Rafael Rubio

Release of Adenosine from Ischemic Brain EFFECT ON CEREBRAL VASCULAR RESISTANCE AND INCORPORATION INTO CEREBRAL ADENINE NUCLEOTIDES

A threefold increase in tissue adenosine levels was produced by ischemia in the dog and the rat brain within 1 minute; adenosine levels increased further with longer periods of ischemia. Inosine and hypoxanthine were also increased by ischemia but to different degrees. The nucleosides and hypoxanthine appeared in cerebrospinal fluid during ischemia in the dog brain, and incubation of adenosine in normal cerebrospinal fluid failed to show the presence of degradative enzymes. Intra-arterially administered adenosine in the dog and the cat produced little or no increase in cerebral blood flow or the diameter of pial arterioles, respectively, when it was given in amounts that reduced arterial blood pressure. However, when it was applied topically to exposed pial arterioles of the cat, adenosine induced dilation that was roughly proportional to the dose used. When U-14C-adenosine was infused into the internal carotid arteries of the dog, no radioactivity was detectable in the cerebrospinal fluid and practically none appeared in the brain tissue. When labeled adenosine was added to dog cerebrospinal fluid, only a few counts appeared in the cerebral venous blood, whereas cerebral tissue was heavily labeled with 84–87% of the radioactivity in the form of adenine nucleotides. In the rat, after intravenous injection of labeled adenosine, the counts per gram of heart were twenty- to thirtyfold greater than those per gram of brain. These observations indicate that intra-arterially administered adenosine probably fails to cross the blood-brain barrier rapidly enough to influence cerebral blood flow but that it can be released from the ischemic brain into the cerebrospinal fluid and be reincorporated from the cerebrospinal fluid into brain nucleotides. Hence, adenosine can conceivably participate in the regulation of cerebral blood flow.

Release of Adenosine by the Normal Myocardium in Dogs and Its Relationship to the Regulation of Coronary Resistance

The evidence supporting the hypothesis that adenosine is the mediator of metabolic regulation of coronary blood flow was obtained from experiments characterized by myocardial hypoxia. If adenosine serves the role of physiological regulator of coronary blood flow, it must also be released by the normal heart. Experiments designed to study this question were performed on 15 open-chest dogs in which adenosine was sought in perfusates of the epicardial surface of the well-oxygenated heart. The pericardial space was perfused with warm (37°C) Tyrodes or Krebs-Henseleit solutions (400 to 1200 ml over 1 to 3 hours), and the perfusates were analyzed for adenosine. With a normal myocardial oxygen supply, adenosine was present in the perfusates in a concentration of 3.1 ± 0.5 x 10>-8M. Partial asphyxia, induced by reducing pulmonary ventilation, significantly (P ≤ 0.02) increased the adenosine concentration of the perfusates to 5.4 ± 0.8 x 10-8M. In four dogs the normal pericardial fluid was found to contain adenos...

Increases in Cerebral Interstitial Fluid Adenosine Concentration during Hypoxia, Local Potassium Infusion, and Ischemia

This study used the brain dialysis technique to test the hypothesis that the adenosine concentration of cerebral interstitial fluid increases during situations in which cerebral oxygen supply is inadequate for oxygen demand. Sealed 300-μm hollow dialysis fibers were implanted in the caudate nucleus of pentobarbital-anesthetized rats and perfused at 2 μl/min with artificial cerebrospinal fluid. In vitro tests indicated the recovery of adenosine, inosine, and hypoxanthine from the external medium to be ∼20% at 2 μl/min and close to 100% at 0.1 μl/min. Three in vivo interventions were tested: hypoxia/hypotension (Pao2 = 41.9 mm Hg; MABP = 42.8 mm Hg; n = 9), local potassium infusion (n = 4), and cerebral anoxia/ischemia (n = 10). These interventions produced 10-, 4-, and 30-fold increases in perfusate adenosine concentration, respectively, as well as increases in perfusate concentrations of inosine and hypoxanthine. A separate group of rats (n = 9) perfused at 0.1 μl/min yielded estimates of cerebral interstitial fluid adenosine, inosine, and hypoxanthine concentrations of 1.26, 3.30, and 7.19 μM, respectively. These results are consistent with the adenosine hypothesis for the regulation of CBF.

Brain adenosine production in the rat during 60 seconds of ischemia.

