The dopamine D3 receptor subtype was identified in rat kidney using both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native D3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D3 receptor was observed only in the cortex. Specific staining was present in proximal and distal tubules, cortical collecting ducts, glomeruli, and renal vasculature. Immunostaining was observed predominantly in the apical portion of both the proximal and distal tubules. Renal arterial staining was prominent in the medial and adventitial layers. Electron microscopic immunocytochemistry revealed immunogold particles in arteriolar smooth muscle cells of the renal vasculature. In proximal and distal tubules and cortical collecting duct, immunogold staining was localized to apical portions of tubule cells. D3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D3 receptor band in infected Sf9 cell membranes, in transfected LTK- cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or nontransfected LTK- cells. The use of receptor subtype-selective antibodies allows for the tissue localization of specific dopamine receptors that are not distinguished by current pharmacological or ligand-binding technology. The rat kidney expresses the D3 receptor at sites previously deemed to have D2-like receptors.

Dopamine (DA) is regarded as an important modulator of enteric function. Recent experiments have suggested that newly cloned DA receptor subtypes are widely expressed in peripheral organs, including the gastrointestinal tract. In the present studies, the D(1A) receptor subtype was identified in rat gut regions through localization of receptor protein by means of light microscopic immunohistochemistry and Western blot analysis and receptor mRNA by RT-PCR and in situ amplification and hybridization (3SR in situ). D(1A) receptor immunoreactivity was shown to have a diverse distribution in the gastrointestinal tract, being present in the gastroesophageal junction, stomach, pylorus, small intestine, and colon. The receptor has a transmural distribution present in both epithelial and muscle layers as well as in blood vessels and lamina propria cells of different gastrointestinal regions. Western blot analysis demonstrated a single 50-kDa band for esophagus, stomach, duodenum, jejunum, and colon. The in situ hybridization signal was localized to the same sites revealed by D(1A) receptor immunoreactivity. RT-PCR revealed an appropriate sized signal in similar regions. This study is the first to identify expression of the central D(1A) receptor throughout the normal mammalian gastrointestinal tract.

In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1Areceptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1A receptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1A receptor protein. D1A receptor mRNA was predominantly localized in the cortex. Specificity of D1A receptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1A receptor mRNA. In addition, we confirmed the presence of renal D1A receptor mRNA by RT-PCR. We conclude that D1A receptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.

Dopamine (DA) has been shown to influence kidney function through endogenous synthesis and subsequent interaction with locally expressed dopamine receptor subtypes (D1, D5 as D1-like and D2, D3, and D4 as D2-like). DA, and DA-receptor specific agonists and antagonists can alter renal water and electrolyte excretion along with renin release when infused systemically or intrarenally. Such effects are brought about by a combination of renal hemodynamic and direct tubular effects evoked along the full length of the nephron. The cellular mechanisms that direct these dopamine-mediated renal electrolyte fluxes have recently been clarified and include alterations in adenylyl cyclase, phospholipase C, and phospholipase A1 activity. The dopaminergic system also interacts directly with the renal kallikrein-kinin, prostaglandin and other neurohumoral systems. Aberrant renal dopamine production and/or dopamine receptor function have been reported in salt-dependent and low-renin forms of human primary hypertension as well as in genetic models of animal hypertension, including the SHR and Dahl SS rat. DA D1 or D3 receptor knockout mice have been shown to develop hypertension.