiometer to measure changes in extracellular pH as a reflection of activation of the Na+/H+ exchanger (NHE), and Western blotting to assess extracellular signal regulated kinase (ERK) activation.Exposure to 100 nM BK resulted in rapid elevation of intracellular Ca2+, caused a ≥ 20% increase in NHE activity, and a ≥ 200% increase in ERK phosphorylation. While BK‐induced Ca2+ signals and NHE activation were similar in orpk cilia (+) and orpk cilia (−) cell lines, BK‐induced ERK phosphorylation was less pronounced in orpk cilia (−) cells. The BK B2 receptor antagonist and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor blocked BK‐induced ERK phosphorylation suggesting a role for the B2 receptor‐induced EGFR transactivation in the mitogenic ERK pathway. We speculate that cilium is required for effective transactivation of EGFR and ERK activation by BK B2 receptors in PKD cells. These studies are important because of proposed role of ERK pathway in pathophysiology of PKD.

First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined applicability domain

A replacement alternative to the rabbit eye irritation test has been sought for many years. First published in 1944 by FDA toxicologist J. H. Draize, the test, now known as the Draize Eye Test, has been used extensively to assess eye safety. It has also been a focal point for concern regarding its animal use. In 1992, Molecular Devices developed the Cytosensor Microphysiometer (CM) technology, an automated potentiometric online measurement of pH changes in cells, and evaluated it also for chemically induced irritation. The method was included in some of the six major validation studies for eye irritation from 1991-1997. The results for CM were inconclusive as were those from other tests evaluated as stand-alone methods to fully replace the animal test. In 2002, the European Centre for the Validation of Alternative Methods (ECVAM) started applying concepts from evidence-based medicine, and opened validation to retrospective meta-analysis. This activity was done in collaboration with US counterpart ICCVAM/NICEATM, and the European Cosmetics Association, Colipa. After a new, comprehensive evaluation of the prior available data, the ECVAM scientific advisory committee (ESAC) has recently accepted the CM as capable of identifying non-irritants for testing limited to water-soluble surfactants and water-soluble surfactant-containing mixtures. This 25-year development is remarkable and instructive in many respects. The authors see this as opening the door, at last, for an end to the use of animals as a standard requirement for eye irritation. Here, several of the people critically involved in this processes have summarized the important aspects of this history.

Epidermal growth factor activates Na+/H+ exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin

Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth factors, and plays significant roles in regulating intracellular pH and cellular volume, proliferation and cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in proton efflux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological inhibitors of Janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF→EGFR→Jak2 activation (independent of EGFR tyrosine kinase activity)→tyrosine phosphorylation of CaM→CaM binding to NHE-1→conformational change of NHE-1→activation of NHE-1; and (2) EGF→EGFR→EGFR kinase activation→association of CaM with NHE-1 (independent of Jak2)→conformational change of NHE-1→activation of NHE-1.

Intracellular Peptides as Natural Regulators of Cell Signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 microm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

The β3-Adrenoceptor Agonist 4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4-hydroxyphenoxy)propyl]amino]ethyl]-phenyl]-benzenesulfonamide (L755507) and Antagonist (S)-N-[4-[2-[[3-[3-(Acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]-ethyl]phenyl]benzenesulfonamide (L748337) Activate Different Signaling Pathways in Chinese Hamster Ovary-K1 Cells Stably Expressing the Human β3-Adrenoceptor

