Xenogeneic cell therapy: current progress and future developments in porcine cell transplantation.

The multitude of distinct cell types present in mature and developing tissues display unique physiologic characteristics. Cellular therapy is a novel technology with the promise of utilizing this diversity to treat a wide range of human degenerative diseases. Intractable diseases, disorders, and injuries are characterized by cell death or aberrant cellular function. Cell transplantation can replace diseased or lost tissue to provide restorative therapy for these conditions. The limited use of cell transplants as a basis for current therapy can, in part, be attributed to the lack of available human cells suitable for transplantation. This has prevented further realization of the promise of cell transplantation as a platform technology. Accordingly, cell-based therapies such as blood transfusions, for which the cells are readily available, are a standard part of current medical practice. Despite numerous attempts to expand primary human cells in tissue culture, current technological limitations of this approach in regard to proliferative capacity and maintenance of the differentiated phenotype has prevented their use for transplantation. Further, use of human stem cells for the derivation of specific cell types for transplantation is an area of future application with great potential, but hurdles remain in regard to deriving and sufficiently expanding these multipotential cells. Thus, it appears that primary cells are at present a superior source for transplantation. This review focuses on pigs as a source of a variety of primary cells to advance cell therapy to the clinic and implement achievement of its full potential. We outline the advantages and disadvantages of xenogeneic cell therapy while underscoring the utility of transplantable porcine cells for the treatment of human disease.

Impaired cyclic AMP-dependent phosphorylation renders CREB a repressor of C/EBP-induced transcription of the somatostatin gene in an insulinoma cell line.

Transcription factor CREB regulates cyclic AMP (cAMP)-dependent gene expression by binding to and activating transcription from cAMP response elements (CREs) in the promoters of target genes. The transcriptional transactivation functions of CREB are activated by its phosphorylation by cAMP-dependent protein kinase A (PKA). In studies of many different phenotypically distinct cells, the CRE of the somatostatin gene promoter is a prototype of a highly cAMP-responsive element regulated by CREB. We now report on a somatostatin-producing rat insulinoma cell line, RIN-1027-B2, in which transcription from the somatostatin gene promoter is paradoxically repressed by CREB. We find that CREB fails to transactivate a CRE-containing somatostatin-chloramphenicol acetyltransferase reporter even when coexpressed with the catalytic subunit of PKA. CAAT box/enhancer-binding protein beta (C/EBP beta) and C/EBP-related activating transcription factor bind to the CRE in the promoter of the somatostatin gene and transactivate transcription. CREB binds competitively with C/EBP beta to the somatostatin CRE in vitro and represses C/EBP beta-induced transcription of the CRE-containing somatostatin-chloramphenicol acetyltransferase reporter. The lack of CREB-mediated transcriptional stimulation is due to the presence of a heat-stable inhibitor of PKA that prevents activation of PKA and subsequent CREB phosphorylation in the nucleus. These findings indicate that dephosphorylated CREB is a negative regulator of C/EBP-activated transcription of the somatostatin gene promoter in RIN-1027-B2 cells.

Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit.

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.

Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.

The regulatory and catalytic subunits of cAMP-dependent protein kinase (PKA) were coexpressed within the same bacterial cell using a polycistronic bacterial T7 expression vector encoding Chinese hamster cDNAs for the type I regulatory (RI) and catalytic alpha (C alpha) subunits of PKA. Basal expression of active RI/C alpha holoenzyme in the BL21(DE3) strain of Escherichia coli caused severe growth inhibition resulting in extremely small colony size. Several lines of evidence demonstrate that this growth inhibition requires active PKA subunits and cAMP: (i) this phenotype is dependent on cAMP since it is not seen in a strain lacking adenylyl cyclase activity, but the growth rate of these transformants is slower when exogenous cAMP is added; (ii) normal growth occurs when wild-type RI cDNA is replaced by a mutant RI cDNA encoding a RI protein with reduced cAMP binding; and (iii) the growth-inhibited phenotype of the transformed BL21(DE3) cells requires soluble, active C alpha protein. Holoenzyme expressed in bacteria is activated by cAMP, which stimulates phosphorylation of an endogenous 50-kDa protein that is missing in four host mutants selected for normal growth after transformation with PKA holoenzyme. A mutant RI cDNA library was generated by PCR random mutagenesis and screened by polycistronic expression in BL21(DE3) cells. The RI cDNA sequence from one revertant has base-pair substitutions creating two amino acid substitutions within the cAMP binding sites. The coexpression of the RI/C alpha subunits in BL21(DE3) bacterial cells provides a system for rapidly selecting mutations in the RI subunits of PKA.

