The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the pre... more The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D 1A receptor in Sprague-Dawley and Wistar-Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D 1A receptor expressed in stably transfected LTK − cells. D 1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription–polymerase chain reaction. D 1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D 1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D 1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D 1A bands in transfected LTK − cells and the atrium but not in nontransfected LTK − cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390 . These results demonstrate that the D 1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.
To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular d... more To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O 2) or low oxygen (1-3% O 2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O 2-treated explants (2.5 { 0.17 mg/kidney, n Å 9) than in 20% O 2 explants (1.5 { 0.09 mg/kidney, n Å 9), P õ 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O 2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.
The dopamine D 3 receptor subtype was identified in rat kidney using both light microscopic immun... more The dopamine D 3 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 D 3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK Ϫ cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D 3 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. D 3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D 3 receptor band in infected Sf9 cell membranes, in transfected LTK Ϫ cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D 2 or D 3 receptor transfected or nontransfected LTK Ϫ 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 D 3 receptor at sites previously deemed to have D 2-like receptors. (Hypertension. 1998;32:886-895.
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the pre... more The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D 1A receptor in Sprague-Dawley and Wistar-Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D 1A receptor expressed in stably transfected LTK − cells. D 1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription–polymerase chain reaction. D 1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold...
Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopam... more Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopamine receptors in the adrenal gland. Dopaminergic ligands have been shown to modulate adrenocortical aldosterone biosynthesis and secretion as well as adrenomedullary catecholamine production and release. Using a combination of light microscopic immunochemistry and in situ amplification and hybridization, the present study sought to determine the site-specific expression of the recently cloned D 1A receptor subtype in rat adrenal gland. Light microscopic immunohistochemistry was conducted using polyclonal antisera raised to the putative rat D 1A receptor. Immunoreactive product was detected using an avidin-biotin immunoperoxidase method. D 1A receptor messenger RNA (mRNA) was detected using a transcription-based isothermal in situ amplification and hy-bridization approach using receptor-specific mRNA oligonucleotide probes. The amplified product was localized using an alkaline phosphatase 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate technique. This combined experimental approach, using both receptor subtype-selective antibodies and oligonucleotide probes, allows for the site-specific localization of the D 1A receptor subtype, which would otherwise not be possible with the pharmacological methods currently available. The D 1A receptor protein and mRNA were expressed solely in the zona glomerulosa of the rat adrenal gland, with no signal evident in any of the other cortical layers or in the medulla. Such a distribution raises the possibility that the D 1A receptor subtype could modulate, at least in part, some of the known effects of dopamine on aldosterone secretion.
To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular d... more To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O 2) or low oxygen (1-3% O 2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O 2-treated explants (2.5 { 0.17 mg/kidney, n Å 9) than in 20% O 2 explants (1.5 { 0.09 mg/kidney, n Å 9), P õ 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O 2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.
The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the pre... more The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D 1A receptor in Sprague-Dawley and Wistar-Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D 1A receptor expressed in stably transfected LTK − cells. D 1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription–polymerase chain reaction. D 1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D 1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D 1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D 1A bands in transfected LTK − cells and the atrium but not in nontransfected LTK − cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390 . These results demonstrate that the D 1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.
To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular d... more To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O 2) or low oxygen (1-3% O 2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O 2-treated explants (2.5 { 0.17 mg/kidney, n Å 9) than in 20% O 2 explants (1.5 { 0.09 mg/kidney, n Å 9), P õ 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O 2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.
The dopamine D 3 receptor subtype was identified in rat kidney using both light microscopic immun... more The dopamine D 3 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 D 3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK Ϫ cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D 3 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. D 3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D 3 receptor band in infected Sf9 cell membranes, in transfected LTK Ϫ cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D 2 or D 3 receptor transfected or nontransfected LTK Ϫ 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 D 3 receptor at sites previously deemed to have D 2-like receptors. (Hypertension. 1998;32:886-895.
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the pre... more The subtype 1A dopamine receptor (D 1A ) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D 1A receptor in Sprague-Dawley and Wistar-Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D 1A receptor expressed in stably transfected LTK − cells. D 1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription–polymerase chain reaction. D 1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold...
Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopam... more Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopamine receptors in the adrenal gland. Dopaminergic ligands have been shown to modulate adrenocortical aldosterone biosynthesis and secretion as well as adrenomedullary catecholamine production and release. Using a combination of light microscopic immunochemistry and in situ amplification and hybridization, the present study sought to determine the site-specific expression of the recently cloned D 1A receptor subtype in rat adrenal gland. Light microscopic immunohistochemistry was conducted using polyclonal antisera raised to the putative rat D 1A receptor. Immunoreactive product was detected using an avidin-biotin immunoperoxidase method. D 1A receptor messenger RNA (mRNA) was detected using a transcription-based isothermal in situ amplification and hy-bridization approach using receptor-specific mRNA oligonucleotide probes. The amplified product was localized using an alkaline phosphatase 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate technique. This combined experimental approach, using both receptor subtype-selective antibodies and oligonucleotide probes, allows for the site-specific localization of the D 1A receptor subtype, which would otherwise not be possible with the pharmacological methods currently available. The D 1A receptor protein and mRNA were expressed solely in the zona glomerulosa of the rat adrenal gland, with no signal evident in any of the other cortical layers or in the medulla. Such a distribution raises the possibility that the D 1A receptor subtype could modulate, at least in part, some of the known effects of dopamine on aldosterone secretion.
To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular d... more To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O 2) or low oxygen (1-3% O 2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O 2-treated explants (2.5 { 0.17 mg/kidney, n Å 9) than in 20% O 2 explants (1.5 { 0.09 mg/kidney, n Å 9), P õ 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O 2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.
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