This study identifies signaling pathways activated by the beta(2)-/beta(3)-adrenoceptor (AR) agonist zinterol, the selective beta(3)-AR agonist L755507, and the selective beta(3)-AR antagonist L748337 in CHO-K1 cells expressing human beta(3)-adrenoceptors. Zinterol and L755507 caused a robust concentration-dependent increase in cAMP accumulation (pEC(50) values of 8.5 and 12.3, respectively), whereas L748337 had low efficacy. Maximal cAMP accumulation with zinterol and L755507 was increased after pretreatment with pertussis toxin, indicating that the human beta(3)-AR couples to G(i) and to G(s). In contrast to cAMP, zinterol, L755507 and L748337 increased phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) with very high potency (pEC(50) values of 10.9, 11.7, and 11.6). These compounds also stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) but with much lower potency than Erk1/2 (pEC(50) values of 5.9, 5.5, and 5.7, respectively). Pertussis toxin completely blocked Erk1/2 and p38 MAPK phosphorylation in response to L748337, demonstrating a requirement for G(i/o) coupling, whereas L755507-stimulated p38 MAPK phosphorylation was not inhibited by pertussis toxin, and Erk1/2 phosphorylation was inhibited by only 30%. We found that high levels of cAMP interfered with agonist-activated p38 MAPK phosphorylation. L748337 increased extracellular acidification rate (ECAR) in the cytosensor microphysiometer with efficacy similar to zinterol and L755507, albeit with lower potency (pEC(50) value of 7.2 compared with zinterol, 8.1, and L755507, 8.6). The ECAR response to L748337 was largely via activation of p38 MAPK, demonstrated by 65% inhibition with 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ67657). We conclude that the beta(3)-AR agonist L755507 couples to both G(s) and G(i) to activate adenylate cyclase and MAPK signaling, whereas the beta(3)-AR antagonist L748337 couples predominantly to G(i) to activate MAPK signaling.

Agonist and antagonist activation of signalling pathways in CHO‐K1 cells stably expressing the human β3‐adrenoceptor

The human β3‐adrenoceptor (hβ3‐AR) couples to Gs to activate adenylyl cyclase, leading to lipolysis or thermogenesis in adipose tissue. hβ3‐AR also couple to Gi, to modulate adenylyl cyclase and activate mitogen‐activated protein kinase (MAPK) pathways. Here, we have examined the action of the β2‐AR agonist zinterol, the selective hβ3‐AR agonist L755507 and the selective hβ3‐AR antagonist L748337 on hβ3‐AR stably expressed in CHO‐K1 cells. Zinterol and L755507 caused dose‐dependent cAMP accumulation (pEC50′ 8.47±0.11 and 12.28±0.03) while L748337 had little effect in this assay. In contrast, in the cytosensor microphysiometer, the maximum extracellular acidification rate (ECAR) responses elicited by L748337 (pEC50 7.14±0.03) were almost identical to those obtained with zinterol and L755507 (pEC50 8.13±0.03 and 8.63±0.10 respectively). This suggested that L748337 can selectively activate pathways other than cAMP. Western blots of p38 MAPK and Erk1/2 phosphorylation showed strong activation in response to both L748337 and to L755507. Activation in response to L748337 was almost completely abolished by PTX pre‐treatment that only slightly affected responses to L755507. We conclude that L755507 and L748337 utilize discrete pathways to activate p38 MAPK and Erk1/2; L755507 by signalling through Gs and L748337 by signalling through Gi.This research was supported by National Health and Medical Research Council Project Grants 236884 and 491190.

In vitro evaluation of leptin fragments activity on the ob receptor

In an attempt to identify regions in the leptin molecule responsible for its bioactivity, we tested six related-leptin peptide fragments denoted: Ac-hLEP(23-47)-NH(2) (I), Ac-hLEP(48-71)-NH(2) (II), Ac-hLEP(72-88)-NH(2) (III), Ac-hLEP(92-115)-NH(2) (IV), Ac-[Ser(117)]-hLEP(116-140)-NH(2) (V), Ac-hLEP(141-164)-NH(2) (VI) and their correspondent disulfide bridged dimer forms. The activity of the fragments was evaluated in comparision to leptin, by their ability to interact with leptin receptor using a cytosensor microphysiometer. Our results indicated that the fragments IV and V and [D-Leu(4)]-OB(3) and its human sequence analog were recognized by leptin receptor present in HP-75 cells, in agreement with the results obtained by other workers, validating that this region of the molecule contain the functional epitope of the leptin molecule.

