David L. Snyder

Effect of aging on A1-adenosine receptor-mediated inhibition of norepinephrine release in the rat heart.

Adenosine inhibits norepinephrine (NE) release from cardiac adrenergic nerves and reduces the postsynaptic beta-adrenergic mediated actions of NE, leading to decreased myocardial force of contraction. The actions of adenosine are mediated by pre- and postsynaptic adenosine A1 receptors (A1-AdoR). We reported that adenosine inhibition of postsynaptic beta-adrenergic receptor-mediated cyclic adenosine monophosphate (cAMP) production declines with age in male F344 rat hearts. In this study, cardiac synaptosomes, isolated intact adrenergic nerve terminals, were used to examine the effect of age on adenosine inhibition of NE release. Cardiac synaptosomes were prepared from the hearts of 6- and 24-month-old male F344 rats, loaded with [3H]NE, and placed in a superfusion system. [3H]NE release was induced by high [K+] exposure in the presence of varying concentrations of adenosine or the specific A1-AdoR agonist, N6-p-sulfophenyladenosine (SPA). [3H]NE release was significantly reduced in old rats compared with young rats. Inhibition of [3H]NE release by adenosine and SPA was significantly greater in young rats compared with old rats. The A1-AdoR antagonist, 8-(p-sulfophenyl)-theophylline, blocked the actions of adenosine on [3H]NE release, and the specific adenosine A2-receptor agonist, cyclopropylcarboxamidoadenosine, had no effect on [3H]NE release. Our data suggest that presynaptic A1-AdoR-mediated inhibition of NE release in the rat heart declines with age.

Influence of age and dietary restriction on norepinephrine uptake into cardiac synaptosomes.

The purpose of this study was to determine whether aging alters neuronal uptake of norepinephrine (NE) in the rat heart and if dietary restriction influenced the effect of age on this system. Cardiac synaptosomes were prepared from 6-, 12- and 24-month-old male F344 rats fed ad libitum (AL) or a diet restricted (DR) to 60% of AL intake. Cardiac synaptosomes were incubated with 50, 100, 200, or 400 nM [3H]NE for 10 min at 37 degrees C with and without desmethylimipramine (DMI), a selective neuronal-uptake blocker. DMI-sensitive [3H]NE uptake was calculated as the difference between samples with and without DMI. NE uptake was adjusted for the number of cardiac synaptosomes in each sample by dividing by the endogenous NE content in each sample. The Vmax for uptake ([3H]NE/min/ng NE) declined significantly between 6 and 12 months in AL rats and between 12 and 24 months in DR rats. Km was not significantly different between age or diet groups. The change in Vmax with age suggests that the number of NE transporters per synaptosome may decline with age and that DR delays this effect of age. There were no differences in the sensitivity to DMI between age or diet groups.

Cardiovascular hypertrophy and increased vascular contractile responsiveness following repeated cocaine administration in rabbits

The effects of repeated cocaine administration on contractile responses were studied in adult rabbits. Repeated cocaine exposure caused a significant increase in the maximal response of the aorta to the agonists norepinephrine and serotonin as well as the receptor- independent stimulus KCl when compared to the saline controls. Cocaine exposure caused a significant increase in the wet weights of both heart and aorta. When the contraction was normalized to the wet weight of the aorta there was no difference between rabbits administered cocaine and saline. Acute cocaine administration caused a time-dependent increase in immunoreactivity of the proto-oncogene c-Fos in the aorta. These results show that repeated cocaine administration leads to the development of cardiovascular hypertrophy.

Prenatal cocaine exposure alters norepinephrine release from cardiac adrenergic nerve terminals

The effect of prenatal cocaine exposure on the development of the cardiac adrenergic nervous system was assessed in neonatal rabbits. Pregnant does received cocaine (4 mg/kg, i.v., bid) or saline during gestational days 8 to 29. Hearts were obtained on postnatal days 10, 20, 30, and 50. Adrenergic nerve function was assessed by measuring 3H-norepinephrine (NE) uptake and 3H-NE release from cardiac synaptosomes. NE uptake increased with postnatal age and was not affected by cocaine exposure. K(+)-induced NE release increased with age, was significantly less in cocaine exposed rabbits compared to saline exposed rabbits at days 10, and 20, but was similar at days 30 and 50. NE release induced by ionomycin, a Ca2+ ionophore, did not change with age, was significantly greater in cocaine exposed rabbits compared to saline exposed rabbits at days 10, 20, and 30, but was similar at day 50. Wet heart weight, heart weight per body weight, and NE content of the hearts were not affected by cocaine exposure. These results suggest that prenatal cocaine exposure delays the development of the mechanisms responsible for Ca2+ influx during K(+)-induced depolarization and increases the neurosecretory response to intracellular Ca2+.

Effect of Aging on the Negative Chronotropic and Anti-β-Adrenergic Actions of Adenosine in the Rat Heart