In rats, cerebral perfusion pressure was altered abruptly by aortic transection to determine the production by ischemic brain of adenosine and its metabolites, inosine and hypoxanthine. Brain samples were obtained after 0, 5, 10, 15, 30, and 60 seconds of ischemia. Also measured were ATP, ADP AMP, phosphocreatine (PCr), lactate, and pyruvate. Blood pressure was monitored continuously, and arterial Po2, PcO2, and pH were measured just prior to induction of ischemia. Adenosine was elevated to 2.30 ± 0.31 (Sti) nmol/g at 5 seconds from a control value of 0.96 ± 0.07. A significant elevation of adenosine continued to 60 seconds (5.50 ± 1.24). Furthermore, inosine showed a progressive upward trend during the entire 60 seconds of ischemia, whereas no change in hypoxanthine occurred between the moment of transection (31.81 ± 2.01 nmol/g) and 60 seconds of ischemia (34.72 ± 2.93). PCr decreased by 1.24 fimol/g within the first 5 seconds. After the onset of hypotension, significant changes did not occur in AMP and ADP until 30 seconds, and in ATP and pyruvate until 60 seconds after aortic transection; lactate was elevated by 10 seconds. The rapid rise of cerebral adenosine within 5 seconds after the onset of ischemia supports a role for adenosine in the regulation of cerebral blood flow. Ore Res 45: 486-492, 1979

Regulation of coronary blood flow

Abstract A very schematic summary of possible factors that affect coronary vascular resistance is illustrated in Fig. 11. These factors are mechanical, myogenic, metabolic, and neural. 64 Mechanical influence is passive and is essentially determined by the extravascular compression which is important only during systole and particularly in the subendocardial layers of the left ventricular myocardium. With respect to the myogenic factor it is controversial whether it plays a role in adjustments of coronary vascular tone. Metabolic processes are possibly integrated at the level of the myocardial cell P O 2 which is the resultant of oxygen supply and need, which in turn depend on the myocardial contractile activity as well as other biochemical processes. A decrease in myocardial P O 2 gives rise to the release of the vasodilator metabolite adenosine. Nevertheless, other chemical factors (H + , K + , osmolarity) known to be released by the heart (which by themselves are poor and transient coronary vasodilators and whose release does not correspond with changes in coronary resistance) may play a role by modulating the coronary sensitivity to adenosine and other factors.

Role of Adenine Nucleotides, Adenosine, and Inorganic Phosphate in the Regulation of Skeletal Muscle Blood Flow

Experiments were performed on isolated frog sartorius muscle and in situ dog skeletal muscle to determine whether adenine nucleotides and their degradation products are released during contraction in concentrations capable of producing arteriolar dilation ATP was not detectable (>10−8M) in the bathing solution of the resting or contracting frog sartorius muscle. Inorganic phosphate (P1) in the muscle bath increased from 9 × 10−5M to 28 × 10−5M with 30 minutes of contraction (2 Hz) or with rest. With the dog hindlirnb preparation, ATP, ADP, and AMP were not detectable (>5 × 10−8M in the venous blood collected after 5 minutes of ischemic contraction whereas P1 was present at a concentration of 3.7 × 10×8M. Arterial blood levels required to elicit detectable vasodilation for ATP, ADP, AMP, and P1 were 28.7 × 10−8M, 27.1 × 10−8M 31.4 × 10−8M and 7.2 × 10−4M respectively. The adenosine concentration in dog muscle increased from 0.7 to 1.5 nmole/g with ischemic contraction, and hypoxanthine and inosine increased from 4.5 to 8.5 nmole/g and 2.0 to 5.5 nmole/g, respectively. The adenosine concentration in venous plasma collected from the hiodlimb immediately after termination of the irchemic contraction period was 2.2 × 10−7MM as compared to 0.4 × 10−7M in control venous and arterial blood samples. Hypoxanthine and inosine concentrations in venous blood increased 22- and 270-fold, respectively, foflowing ischemic contraction. The calculated interstitial fluid adenosine concentration was twice the arterial concentration of adenosine required to elicit maximal arteriolar dilation. These findings suggest that adenosine may play a role in the metabolic regulation of skeletal muscle blood flow, whereas ATP, ADP, AMP, and P1 may not.

Inhibition of slow action potentials of guinea pig atrial muscle by adenosine: A possible effect on Ca2+ influx ☆

Adenosine decreases the force of contraction in atrial muscle, the uptake of Ca2+, the plateau phase and duration of the action potential. In contrast to acetylcholine which increases K+ permeability the effects of adenosine are antagonized by caffeine and are not blocked by atropine. It has been suggested that these effects of adenosine are mediated by a depression of the cell membrane to Ca2+ permeability. In order to test this hypothesis we attempted to determine whether adenosine had inhibitory effects on the slow action potential of potassium-depolarized (20 mm) atrial muscle treated with norepinephrine (5 × 10−5m). With the slow action potentials it appears that Ca2+ carries the inward current since (1) the overshoot varies according to the Nernst equation upon changes in extracellular [Ca2+], (2) changes in inward ionic flow associated with the action potential are paralleled by changes in developed tension. Adenosine at micromolar concentrations reduced within seconds the rate of rise and amplitude of the action potential. The action potentials lost their all-or-none nature and appeared graded with adenosine. The muscle became completely inexcitable at concentrations as low as 10.2 μm. These effects of adenosine could either be reversed within seconds by enzymatic degradation of adenosine or by raising the extracellular [Ca2+]. These findings suggest that adenosine depresses the membrane permeability to Ca2+ either directly or through an indirect membrane stabilizing effect mediated by permeability changes in Na+ or K+ ions. This effect of adenosine possibly occurs on the extracellular side of the membrane since adenosine can only exist extracellularly and large and charged adenosine derivatives (ATP, ADP, AMP, c-AMP, NAD and NADP) that probably do not penetrate the cell cause similar effects.

