Molecular and functional identification of β-adrenergic receptors in distal convoluted tubule cells

Frank A. Gesek, Kenneth White

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Renal nerve stimulation or circulating catecholamines activate the β- adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of β-adrenergic receptors have been characterized: β1, β2, and β3. β-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of β-adrenergic receptors in DCT cells and 2) examine functional responses to β-receptor activation on adenosine 3',5'- cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of β-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for β1-, β2-, and β3-receptor subtypes. Products of the appropriate sizes were obtained with β1- and β2-primers. No product was observed with primers to the β3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse β1- and β2-receptor sequence. Receptor binding of [3H]dihydroalprenolol was 123 ± 13 fmol/mg protein, and a 3:1 ratio of β1- to β2-receptors was observed with DCT cell membranes. Isoproterenol, a β-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 ± 23 compared with a basal rate of 256 ± 12 nmol·min-1·mg protein-1 and was blocked with propranolol and β1- and β2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more β1-than β2-receptors and express no detectable β3-adrenergic receptors. β- Receptors couple to adenylyl cyclase, and activation of β-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume272
Issue number6 41-6
StatePublished - 1997
Externally publishedYes

Fingerprint

Adrenergic Receptors
Isoproterenol
Propranolol
Dihydroalprenolol
Adenylyl Cyclases
Cyclic AMP
Adrenergic Agents
Catecholamines
Proteins
Complementary DNA
Cell Membrane
RNA
Kidney

Keywords

  • Adenylyl cyclase
  • Adrenergic receptor
  • Calcium transport
  • Distal convoluted tubule
  • Isoproterenol
  • Propranolol
  • Sodium transport

ASJC Scopus subject areas

  • Physiology

Cite this

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title = "Molecular and functional identification of β-adrenergic receptors in distal convoluted tubule cells",
abstract = "Renal nerve stimulation or circulating catecholamines activate the β- adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of β-adrenergic receptors have been characterized: β1, β2, and β3. β-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of β-adrenergic receptors in DCT cells and 2) examine functional responses to β-receptor activation on adenosine 3',5'- cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of β-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for β1-, β2-, and β3-receptor subtypes. Products of the appropriate sizes were obtained with β1- and β2-primers. No product was observed with primers to the β3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse β1- and β2-receptor sequence. Receptor binding of [3H]dihydroalprenolol was 123 ± 13 fmol/mg protein, and a 3:1 ratio of β1- to β2-receptors was observed with DCT cell membranes. Isoproterenol, a β-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 ± 23 compared with a basal rate of 256 ± 12 nmol·min-1·mg protein-1 and was blocked with propranolol and β1- and β2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more β1-than β2-receptors and express no detectable β3-adrenergic receptors. β- Receptors couple to adenylyl cyclase, and activation of β-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.",
keywords = "Adenylyl cyclase, Adrenergic receptor, Calcium transport, Distal convoluted tubule, Isoproterenol, Propranolol, Sodium transport",
author = "Gesek, {Frank A.} and Kenneth White",
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volume = "272",
journal = "American Journal of Physiology",
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publisher = "American Physiological Society",
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TY - JOUR

T1 - Molecular and functional identification of β-adrenergic receptors in distal convoluted tubule cells

AU - Gesek, Frank A.

AU - White, Kenneth

PY - 1997

Y1 - 1997

N2 - Renal nerve stimulation or circulating catecholamines activate the β- adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of β-adrenergic receptors have been characterized: β1, β2, and β3. β-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of β-adrenergic receptors in DCT cells and 2) examine functional responses to β-receptor activation on adenosine 3',5'- cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of β-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for β1-, β2-, and β3-receptor subtypes. Products of the appropriate sizes were obtained with β1- and β2-primers. No product was observed with primers to the β3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse β1- and β2-receptor sequence. Receptor binding of [3H]dihydroalprenolol was 123 ± 13 fmol/mg protein, and a 3:1 ratio of β1- to β2-receptors was observed with DCT cell membranes. Isoproterenol, a β-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 ± 23 compared with a basal rate of 256 ± 12 nmol·min-1·mg protein-1 and was blocked with propranolol and β1- and β2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more β1-than β2-receptors and express no detectable β3-adrenergic receptors. β- Receptors couple to adenylyl cyclase, and activation of β-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.

AB - Renal nerve stimulation or circulating catecholamines activate the β- adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of β-adrenergic receptors have been characterized: β1, β2, and β3. β-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of β-adrenergic receptors in DCT cells and 2) examine functional responses to β-receptor activation on adenosine 3',5'- cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of β-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for β1-, β2-, and β3-receptor subtypes. Products of the appropriate sizes were obtained with β1- and β2-primers. No product was observed with primers to the β3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse β1- and β2-receptor sequence. Receptor binding of [3H]dihydroalprenolol was 123 ± 13 fmol/mg protein, and a 3:1 ratio of β1- to β2-receptors was observed with DCT cell membranes. Isoproterenol, a β-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 ± 23 compared with a basal rate of 256 ± 12 nmol·min-1·mg protein-1 and was blocked with propranolol and β1- and β2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more β1-than β2-receptors and express no detectable β3-adrenergic receptors. β- Receptors couple to adenylyl cyclase, and activation of β-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.

KW - Adenylyl cyclase

KW - Adrenergic receptor

KW - Calcium transport

KW - Distal convoluted tubule

KW - Isoproterenol

KW - Propranolol

KW - Sodium transport

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