Use of the cytosensor microphysiometer to predict results of a 21-day cumulative irritation patch test in humans

The cytosensor™ microphysiometer (μϕ) was investigated as a rapid, relatively inexpensive test to predict performance of skin cleansing wipes on the human 21-day cumulative irritation patch test (21CIPT). It indirectly measures metabolic rate changes in L929 cells as a function of test article dose, by measuring the acidification rate in a low-buffer medium. The dose producing a 50% reduction in metabolic rate (MRD 50), relative to the baseline rate, is used as a measure of toxicity. The acute toxicity of the μϕ assay can be compared to the chronic toxicity of the 21CIPT, which is based largely on the exposure of test agents to the epidermal cells, resulting in damage and penetration of the stratum corneum leading to cell toxicity. Two series of surfactant-based cleansing wipe products were tested via the μϕ assay and 21CIPT. The first series, consisting of 20 products, was used to determine a prediction model. The second series of 38 products consisted of routine product development formulas or marketed products. Comparing the results from both tests, samples with an MRD 50 greater than 50 mg/ml provided a 21CIPT score consistent with a product that performs satisfactorily in the market. When the MRD 50 was greater than 78 mg/ml, the 21CIPT score was usually zero. The μϕ may be more sensitive than the 21CIPT for ranking minimally irritating materials. The μϕ assay is useful as a screen for predicting the performance of a wet wipes formula on the 21CIPT, and concurrently reduces the use of animals for safety testing in a product development program for cleansing wipes.

Agonist effects of zinterol at the mouse and human β3-adrenoceptor

The present study investigates the action of zinterol at beta(3)-adrenoceptors. We used mouse primary brown adipocytes and Chinese hamster ovary (CHO-K1) cells expressing the mouse or human beta(3)-adrenoceptor. Zinterol was a full agonist at increasing cyclic AMP levels in primary brown adipocytes (which express beta(1)- and beta(3)-adrenoceptors but not beta(2)-adrenoceptors), and this effect was almost totally abolished in adipocytes derived from beta(3)-adrenoceptor knock-out (KO) mice. Zinterol was also a full agonist at increasing another biological end-point, glucose uptake in brown adipocytes. This effect was reduced in adipocytes derived from beta(3)-adrenoceptor KO mice, with the remaining response sensitive to beta(1)-adrenoceptor antagonism. To determine whether the effect of zinterol on beta(3)-adrenoceptors in primary brown adipocytes can be replicated in a recombinant system, we used CHO-K1 cells expressing the mouse or human beta(3)-adrenoceptor. Zinterol was a full agonist at mouse and human receptors with respect to increasing cyclic AMP levels, with pEC(50) values similar to that of the selective beta(3)-adrenoceptor agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate (CL316243) at the mouse receptor. At the human receptor, zinterol was more potent at increasing cyclic AMP levels than CL316243. In cytosensor microphysiometer studies, zinterol was a full agonist for increases in extracellular acidification rates at the mouse and human beta(3)-adrenoceptor. Therefore, we have shown that zinterol is a potent, high-efficacy beta(3)-adrenoceptor agonist at the endogenous mouse beta(3)-adrenoceptor in primary brown adipocytes and at the cloned mouse and human beta(3)-adrenoceptor expressed in CHO-K1 cells. Zinterol is therefore one of few beta-adrenoceptor agonists with high potency and efficacy at the human beta(3)-adrenoceptor.

Epidermal Growth Factor Activates Na+/H+ Exchange in Podocytes through a Mechanism that Involves Janus Kinase and Calmodulin

Previous studies from our laboratory have shown that the NHE-1 can be activated by G protein-coupled receptors or hypertonic medium, through janus kinase 2 (Jak2)-dependent phosphorylation of calmodulin (CaM) and subsequent interaction between CaM and NHE-1. Therefore, we wanted to determine whether the EGF receptor regulated NHE-1 activity through Jak2 and CaM. EGF induced concentration-dependent increases in proton efflux in renal podocytes as accessed using a Cytosensor microphysiometer. Stimulation of proton efflux by EGF in podocytes was diminished in the presence of MIA or a sodium-free solution, which demonstrates that EGF stimulates a sodium-dependent proton efflux that can be attenuated by a selective NHE-1 inhibitor in podocytes. Furthermore, inhibitors of Jak2 and calmodulin attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2, CaM, and NHE-1. We found that there were increased amounts of NHE-1 and calmodulin assembled with Jak2 upon EGF stimulation. The results suggest that EGF induces NHE-1 activity in podocytes through Jak2 and increased interaction between CaM and NHE-1. This work was supported by Veterans Affairs Merit Award and NIH R01DK052448.