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The plasma membrane calmodulin-dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca2+ homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na+/Ca2+ exchanger. We approached the question of the physiological function of the calcium pump using a transgenic "gain of function" model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the L-type Ca2+ current, or fast [Ca2+]i transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 micromol/L phenylephrine. A 1.4-fold- and 2.0-fold-higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the alpha- and beta-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of alpha- and beta-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca2+-pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.

To examine whether stimulation of alpha-adrenergic receptors may affect the oxidative pentose phosphate pathway (PPP) in the rat heart, norepinephrine (NE) and the alpha-adrenergic agonist norfenephrine were used. NE was administered as a continuous intravenous infusion in awake rats for 3 days. It stimulated the activity of cardiac glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of the oxidative PPP, in a dose-dependent manner. With the highest dose (0.2 mg.kg-1.hr-1), there was also a time-dependent enhancement. The increase observed after 48 hours was attenuated partially by the beta-receptor blocker metoprolol and the alpha-receptor blocker prazosin. It was entirely abolished when both drugs were administered. Carvedilol, a beta-adrenergic blocker and vasodilator with alpha 1-blocking activity (0.5 mg.kg-1.hr-1), prevented the NE-induced increase in cardiac G-6-PD activity, in functional parameters (heart rate, left ventricular systolic pressure, and left ventricular dP/dtmax), and in the heart weight/body weight ratio. The alpha-adrenergic stimulator norfenephrine increased myocardial G-6-PD activity; prazosin prevented this stimulation. NE and norfenephrine also elevated the available pool of cardiac 5-phosphoribosyl-1-pyrophosphate. G-6-PD activity was enhanced in cardiac myocytes freshly isolated from the left ventricle of rats that had received NE infusion for 3 days (12.3 +/- 1.4 units/g protein) compared with control rats (1.5 +/- 0.4 units/g protein). The activity of 6-phosphogluconate dehydrogenase, one of the enzymes in the oxidative PPP, was elevated only moderately from 12.7 +/- 0.7 to 19.1 +/- 1.4 units/g protein. Combined alpha- and beta-receptor blockade with carvedilol attenuated these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

In closed-chest rats, isoproterenol (ISO, 25 mg/kg), 5 hours after subcutaneous administration, increased heart rate by 53%, left ventricular (LV) dP/dtmax by 80%, and cardiac output by 37%. LV systolic pressure (LVSP, -10%), mean arterial pressure (MAP, -12%), and total peripheral resistance (TPR, -36%) were diminished. In separate experiments, continuous intravenous infusion of adenine (50 mg/kg/hr) for 5 hours reduced heart rate (-11%), LVSP (-16%), MAP (-20%), TPR (-33%), and LV dP/dtmax (-20%). Cardiac output was increased (+20%). Inosine has been shown to have similar effects, except for a decline in cardiac output. Adenine (50 mg/kg/hr) attenuated the ISO-induced increase in heart rate and LV dP/dtmax and aggravated the decline in LVSP, MAP, and TPR. The increase in cardiac output was not changed. Inosine (200 mg/kg/hr) modified the ISO effects to a similar extent. Ribose (200 mg/kg/hr) added to the adenine infusion did not have functional effects. However, it aggravated the modifying influence of inosine on LVSP, LV dP/dtmax, and MAP. ISO reduced the cardiac ATP content (mumol/g) from a control value of 5.02 +/- 0.06 (n = 12) to 3.51 +/- 0.13 (n = 10). Adenine (3.56 +/- 0.21, n = 7) and ribose (3.64 +/- 0.11, n = 9) alone did not affect it, but inosine attenuated it (4.33 +/- 0.08, n = 8). Adenine and inosine in combination with ribose abolished the ISO-induced ATP decline (5.18 +/- 0.23, n = 7, and 4.76 +/- 0.10, n = 8, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of angiotensin converting enzyme inhibition on the development of left ventricular (LV) hypertrophy due to stenosis of the aortic arch was studied in female Sprague-Dawley rats. The aortic arch was banded to an outer diameter of 1.0 mm. After 14 days, LV and right ventricular functional parameters and transstenotic pressure gradient were measured in anesthetized rats. In addition, regional heart weights were determined, and myocytes of three different heart regions were isolated and subjected to morphometric analysis. To inhibit the angiotensin converting enzyme, ramipril was administered orally by gavage in a single daily dose of 1 mg/kg. Rats with aortic stenosis showed a marked increase in LV systolic pressure, mean prestenotic aortic pressure, and LV stroke work compared with sham-operated rats and demonstrated a systolic transstenotic pressure gradient of 82 mm Hg. This increase in LV hemodynamic load was paralleled by the development of LV hypertrophy as determined by a 37% increase in LV weight and by a 20% increase in cell volume of isolated LV myocytes. Concomitant ramipril treatment did not significantly affect LV functional parameters. The transstenotic pressure gradient was the same as in untreated rats with aortic stenosis. Likewise, the weight gain of the LV as well as the development of cellular hypertrophy of the LV were not influenced. Thus, in this model, angiotensin converting enzyme inhibition did not reduce the development of LV hypertrophy independent of the hemodynamic load.