Primary angioplasty versus systemic thrombolysis in anterior myocardial infarction.

OBJECTIVES This study compares the efficacy of primary angioplasty and systemic thrombolysis with t-PA in reducing the in-hospital mortality of patients with anterior AMI. BACKGROUND Controversy still exists about the relative benefit of primary angioplasty over thrombolysis as treatment for AMI. METHODS Two-hundred and twenty patients with anterior AMI were randomly assigned in our institution to primary angioplasty (109 patients) or systemic thrombolysis with accelerated t-PA (111 patients) within the first five hours from the onset of symptoms. RESULTS Baseline characteristics were similar in both groups. Primary angioplasty was independently associated with a lower in-hospital mortality (2.8% vs. 10.8%, p = 0.02, adjusted odds ratio 0.23, 95% confidence interval 0.06 to 0.85). During hospitalization, patients treated by angioplasty had a lower frequency of postinfarction angina or positive stress test (11.9% vs. 25.2%, p = 0.01) and less frequently underwent percutaneous or surgical revascularization after the initial treatment (22.0% vs. 47.7%, p < 0.001) than did patients treated by t-PA. At six month follow-up, patients treated by angioplasty had a lower cumulative rate of death (4.6% vs. 11.7%, p = 0.05) and revascularization (31.2% vs. 55.9%, p < 0.001) than those treated by t-PA. CONCLUSIONS In centers with an experienced and readily available interventional team, primary angioplasty is superior to t-PA for the treatment of anterior AMI.

Differential effects of adenosine and nitroglycerin on the action potentials of large and small coronary arteries.

We used intracellular microelectrodes to study some membrane electrical properties of isolated large (>1.0 nun) and small (<500 /un) coronary arteries of the dog. The resting membrane potential (Em) was not significantly different in large arteries and small arteries (average of -56 mV and -53 mV, respectively) nor was the input resistance (9 MΩ and 10 MΩ, respectively). Spontaneous action potentials were not present in vessels of either size, and action potentials could not be induced by electrical stimulation. Addition of tetraethylammonium ion (TEA, 10 mM) rapidly induced overshooting action potentials on electrical stimulation in both large and small arteries. The amplitude of the action potential increased as a function of log [Ca1+]o, the slope of the curve being 30 mV/decade in the large and small arteries, thus demonstrating that CaI+ carries most of the inward current during the action potential. Verapamil (10−6 M) blocked these Ca1+-dependent action potentials in both the large and small coronaries. Adenosine (1CT6 M) blocked the action potential in the small coronary arteries but had no effect on the action potential in the large coronary arteries. In contrast, nitroglycerin (10−6M) blocked the action potential in the large coronary arteries but not in the small arteries. The results of this study demonstrate that adenosine blocks Ca1+ inward current preferentially in small coronary arteries, and nitroglycerin blocks the Ca1+ current preferentially in large coronary arteries. These observations are consonant with a role for adenosine in the metabolic regulation of coronary blood flow in the small coronary vessels. cire Res 44:176-182, 1979

Relationship between coronary flow and adenosine production and release.

Hearts from 5 guinea pigs were excised and simultaneously perfused for 30 min with Krebs-Henseleit solution at 37°C and equilibrated with 95% O2 and 5% CO2. The coronary flow at the end of the equilibration period was taken as the control flow (100%). Thereafter the hearts were perfused for 10 min with solutions equilibrated with various oxygen mixtures (95, 60, 30, 15, 10 and 5% O2). Experiments were performed on two groups of 5 hearts at each oxygen level. Coronary flow was measured and the perfusates were collected for adenosine analysis. At the end of 10 min the hearts were instantaneously frozen and myocardial adenosine and adenine nucleotides were determined. As the PO2 of the gas phase was reduced, per cent change in coronary flow, tissue adenosine and the rate of adenosine release into perfusates were the same for 95 and 60% PO2 but increased in a parallel fashion at the lower PO2 values. However, oxygen consumption remained relatively constant and the estimated energy charge (E = 12ADP + ATPATP + ADP + AMP) decreased slightly. The striking parallelism between coronary flow and adenosine production suggests that the decrease in coronary resistance associated with reduction of oxygen tension in the perfusing fluid may be attributed to the release of adenosine by myocardial cells. Since 5′-nucleotidase is activated in vitro by a decrease in E, it is possible that the small change in the balance of the myocardial adenine nucleotides may control adenosine production.