Different effects of alpha- and beta-adrenergic stimulation on cytosolic pH and myofilament responsiveness to Ca2+ in cardiac myocytes.
alpha-Adrenergic stimulation (alpha-AS) and beta-adrenergic stimulation (beta-AS) of the myocardium are associated respectively with an increase and a decrease in myofilament responsiveness to Ca2+. We hypothesized that changes in cytosolic pH (pH(i)) may modulate these opposite actions of alpha-AS and beta-AS. The effects of alpha-AS (50 microM phenylephrine and 1 microM nadolol) and beta-AS (0.05 microM isoproterenol) on contraction and either cytosolic Ca2+ (Cai) or pH(i) were assessed in adult rat ventricular myocytes bathed in bicarbonate buffer (pH 7.36 +/- 0.05). In cells loaded with the ester derivative (AM form) of indo-1, the 410/490-nm ratio of emitted fluorescence indexed Cai. Myofilament responsiveness to Ca2+ was assessed by the relaxation phase of the length-indo-1 fluorescence relation during a twitch. alpha-AS and beta-AS shifted this relation in opposite directions, indicating that alpha-AS increased and beta-AS decreased myofilament responsiveness to Ca2+. In addition, the positive inotropic action of alpha-AS was associated with an increased Cai transient amplitude in 50% of the myocytes (n = 12), whereas beta-AS always increased Cai (n = 5). In cells loaded with the fluorescent pH(i) probe SNARF-1 AM, the emitted 590/640-nm fluorescence is a measure of pH(i). The effect of alpha-AS on the extent of cell shortening during the twitch (ES) was expressed as the percentage of resting cell length. Both ES and pH(i) were assessed in myocytes bathed in 1.5 mM [Ca2+] and stimulated at 0.5 Hz (control ES, 7.4 +/- 1.5%; control pH(i), 7.11 +/- 0.05; n = 10). alpha-AS enhanced both ES (delta ES, 1.8 +/- 0.6%; p less than 0.05) and pH(i) (delta pH(i), 0.06 +/- 0.01; p less than 0.005), and there was a significant correlation between delta ES and delta pH(i) (r = 0.76, p less than 0.05). A similar effect of alpha-AS on pH(i) was observed in the absence of electrical stimulation (n = 8). The alpha-AS-induced enhancement of ES and pH(i) was abolished by 10 microM ethylisopropylamiloride, a Na(+)-H+ exchange inhibitor (n = 7). In additional experiments, myocytes were preincubated either with 0.2 microM 4 beta-phorbol 12-myristate 13-acetate (n = 8) or with 5 nM staurosporine (n = 8), which have been shown to downregulate and inhibit Ca(2+)-activated phospholipid-dependent protein kinase C, respectively. In either group, alpha-AS had no effect on pH(i) and decreased ES to approximately 60% of control.(ABSTRACT TRUNCATED AT 400 WORDS)
Gambassi
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Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.
Spurgeon
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HA
Lakatta
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EG
Blank
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Capogrossi
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MC
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects*; D005453:Fluorescence; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology*; D008297:Male; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D009248:Nadolol / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013268:Stimulation, Chemical
D010656:Phenylephrine; D009248:Nadolol; D007545:Isoproterenol; D002118:Calcium