Insulinoma-derived pseudo-islets for diabetes research.

The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion, and isolation of the islets from nonendocrine tissue. This process is time consuming, expensive, and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized ß cell lines reflect several aspects of primary ß cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research, which depend on islet morphology and/or functional assessment. In this manuscript, we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 µm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.

Adrenergic receptor stimulation suppresses oxidative metabolism in isolated rat islets and Min6 cells.

Insulin secretion is stimulated by glucose metabolism and inhibited by catecholamines through adrenergic receptor stimulation. We determined whether catecholamines suppress oxidative metabolism in ß-cells through adrenergic receptors. In Min6 cells and isolated rat islets, epinephrine decreased oxygen consumption rates compared to vehicle control or co-administration of epinephrine with α2-adrenergic receptor antagonist yohimbine. Epinephrine also decreased forskolin-stimulated oxygen consumption rates, indicating cAMP dependent and independent actions. Furthermore, glucose oxidation rates were decreased with epinephrine, independent of the exocytosis of insulin, which was blocked with yohimbine. We evaluated metabolic targets through proteomic analysis after 4 h epinephrine exposure that revealed 466 differentially expressed proteins that were significantly enriched for processes including oxidative metabolism, protein turnover, exocytosis, and cell proliferation. These results demonstrate that acute α2-adrenergic stimulation suppresses glucose oxidation in ß-cells independent of nutrient availability and insulin exocytosis, while cAMP concentrations are elevated. Proteomics and immunoblots revealed changes in electron transport chain proteins that were correlated with lower metabolic reducing equivalents, intracellular ATP concentrations, and altered mitochondrial membrane potential implicating a new role for adrenergic control of mitochondrial function and ultimately insulin secretion.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

BACKGROUND: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived ß cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high ß cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic ß cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to ß cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and ß cell function and leads to increased inflammatory signaling.

Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

PURPOSE: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. METHODS: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. RESULTS: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling. CONCLUSIONS: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Trigonal scaffolds for multivalent targeting of melanocortin receptors.

Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo.

PURPOSE: Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. METHODS: Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. RESULTS: Inhibition of the Ca(2+)-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.

Increased redox-sensitive green fluorescent protein reduction potential in the endoplasmic reticulum following glutathione-mediated dimerization.

As the endoplasmic reticulum (ER) is the compartment where disulfide bridges in secreted and cell surface proteins are formed, the disturbance of its redox state has profound consequences, yet regulation of ER redox potential remains poorly understood. To monitor the ER redox state in live cells, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. One particular green fluorescent protein (GFP)-based redox sensor, roGFP1-iL, could detect oxidizing changes in the ER despite having a reduction potential significantly lower than that previously reported for the ER. We have confirmed these observations and determined the mechanisms by which roGFP1-iL detects oxidizing changes. First, glutathione mediates the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitates dimerization of roGFP1-iL, which shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule's reduction potential compared with that of a dithiol redox buffer. We conclude that the glutathione redox couple in the ER significantly increased the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in the ER with a high degree of reliability in real time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggests a range of ER redox potentials consistent with those determined by earlier approaches that did not involve fluorescent sensors.

Multivalent Interactions: Synthesis and Evaluation of Melanotropin Multimers - Tools for Melanoma Targeting.

In order to develop agents for early detection and selective treatment of melanomas, high affinity and high specificity molecular tools are required. Enhanced specificity may be obtained by simultaneously binding to multiple cell surface targets via the use of multimeric analogs of naturally occurring ligands. Trimers targeting overexpressed melanocortin receptors have been found to be potential candidates for this purpose. In the present letter, we describe the synthesis and study of multimers based on a dendrimer-like scaffold. The binding affinity and activity results revealed that dendrimers promote multivalent interactions via statistical and/or cooperative effects on binding. Moreover, viability studies showed no significant toxicity at micromolar concentrations, which will allow these molecular complexes to be used in vivo. Finally, imaging studies showed effective internalization for all the molecules confirming their potential as delivery agents.

Synthesis and evaluation of cholecystokinin trimers: a multivalent approach to pancreatic cancer detection and treatment.

In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R.

Heterobivalent ligands target cell-surface receptor combinations in vivo.

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.

Design, synthesis, and biological studies of efficient multivalent melanotropin ligands: tools toward melanoma diagnosis and treatment.

To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.

Cell-specific targeting by heterobivalent ligands.

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Fluorescent and lanthanide labeling for ligand screens, assays, and imaging.

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening -studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.

Na(+)-K(+)-ATPase and Ca(2+) clearance proteins in smooth muscle: a functional unit.

The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.

Enhanced targeting with heterobivalent ligands.

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human delta-opioid receptor (deltaOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-alpha-MSH, and Eu-CCK8 for the deltaOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a deltaOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers.

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase alpha 2-isoform gene-ablated mice.

Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.

Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells.

BACKGROUND: In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates. RESULTS: Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions. CONCLUSION: Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.

Transgenic mice expressing Na+-K+-ATPase in smooth muscle decreases blood pressure.

The Na(+)-K(+)-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na(+) in exchange for two K(+). An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA alpha(1)- or alpha(2)-isoform (alpha(1 sm+) or alpha(2 sm+) mice) driven by the smooth muscle-specific alpha-actin promoter SMP8. Interestingly, both alpha-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total alpha-isoform protein was increased from 1.5-fold (alpha(1 sm+) mice) to 7-fold (alpha(2 sm+) mice). The increase in total NKA alpha-isoform protein was accompanied by a 2.5-fold increase in NKA activity in alpha(2 sm+) gastric antrum. Immunocytochemistry of the alpha(1)- and alpha(2)-isoforms in alpha(2 sm+) aortic smooth muscle cells indicated that alpha-isoform distributions were similar to those shown in wild-type cells. alpha(2 sm+) Mice (high expression) were hypotensive (109.9 +/- 1.6 vs. 121.3 +/- 1.4 mmHg; n = 13 and 11, respectively), whereas alpha(1 sm+) mice (low expression) were normotensive (122.7 +/- 2.5 vs. 117.4 +/- 2.3; n = 11 or 12). alpha(2 sm+) Aorta, but not alpha(1 sm+) aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the alpha-isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased alpha-isoform expression.