Solubilization of high-affinity, guanine nucleotide-sensitive mu-opioid receptors from 7315c cell membranes.

High-affinity mu-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD = 0.304 +/- 0.06 nM; Bmax = 154 +/- 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 +/- 5% were recovered in the solubilized fraction. Both mu-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, delta- and kappa-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of < 1 microM. The mu-selective ligand [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD = 0.79 nM; Bmax = 108 +/- 17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites, 43 +/- 3% were recovered in the solubilized material. Guanosine 5'-O-(3-thiotriphosphate), GTP, and guanosine 5'-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin binding in a concentration-dependent manner. Finally, mu-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate.

Reconstitution of the solubilized mu-opioid receptor coupled to a GTP-binding protein.

A mu-opioid receptor-GTP binding protein (mu-opioid receptor-G-protein) complex from the 7315c cell was solubilized with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) and reconstituted into phospholipid vesicles. Pretreatment of the tissue with either [3H]etorphine or morphine greatly improved recovery of the receptor and maintained it in a GTP-sensitive state. GTP sensitivity was consistent with the hypothesis that a receptor-G-protein complex had been obtained. Other evidence consistent with this hypothesis was that recovery of the solubilized, prelabelled receptor was decreased by approximately 70% by pretreatment of 7315c cells with pertussis toxin. The reconstituted receptor was mu-selective: DAGO (Tyr-D-Ala-Gly-Met-Phe- NH(CH2)2OH), but not ICI 174864 or U50488-H, displaced [3H]etorphine binding with high affinity. The affinity of the reconstituted receptor for [3H]etorphine (1.25 +/- 0.20 nM) was similar to that observed for the membrane-associated receptor (0.53 +/- 0.25 nM). GTP gamma S decreased this affinity 3-fold without changing the number of binding sites. The potencies of GTP gamma S and GTP in diminishing [3H]etorphine binding were similar in the membrane and vesicle preparations, but were 10-fold lower than the potencies observed in diminishing binding to the solubilized receptor. The ability to reconstitute a functional mu-opioid receptor-G-protein complex will facilitate further study of the structure and function of the receptor and the specific identification of the associated GTP-binding protein(s).

Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi.

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein.

In a crude membrane preparation of rat 7315c cells, GTP was found to enhance thyrotropin-releasing hormone- (TRH) stimulated inositol triphosphate (IP3) formation with a potency of 0.97 +/- 0.1 microM. TRH stimulation of IP3 formation was inhibited by high GDP concentrations. Neither nucleotide had any effect in the absence of TRH. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) stimulated IP3 formation in the absence of TRH; the apparent affinity of GTP gamma S was 0.16 +/- 0.05 microM. GTP blocked GTP gamma S stimulation of IP3 formation in a concentration-dependent manner. The apparent affinity of GTP for the site of action shared by GTP gamma S was calculated to be 0.98 +/- 0.3 microM. TRH was able to reverse inhibition of GTP gamma S-stimulated IP3 formation by GTP but could not reverse inhibition by GDP. A lag in the rate of IP3 formation in response to GTP gamma S was abolished by addition of TRH. These data support the proposal that activation of the TRH receptor enhances turnover of guanine nucleotides at the binding protein coupling the receptor to phospholipase C. In addition, GTP gamma S diminished high affinity [3H]Me-TRH binding. The potency of GTP gamma S at decreasing [3H]Me-TRH binding was 0.092 +/- 0.03 microM. GTP gamma S (0.1 microM) decreased the affinity of the TRH receptor for [3H]Me-TRH from 2 to 100 nM. Maximally effective concentrations of GTP gamma S, Gpp(NH)p, GTP, and GDP decreased specific [3H]Me-TRH binding by 80%. Pretreatment of cells with pertussis toxin (30 ng/ml for 24 h) failed to affect TRH receptor affinity or the potency or efficacy of GTP gamma S in diminishing [3H]Me-TRH binding, supporting the identification of Gp (a GTP-binding protein associated with phospholipase C and Ca2+-mobilizing receptors) as distinct from Gi (an inhibitory GTP-binding protein). In contrast to its lack of effect on TRH receptor binding, 3-h pertussis toxin treatment decreased agonist affinity of the mu-opiate receptor and abolished the ability of GTP gamma S to shift the affinity of the mu-opiate receptor for its agonist. The affinities calculated for GTP, GDP, GTP gamma S, and Gpp (NH)p for the G-protein regulating receptor affinity and IP3 formation are nearly identical for each guanine nucleotide tested, suggesting the same G-protein regulates both activities.