The effect of aging on the antiadrenergic actions of adenosine was studied in vitro and in vivo by using adult (6-month-old) and old (24-month-old) male Fischer 344 rats. In anesthetized animals, adenosine (0.01-0.1 micromol/kg), given as a rapid bolus into the right atrium, exerted a negative chronotropic effect manifested by a dose-dependent transient prolongation of sinus cycle length (SCL). This effect was similar in both age groups (n = 6, each; i.e., the percentage maximal prolongation of SCL (%deltaSCL) ranged from 12 +/- 2% to 63 +/-14% in the adult and from 20 +/- 7% to 57 +/- 15% in the old rats. In the presence of isoproterenol (0.2 microg/kg/min), the negative chronotropic action of adenosine was potentiated in the adult rats much more than in the old rats [i.e., %deltaSCL ranged from 60 +/- 28% to 183 +/- 48% vs. 40 +/- 12% to 70 +/- 13%, respectively (p < 0.05, adult vs. old)]. In the isolated perfused hearts, isoproterenol (1 microM for 1 min) exerted similar chronotropic and inotropic effects in adult (n = 9) and old hearts [n = 6; i.e., heart rate, left ventricular pressure (LVP), and LVdp/dt increased by 56 +/- 3%, 17 +/- 1%, and 37 +/- 2%, and 57 +/- 2%, 17 +/- 1%, and 35 +/- 3%, respectively, in the absence of, and by 27 +/- 2%, 7 +/- 1%, and 19 +/- 2% and 41 +/- 3%, 12 +/- 1%, and 25 +/-2% in the presence of adenosine (5 microM for 1 min)]. Adenosine administration after isoproterenol caused only an insignificant increase in coronary blood flow. Finally, the adenosine attenuation of either isoproterenol- or forskolin-induced production of 3,5-cyclic adenosine monophosphate (cAMP) was significantly less in atrial membranes isolated from old versus adult rats (n = 6, each). It was concluded that in the old Fischer 344 rat hearts, the antiadrenergic action of adenosine is attenuated as compared with its action in adult rat hearts.

Effect of age on cardiac norepinephrine release in the female rat

We previously demonstrated an age-related decline in K+- induced norepinephrine (NE) release from cardiac synaptosomes prepared from 6- and 24- month- old male F344 rats. The purpose of the present study was to determine if the age- related decrease in NE release seen in male F344 rats is also present in female F344 rats. K+- induced NE release was assessed in cardiac synaptosomes prepared from 6-, 12-, 18-, and 24- month- old male and female F344 rats. NE release was significantly greater in young male rats, compared to old male rats. However, no age- related decrease in NE release was observed in the female rats. In contrast to previous observations in male rats, raising extracellular [Mg2+], an inorganic Ca2+ channel blocker, reduced NE release to the same extent in all female ages. Omega- conotoxin, an organic Ca2+ channel blocker, also decreased NE release to the same extent in all female ages. These studies suggest that in contrast to aging male rats, cardiac adrenergic nerve terminals of aging female rats maintain their capacity to release NE. (Aging Clin. Exp. Res. 7: 210–217, 1995)

Prenatal cocaine exposure affects the development of aortic adrenergic innervation and contractile responses

This study examines the effects of prenatal cocaine administration on the development of vascular sympathetic innervation and contractile responsiveness. Rabbits received cocaine (4 mg/kg, iv, bid) or saline during gestational days 8 to 29. Aortas were obtained on postnatal days 10, 20, 30 and 50. Vascular smooth muscle responsiveness was assessed by measuring aortic contractile responses to norepinephrine (NE) and to other vasoconstrictors. Vascular adrenergic innervation was evaluated by measuring desipramine sensitive [3H]-NE uptake into aortic ring segments and aortic NE content. [3H]-NE uptake and NE content were reduced at postnatal days 10 and 20 in the rabbits exposed prenatally to cocaine. Differences were not observed at postnatal days 30 or 50. The contractile response to NE was reduced in rabbits exposed to cocaine prenatally. Maximal response and potency were decreased at postnatal day 10 and potency was still decreased at day 20, but not at the older ages. Contractile responses to serotonin (5-HT) and angiotensin II (AII) were not affected by prenatal cocaine exposure. These results suggest that prenatal cocaine exposure delays the development of aortic adrenergic innervation and alpha adrenoceptor responsiveness.

Decrease in Norepinephrine Release from Cardiac Adrenergic Nerve Terminals after Ischemia and Reperfusion a

Occlusion of the coronary arteries causes adrenergc denervation of the infarcted myocardium and surrounding regions.’ Little is known about the extent of functional decline in the surviving adrenergc nerve terminals in the ischemic and non-ischemic portions of the myocardium or about the ability of these nerves to eventually recover full function. Injury and recovery may also be influenced by age and gender. Therefore experiments were performed to assess changes in cardiac adrenergic function after ligation and reperfusion of the left anterior descending artery (LAD) in young (6 months old) and old (24 months old) male and female F344 rats. K+-induced 3H-norepinephrine (NE) release fiom cardiac synaptosomes (SYN) and coronary arteries prepared from these hearts was used to measure adrenergc function. Male and female F344 rats were maintained in separate rooms under barrier conditions. Myocardial ischemia and reperfusion (1-9 was induced by ligation of the LAD as described by Lefer et aL3 Under sodium pentobarbital anesthesia (40 mg/kg) the chest was opened, a suture with slip knot was placed around the LAD, and the chest was closed. Ischemia was maintained for 10 min and was followed by 60 min of reperfusion before removing the heart. SYN were prepared from the ischemic and nonischemic regions of the heart. SYN and segments of the LAD and left circumflex artery (LCX) were incubated with 300 nM 3H-NE and then placed in individual chambers in a superfusion system. Preparations were also obtained from decapitated, unanesthetized, and unoperated control rats of the same ages and gender. 3H-NE release was induced by changing the superfusion buffer fbr 2 min to a high K+ buffer (peak chamber concentration of 45 mM K+). Methods are described in detail in Snyder et aL2 In the control groups, gender and age differences were observed in the net fractional release of 3H-NE from rat coronary arteries and SYN (TABLE 1). Old males showed significant decreases in release from the LAD, LCX, and SYN when compared to young males. These results agree with our previous findings.2 In contrast, female rats showed no significant age-related changes in release from the LAD, LCX, or SYN.