Chronic (−)-isoprenaline infusion down-regulates β₁- and β₂-adrenoceptors but does not transregulate muscarinic cholinoceptors in rat heart

Abstract

Regulation of β-adrenoceptor (β-ar) subtypes and transregulation of muscarinic cholinoceptors (mAchr) was examined in regions of rat heart after chronic infusion of (−)-isoprenaline (450 μ/kg per hour) for 14 days. Following (−)-isoprenaline infusion systolic blood pressure was reduced for 10 days but then gradually returned to control levels, whereas heart rate was increased for 7 days before declining to a level significantly above control. Heart weight to body weight ratio was increased in (−)-isoprenaline treated rats. β-ar subtype densities were measured by quantitative autoradiography with [¹²⁵I]-cyanopindolol (CYP) in sinoatrial node (SA), atrioventricular node (AV), bundle of His (BH), left (LB) and right (RB) bundle branches, interventricular (IVS) and interatrial (IAS) septa, right atria (RA), apex (AX) and mitral valve (MV). β₁-ars were reduced by 59.1–74.2% in the AV conducting regions, 53.4% in the SA node and 43.3–53.4% in myocardial areas. β₂-ars were markedly reduced in myocardial regions (93.2–98.5%) and in pacemaker and conducting regions (87.7–97.8%). No changes in mAchr densities measured using [³H]-N-methyl scopolamine (NMS) occurred in the AV node, BH, LB, RB, IVS and IAS following (−)-isoprenaline infusion.

Densities of β₁- and β₂-ars and mAchrs were also measured in ventricular homogenates from control and (−)-isoprenaline treated animals. β-ar levels were significantly reduced (P < 0.05) in treated animals and the ratio of β₁- to β₂-ars increased after treatment. mAchr density in ventricular homogenates measured using either [³H]-NMS or [³H]-quinuclidinyl [phenyl-4-³H]benzilate (QNB) was unchanged. Homogenates of left and right ventricle also showed no change using [³H]-NMS.

Organ bath studies were used to investigate the effect of (−)-isoprenaline infusion on negative inotropic and chronotropic effects of the non-selective muscarinic receptor agonist bethanechol in left and right atria, respectively. Lower concentrations of bethanechol (3 × 10⁻¹⁰ to 10⁻⁶ M) produced a negative inotropic response in isolated electrically driven left atria from (−)-isoprenaline treated rats, but not from control rats, with the slope of the curves being significantly different between groups (ANCOVA, P = 0.037). At concentrations of bethanechol from 10⁻⁶ to 3 × 10⁻⁴ M the negative inotropic response was not changed between (−)-isoprenaline treated and control animals. Bethanechol also produced a negative chronotropic response at lower concentrations (10⁻¹⁰ to 10⁻⁶ M) in (−)-isoprenaline treated rats, but not in controls. A second, steeper phase of the negative chronotropic response occurred at concentrations of bethanechol greater than 10⁻⁶ M and was also seen in control rats.

Expression of M₂ (cardiac) mAchrs (m₂Achr) in left and right ventricular tissues measured using a quantitative non-competitive polymerase chain reaction (PCR) assay showed a significant (P = 0.001) 28.5% increase in expression in left ventricle and a significant (P = 0.003) 21.5% decrease in expression in right ventricle after (−)-isoprenaline treatment, compared to controls. There was no significant difference in total ventricular m₂Achr expression between the two groups of rats. The results suggest that chronic β-ar stimulation down-regulates both β₁- and β₂-ars, and appears to differentially transregulate m₂Achr expression, but not mAchr protein. Following (−)-isoprenaline infusion, muscarinic receptor mediated responses were sensitised, with no change in receptor densities, suggesting changes occur in the cell signalling system beyond the level of the receptor.