Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits.

The striatal cellular coexpression patterns for the D(1A) and D2 dopamine (DA) receptor subtypes and the ionotropic excitatory amino acid (EAA) subunits of the N-methyl-D-aspartate (NMDA-R1) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (GluR1 and GluR2/3) receptor subunits were examined morphologically. Their coincidence was assessed by visualization of mRNA transcripts, localization of encoded receptor proteins, and binding analysis using concurrently paired methods of fluorescence detection. The findings indicated that 1) mRNA transcripts for both receptor systems were detected in the medium-sized neuron population, and the distribution of receptor message closely reflected protein and binding patterns, with the exception of the GluR1 subunit; 2) both DA receptor mRNA transcripts were coexpressed with each ionotropic EAA receptor subunit examined and with each other, and NMDA and AMPA receptor subunits also showed coincident expression; 3) D(1A) DA receptor protein was detected in neurons which coexpressed EAA subunit proteins; and 4) GluR2/3 and NMDA-R1 subunit proteins were coexpressed in medium-sized neurons which also demonstrated D2 DA receptor binding sites. These findings suggest morphological receptor "promiscuity" since the coexpression patterns between DA and EAA receptors were found in all permutations. The results provide a spatial framework for physiological findings describing functional interactions between the two DA receptor types and between specific DA and EAA receptors in the striatum.

Cellular distribution of the rat D4 dopamine receptor protein in the CNS using anti-receptor antisera.

A polyclonal antiserum was generated against a unique peptide fragment in the rat D4 dopamine (DA) receptor. The titer was monitored using solid-phase ELISA and once it was established, specificity was assessed using Chinese Hamster Ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D4 DA receptor. Immunofluorescent staining produced by incubation with the anti-D4 DA receptor antiserum was selective for D4 DA receptor-transfected CHO cells, and was expressed at their cell membranes and cytoplasm. Attenuated staining for D4 DA receptor protein was visible in untransfected, K1 CHO cells, and in D2 or D3 DA receptor-transfected CHO cells. The regional and cellular CNS distribution patterns for the D4 DA receptor subtype were examined, and illustrated significant protein levels within the frontal (FCx) and parietal cortices. Lesser amounts of receptor protein staining occurred in the thalamus, globus pallidus, hippocampus, cerebellar vermis, and very low expression was detected in the striatum (CPu). D4 DA receptor protein staining was correlated with the cellular expression of its mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D4 DA receptor transcripts and encoded proteins in identified neurons of the FCx and CPu showed variations in receptor expression in these identified basal ganglia pathways.

Cellular distribution of the rat D1B receptor in central nervous system using anti-receptor antisera.

Polyclonal antisera have been generated against two unique polypeptide fragments in the rat D1B dopamine (DA) receptor, as deduced from the cDNA sequence. Antisera titers were monitored using solid-phase ELISA. Once the titers were established, antisera specificity was determined using Chinese Hamster ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D1B DA receptor. Immunoreactivity following staining with either anti-D1B DA receptor antisera was equivalent, selective for the D1B DA receptor-transfected CHO cells, and expressed at their membrane and within the cell cytoplasm. Minimal immunofluorescent staining for D1B DA receptor proteins was detected in untransfected CHO cells, or in D1A DA receptor-transfected CHO cells. The regional and cellular distribution patterns for the D1B DA receptor subtype were examined in various brain areas and illustrated significant protein levels within the frontal and parietal cortices and in the hippocampus and dentate gyrus. Lesser amounts of receptor protein staining were seen in the dorsal striatum, olfactory tubercle, and cerebellar vermis. D1B DA receptor protein staining was correlated with the cellular expression of D1B DA receptor mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D1B DA receptor transcripts and encoded proteins in identified neurons of the frontal cortex and striatum showed variations in receptor expression in these identified basal ganglia pathways.

The relationship of body mass index to intra-abdominal pressure as measured by multichannel cystometry.

The aim of the study was to identify the possible relationship between body mass index and intra-abdominal pressure as measured by multichannel cystometry. A retrospective chart review of patients presenting for urodynamic evaluation between January 1995 and March 1996 was carried out. Variables identified included weight, height, intra-abdominal pressure and intravesical pressure as recorded on multi-channel cystometrogram at first sensation in the absence of detrusor activity. Body mass index was defined as weight in kilograms divided by height in square meters. Intra-abdominal pressure was measured intravaginally except in those cases of complete procidentia or severe prolapse, where it was measured transrectally. Adequate data were available on 136 patients. The mean age was 60.6 years (range 30-91); mean body mass index was 27.7 kg/m2 (range 12.7-47.7); and mean intra-abdominal pressure was 27.5 cmH2O (range 9.0-48.0). A strong association between intra-abdominal pressure and body mass index was demonstrated, with a Pearson coefficient correlation value of 0.76 (P<0.0001). Strong correlation was still demonstrated when those patients who had had the intra-abdominal pressure measured transrectally were separated out, thus eliminating any possible confounding factors between measurements of intra-abdominal pressure measured transvaginally versus transrectally. In addition a strong correlation between intravesical pressure and body mass index was also demonstrated, with a Pearson coefficient correlation value of 0.71 (P<0.0001). Of the 136 patients, 65 (47.8%) were ultimately diagnosed as having genuine stress urinary incontinence (GSUI), 35 (25.7%) with GSUI and a low-pressure urethra (maximum urethral closure pressure of less than 20 cmH2O), and 18 (13.2%) with detrusor instability. The remaining 13.2% had severe prolapse. Our data demonstrate a significant correlation between body mass index and intra-abdominal pressure. These findings suggest that obesity may stress the pelvic floor secondary to chronic state of increased pressure, and may represent a mechanism which supports the widely held belief that obesity is a common factor in the development and recurrence of GSUI.

Co-expression of receptor mRNA and protein: striatal dopamine and excitatory amino acid subtypes.

Dopamine (DA) is known to modulate the post-synaptic response of the excitatory amino acid (EAA) neurotransmitters in the striatum. Thus the intrinsic neurons in this nucleus are potential sites of cross-interaction between these two systems. The recent isolation of 5 different DA receptor subtypes and more than 20 EAA subunits argues for a complicated functional role for the protein products encoded by these transcripts. The simultaneous detection of cellular mRNA distributions and translated protein products was an initial step to determine differences in post-translational expression at the cellular level of resolution for two of these receptors. The cloned D2 DA receptor subtype and the ionotropic GluR1 EAA receptor subunit were examined by fluorescence in situ transcription (FIST) following hybridization of specific cDNA primers, complementary to the mRNA transcripts encoding these receptors. Nascent extension of the annealed primer using reverse transcriptase was detected after incorporation of fluorescently labeled dUTP. Protein products were visualized by standard immunofluorescence after incubation with anti-peptide antisera that were selective for each receptor protein. The experimental data corroborate previous work describing the regional expression of ligand binding and in situ hybridization detected with radiolabeled probes for the DA and EAA receptor systems in the striatum. The dual fluorescence method can be completed within 2 days and may be adapted to cellular localization of many novel mRNA/protein combinations to examine post-translational processing within thin tissue slices.