[Alpha but not beta-adrenergic stimulation has a positive inotropic effect associated with alkalinization of intracellular pH]

There is increasing evidence that alpha-adrenoceptors also exist in the myocardium and that an increase in force of contraction may be produced by stimulation of these sites. This positive inotropism seems to be dependent either on an increased amount of Ca++ released into the cytosol with each action potential or on increased myofilament responsiveness. In contrast, beta-adrenergic stimulation reduces the sensitivity of the contractile proteins and the positive inotropic effect is due to the activation of L-type calcium channels on the sarcolemma. We used single, isolated, enzymatically dissociated, adult rat ventricular myocytes. Cells were loaded either with the ester derivative of the Ca++ probe Indo-1 or with the intracellular pH probe Snarf-1 and at the same time we measured the contractile parameters and monitored the fluorescence as an index of intracellular calcium concentration or pH value. The single cells (bicarbonate buffer continuously gassed with O2 95%, CO2 5%, Ca++ 1.5 mM, field stimulation 0.5 Hz) were exposed to phenylephrine (50 microM) and nadolol (1 microM). Alpha-adrenergic stimulation increased twitch amplitude (delta ES = 1.93 +/- 0.77, n = 8; p less than 0.05) and showed only a slight increase in Ca++ transient. On the other end, the positive inotropic effect (delta ES = 2.84 +/- 0.86, n = 4; p less than 0.02) obtained with beta-adrenergic stimulation (isoproterenol 50 nM, bicarbonate buffer, Ca++ 0.5 mM, field stimulation 0.2 Hz) was always associated with a large increase in intracellular Ca++ concentration. Isoproterenol did not change intracellular pH (delta pH = 0.006 +/- 0.006, n = 4; NS) while phenylephrine increased it significantly (delta pH = 0.055 +/- 0.011, n = 8; p less than 0.002). Moreover, there was a statistically significant correlation between delta ES and delta pH (R2 = 0.532; p less than 0.05) when phenylephrine was present. This alkalinization as well as the increased contractility was antagonized by treatment with ethyl isopropyl-amiloride (10 microM), a selective Na+/H+ inhibitor (delta ES = 0.09 +/- 0.07, n = 6; NS and delta pH = -0.001 +/- 0.011, n = 6; NS). Thus, alpha-adrenergic stimulation in isolated cardiac cells exerts a positive inotropic effect and this is associated with a significant intracellular pH alkalinization. In contrast, the marked inotropic action of beta-stimulation does not involve any intracellular pH modulation. Therefore, it seems likely that, in myocardial cells, an increased myofilament responsiveness due to the alkalinization could represent a possible mechanism for the positive inotropic effect mediated by alpha-adrenergic stimulation

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

Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes

alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50 microM phenylephrine and 1 microM propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe into-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Cao exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1 microM prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca(2+)-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1 microM 4 beta-phrobol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC.(ABSTRACT TRUNCATED AT 400 WORDS

Opposing effects of alpha 1-adrenergic receptor subtypes on Ca2+ and pH homeostasis in rat cardiac myocytes

We examined the effect of alpha 1-adrenergic receptor (AR) subtypes on contraction, cytosolic Ca2+ concentration ([Ca2+]i), and cytosolic pH (pHi) of rat ventricular myocytes loaded with the Ca2+ indicator indo 1 or the pH indicator carboxyseminaphthorhodafluor-1. Nonselective alpha 1-AR stimulation was effected with phenylephrine plus nadolol. alpha 1-AR subtype stimulation was achieved with alpha 1-AR and chloroethylclonidine (CEC) or with alpha 1-AR and WB-4101. Cells were in bicarbonate buffer with 0.5 mM Ca2+ and were electrically stimulated at 0.5 Hz. Results show that 1) nonselective alpha 1-AR stimulation increased twitch and [Ca2+]i transient amplitudes, myofilament response to Ca2+, and pHi; 2) alpha 1-AR plus CEC increased twitch and [Ca2+]i transient amplitudes and also enhanced myofilament response to Ca2+ via cytosolic alkalinization; 3) alpha 1-AR plus WB-4101 decreased twitch and [Ca2+]i transient amplitudes and also pHi; and 4) cytosolic acidification due to alpha 1-AR plus WB-4101 was abolished by protein kinase C inhibition (staurosporine pretreatment) or downregulation (prolonged exposure to phorbol esters). In summary, the net effects of alpha 1-adrenergic stimulation on contraction, [Ca2+]i, and pHi are due to opposing WB-4101- and CEC-sensitive alpha 1-AR subtype signaling pathways

Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo-1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai