Effect of high retinoid doses on RAR-beta mRNA expression in female B6D2F1 mice.

The expression of retinoid receptors is altered during the development of several types of cancer. In the present study, we determined the influence of high dietary concentrations of 4-hydroxyphenylretinamide (4-HPR) and 13-cis-retinoic acid (13-cis-RA) on RAR-beta mRNA expression in female mice. Expression of liver and lung RAR-beta RNA increased with increasing levels of dietary retinoid (both 4-HPR and 13-cis RA). Bladder RAR-beta mRNA levels, however, were significantly decreased in mice fed 13-cis RA or 4-HPR. These results suggest that feeding high levels of retinoids to mice results in tissue-specific elfects on expression of RAR-beta mRNA.

Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells.

The bioavailability of drugs administered topically or orally depends on their metabolism by epithelial enzymes such as the cytosolic sulfotransferases (SULT). Reverse transcriptase-polymerase chain reaction (RT-PCR) methods were established to detect expression of 8 SULT genes and 4 arylsulfatase (ARS) genes in human tissues of epithelial origin and in cultures of normal and transformed (cancer) cells. The results indicate: (i) SULT 1A1, 1A3, ARSC, and ARSD genes are ubiquitously expressed; (ii) expression is frequently similar between cell lines and corresponding tissues; (iii) SULT gene expression in normal cultured cells is generally comparable to the expression in associated transformed (cancer) cell lines; (iv) SULT 1A1 promoter usage is mainly tissue specific; however, both promoters are frequently used in SULT 1A3 expression; and (v) the expression profile of SULT 1A1, 1A3, 1E1, and 2B1a/b suggests that one or more of these isoforms may be involved in the cutaneous sulfoconjugation of minoxidil and cholesterol.

Dopamine D4 receptor isoform mRNA and protein are expressed in the rat cortical collecting duct.

We reported previously [Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F391-F400, 1996] that dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+ transport in the rat cortical collecting duct (CCD) apparently through a D4 dopamine receptor. The present experiments used RT-PCR of total RNA extracted from microdissected rat CCD to determine whether the D4 and D1A dopamine receptor isoforms are expressed. Specific primers were used to amplify three regions of the D4 cDNA. All three gave products with 98-100% nucleotide identity to the known rat D4 sequence; however, there was an extra 6-bp insert at the 3' end of the second transmembrane region that was identical to the human and mouse sequences but which had not been documented in the rat sequence. D4 receptor protein was also localized exclusively to the CCD and medullary collecting ducts by immunohistochemistry. Two regions of the D1A dopamine receptor message were also amplified by RT-PCR of RNA from rat CCD and were verified by sequencing and immunohistochemistry. We conclude that both D4 and D1A dopamine receptors are expressed in the rat CCD, but the physiological effects are attributable to a D4 receptor.

Expression of multiple alpha-adrenoceptor isoforms in rat CCD.

In the rat cortical collecting duct (CCD), epinephrine inhibits vasopressin (AVP)-dependent water permeability and Na+ reabsorption. Although inhibition is reversed by the alpha2-adrenoceptor (AR) antagonist yohimbine, suggesting the epinephrine effect is primarily mediated by an alpha2-AR [C. T. Hawk, L. H. Kudo, A. J. Rouch, and J. A. Schafer. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F449-F460, 1993], there are also suggestions of an effect at an additional receptor, perhaps an alpha1-AR. For the present experiments, we used RT-PCR of total RNA extracted from 1 to 5 mm of microdissected CCDs from rat kidney to identify the alpha-AR isoforms expressed. Specific primers for the alpha2-ARs amplifying from the 6th transmembrane (TM) to the 3'-untranslated regions, revealed the presence of alpha2A and alpha2B. Western blot analysis also indicated the presence of alpha2B-AR at the protein level. Degenerate alpha1-AR primers that amplify from conserved regions of TM-1 to TM-5, as well as specific primers that amplify either the same region (alpha1B), the carboxy terminus (alpha1A), or within the third cytoplasmic loop (alpha1D), indicated the presence of all three alpha1-ARs. Measurement of transepithelial voltage in isolated perfused renal tubules indicated a small inhibitory effect mediated by alpha1-ARs. Although the functional effects of epinephrine on AVP-dependent transport processes appear to be mediated predominantly by an alpha2-AR, a small contribution to the overall alpha-AR effect may be due to simultaneous activation of an alpha1-AR.