The oxidative pentose phosphate pathway is poorly developed in the rat heart compared with other organs, since the activity of glucose-6-phosphate dehydrogenase (G-6-PDH), the first and rate-limiting enzyme of the oxidative pentose phosphate pathway, is low. As a consequence, the available pool of 5-phosphoribosyl-1-pyrophosphate and the rate of adenine nucleotide biosynthesis are limited. Isoproterenol, 24 hours after subcutaneous administration at 0.1, 1, and 25 mg/kg, stimulated the activity of G-6-PDH in whole hearts dose-dependently from 4.3 +/- 0.16 (control) to 6.6 +/- 0.35, 10.3 +/- 0.82, and 11.5 +/- 0.56 units/g protein, respectively. The activity of 6-phosphogluconate dehydrogenase, another of the enzymes in the oxidative pentose phosphate pathway, remained unchanged. G-6-PDH activity started to increase 12 hours after isoproterenol application, when the glycogenolytic and functional response was over, and reached a peak value between 24 and 48 hours. This stimulating effect was also demonstrated in cardiac myocytes that were isolated 28 hours after isoproterenol application. beta-receptor blockade with atenolol reduced the isoproterenol-induced increase in cardiac G-6-PDH activity by 90%. Cycloheximide, which inhibits translation, and actinomycin D, which interferes with transcription, attenuated it by 83% and 78%, respectively. These results indicate that cardiac beta-adrenergic receptors and enzyme protein synthesis are involved in this effect. Other beta-sympathomimetic agents such as dopamine, dobutamine, fenoterol, salbutamol, and terbutaline also stimulated myocardial G-6-PDH activity in a time- and dose-related manner. The calcium antagonist D 600 (gallopamil) reduced the isoproterenol-elicited stimulation by 65%, and verapamil blunted the fenoterol-induced increase by 50%. This suggests that Ca2+ ions also contribute to the stimulation of the cardiac oxidative pentose phosphate pathway.

The role of alpha- and beta-adrenoceptors in the development of catecholamine-induced cardiac hypertrophy in vivo was investigated. Rats received a constant intravenous infusion of norepinephrine or sodium chloride (control) for 3 days. The norepinephrine infusion was combined with the alpha-blocker prazosin, the beta-blocker metoprolol, or both blockers. For modulation of the work load of the heart, the calcium channel blocker verapamil was added to the norepinephrine infusion. A further group of animals was treated with the alpha-adrenergic stimulator norfenephrine, which also was combined with prazosin or verapamil. Norepinephrine induced significant increases in mean aortic pressure, left ventricular dP/dtmax, heart rate, and total peripheral resistance. The left ventricular weight/body weight ratio was significantly elevated and was accompanied by an increase in the RNA concentration and the RNA/DNA ratio. Prazosin as well as metoprolol partially antagonized the increase in left ventricular weight and RNA concentration, whereas simultaneous prazosin and metoprolol treatment prevented the norepinephrine-induced alterations. Although combination of norepinephrine with verapamil resulted in considerable reduction of all functional parameters, the development of cardiac hypertrophy and the elevated RNA/DNA ratio were not significantly influenced. Stimulation of alpha-receptors with norfenephrine elicited an increase in total peripheral resistance and in left ventricular weight, which was abolished by prazosin. Verapamil did not affect the norfenephrine-induced cardiac hypertrophy, although it normalized essentially all functional parameters. Thus, the rapid development of cardiac hypertrophy in the norepinephrine model seems to be directly mediated by stimulation of myocardial alpha- and beta-adrenoceptors rather than by hemodynamic changes.