Differential expression of PKC isoforms in fresh and cultured rabbit CCD.

Growth of rabbit cortical collecting duct (CCD) cells in primary culture results in a phenotype in which there is a stable stimulation of Na+ transport by arginine vasopressin. Our objective was to determine whether this altered phenotype was associated with altered expression of protein kinase C (PKC) isoforms. Western blot analysis of extracted proteins and reverse transcription-polymerase chain reaction analysis of extracted RNA showed expression of PKC-alpha, -epsilon, and -zeta in microdissected CCD segments and in fresh and cultured immunodissected CCD cells. These techniques also suggested that the rabbit CCD expresses PKC-eta- and -theta-like isoforms, which have not been identified in this species. Growth of CCD cells in primary culture produced no apparent change in the level of expression of PKC-alpha, -epsilon, or -zeta; however, the theta-like isoform was strongly expressed in fresh CCDs but only weakly expressed in cultured CCDs. The putative eta-isoform was more heavily expressed in cultured than in fresh CCD cells. The consequences of altered expression of these two isoforms in fresh and cultured rabbit CCD remain unknown, but the possibility exists that they are involved in the altered arginine vasopressin response of cultured cells.

Biochemistry of cytosolic sulfotransferases involved in bioactivation.

Numerous studies have indicated that two classes of cytosolic STs are involved in the bioactivation of procarcinogens and drugs to reactive electrophiles, especially in rodent tissues. These two classes of STs are the hydroxysteroid STs, which are involved in the conjugation of hydroxymethyl PAHs, and the phenol STs involved in the sulfation of alkenylbenzenes and N-hydroxyarylamines. Purification studies of rat liver STs have clearly indicated that specific isoforms of hydroxysteroid and phenol STs are capable of sulfating procarcinogens in vitro. Rat liver STa and BAST I are structurally similar hydroxysteroid STs, which have been shown to sulfate and bioactive HMBA. Molecular cloning studies of the rat hydroxysteroid STs indicate that these enzymes are probably part of a family of closely related genes. The single human hydroxysteroid ST that has been characterized is very similar to the rat enzymes, but its role in the bioactivation of hydroxymethyl PAHs has not been established. Phenol STs have been demonstrated to have an important role in the bioactivation of alkenylbenzenes and N-hydroxyarylamines. Purification of rat phenol STs has identified several different forms, but only some appear to be involved in bioactivation of procarcinogens. Four isoforms (HAST I and II, AST III and IV) are apparently responsible for the majority of N-hydroxyarylamine sulfation. The relationship between these enzymes has not been established but they may represent similar enzymes. Different isoforms of rat phenol ST are also involved in the bioactivation of procarcinogens and drugs. However, the role of these phenol STs, PST-1, Mx-ST, and paracetamol ST, in carcinogenesis requires further study. In human tissues, only two phenol STs, P-PST and M-PST, have been identified. The role of these enzymes or unidentified STs in the sulfation of N-hydroxyarylamine procarcinogens has not yet been established. Initial reports of the molecular cloning and expression of the rat and human phenol ST genes will provide a valuable mechanism for the characterization of roles of the individual enzymes in bioactivation.

Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase.

A cDNA encoding the human liver phenol-sulfating form of phenol sulfotransferase (P-PST) has been isolated and characterized from a lambda Uni-Zap XR human liver cDNA library. P-PST is the major form of phenol sulfotransferase involved in drug and xenobiotic metabolism in human liver. P-PST is also responsible for the sulfation and activation of minoxidil to its therapeutically active sulfate ester. The full length cDNA, P-PST-1, is 1206 base pairs in length and encodes a 295-amino acid protein with a molecular mass of 34,097 Da. The translation sequence of P-PST-1 is 96% similar to the amino acid sequences of five peptides derived from the purified protein. In vitro transcription and translation of P-PST-1 generated a protein that comigrates with immunoreactive P-PST from human liver. Significant increases in sulfotransferase activity toward two P-PST-specific substrates, minoxidil and 4-nitrophenol, were detected in cytosol prepared from COS-7 cells transfected with P-PST-1 in the expression vector p-SV-SPORT-1. Northern blot analysis of human liver RNA detected a transcript of approximately 1300 nucleotides in length. Characterization of P-PST at the molecular level provides insight into the structure and heterogeneity of this major class of drug-metabolizing enzymes.