Pasquale Bernardi

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.

Opioid Peptide Modulation of Circulatory and Endocrine Response to Mental Stress in Humans

Healthy subjects were classified according to their percent increase in systolic blood pressure (SBP) after mental arithmetic test (MAT) as low (delta SBP 9.3-15.1%, n = 15) and high (delta SBP 35.1-45.4%, n = 15) responders. During MAT, low responders showed significantly (p < 0.01) increased plasma levels of beta-endorphin, cortisol, catecholamines, and atrial natriuretic factor (ANF) and decreased levels of endothelin-1, whereas high responders showed increased (p < 0.01) levels of Metenkephalin, dynorphin B, and catecholamines. Pretreatment with naloxone hydrochloride enhanced (p < 0.01) SBP, heart rate, noradrenaline, cortisol, and endothelin-1 levels, and reduced (p < 0.01) ANF in low responders in response to MAT, whereas it decreased (p < 0.01) hemodynamic parameters, noradrenaline, and endothelin-1 in high responders. The individual differences in hemodynamic and endocrine responses to MAT may depend on a different activation of the endogenous opioid system.

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.

ENDOGENOUS OPIOID SYSTEM AND ATRIAL NATRIURETIC FACTOR IN NORMOTENSIVE OFFSPRING OF HYPERTENSIVE PARENTS AT REST AND DURING EXERCISE TEST

Objective: To investigate the effects of the endogenous opioid system on plasma atrial natriuretic factor (ANF) levels during sympathetic hyperactivity. Design: We studied the young normotensive offspring of parents who both had essential hypertension, who are characterized by a hyperactive sympathetic nervous system. Methods: We assessed plasma β-endorphin, met-enkephalin, dynorphin B, ANF and noradrenaline levels, blood pressure and heart rate values in eight normotensive offspring and in 10 young normotensive subjects with no family history of hypertension (controls) at rest and during two exercise tests: the first test performed with the infusion of placebo (1.5 ml/min saline) and the second test with the infusion of an opioid antagonist (9.5 μg/kg per min naloxone hydrochloride). ANF and opioids were radioimmunoassayed after chromatographic pre-extraction. Results: At rest plasma met-enkephalin, dynorphin B, ANF and noradrenaline values in the normotensive offspring were significantly higher than in the controls. Exercise with placebo significantly raised all hormonal and haemodynamic parameters in the two groups. This increase was significantly higher in the normotensive offspring than in the controls. Naloxone did not modify any parameter in either group at rest, but it enhanced further the rise in plasma noradrenaline levels induced by exercise in both groups. A similar effect of naloxone during exercise was observed for plasma ANF levels in the normotensive offspring. Conclusions: Our findings show that plasma met-enkephalin, dynorphin B, ANF and noradrenaline levels at rest and during exercise are higher in normotensive offspring than in controls. The effects of naloxone indicate that in normotensive offspring at rest the opioid system does not affect ANF release, whereas during exercise it attenuates ANF hypersecretion, possibly by reducing noradrenaline release.

Pressor Effects of Endogenous Opioid System During Acute Episodes of Blood Pressure Increases in Hypertensive Patients

To investigate the involvement of endogenous opioids in acute increases in blood pressure and their functional relationship with atrial natriuretic factor and endothelin-1, we assessed plasma levels of beta-endorphin, met-enkephalin, dynorphin B, catecholamines, atrial natriuretic factor, and endothelin-1 before and after administration of the opioid antagonist naloxone hydrochloride (8 mg i.v.) in 28 hypertensive patients with a stress-induced acute increase in blood pressure. Ten patients with established mild or moderate essential hypertension and 10 normotensive subjects served as control groups. Opioids, atrial natriuretic factor, and endothelin-I were radioimmunoassayed after chromatographic preextraction; catecholamines were determined by high-performance liquid chromatography with electrochemical detection. Patients with an acute increase in blood pressure (systolic, 203.2 +/- 2.2 mm Hg; diastolic, 108.4 +/- 1.3) had plasma opioid, catecholamine, and atrial natriuretic factor levels significantly (P < .01) higher than hypertensive control patients (systolic pressure, 176.4 +/- 1.0 mm Hg; diastolic, 100.0 +/- 1.4), who had a hormonal pattern similar to that of normotensive subjects (systolic pressure, 123.2 +/- 1.5 mm Hg; diastolic, 75.0 +/- 2.0). Endothelin-1 did not differ in any group. In patients with an acute increase in blood pressure, naloxone significantly (P < .01) reduced blood pressure, heart rate, opioids, catecholamines, and atrial natriuretic factor 10 minutes after administration. Naloxone effects on blood pressure, heart rate, opioids, and catecholamines wore off within 20 minutes. In control groups, naloxone failed to modify any of the considered parameters. Our findings suggest that pressor effects of opioid peptides mediated by the autonomic nervous system during stress-induced acute episodes of blood pressure increase in hypertensive patients.

HETEROGENEITY OF IMMUNOREACTIVE DYNORPHIN B - LIKE MATERIAL IN HUMAN, RAT,RABBIT AND GUINEA-PIG HEART

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.

Endogenous Opioid Peptides and Mental Stress in Congestive Heart Failure Patients

Two groups of patients with acute congestive heart failure (CHF), New York Heart Association class III, presenting elevated plasma values of beta-endorphin, norepinephrine, atrial natriuretic factor (ANF) and endothelin-1, underwent the Mental Arithmetic Test (MAT) during placebo (n = 10) and naloxone hydrochloride (n = 10) infusion. The MAT during placebo significantly (p < 0.01) increased blood pressure, heart rate, plasma levels of Met-enkephalin, dynorphin B, beta-endorphin, norepinephrine, ANF and endothelin-1. The increases in norepinephrine, ANF and hemodynamics after the MAT during naloxone infusion were higher (p < 0.01) than those during placebo; thus, the transient upregulation of the endogenous opioid system during stress in CHF patients attenuates the hemodynamic response by reducing norepinephrine release.

Opioid peptide response to spinal cord stimulation in chronic critical limb ischemia

Twelve patients with chronic critical limb ischemia in whom a spinal cord stimulation (SCS) system had been implanted for at least one year had increased microvascular flow and achieved healing of trophic acral lesions. After switching off the system, the clinical improvement persisted for 10 days and the neurohormonal pattern showed high plasma values of beta-endorphin and Met-enkephalin, normal dynorphin B, endothelin-1 and catecholamines, and low nitric oxide. Met-enkephalin levels were further increased (P < 0.01) immediately after switching on the electrical stimulation again. The persistence of high plasma opioid levels after switching off the spinal cord stimulation explains the absence of subjective complaints and suggests an involvement of opioids in the regulation and improvement of the microcirculation.

Mechanisms of hypertension in patients with chronic obstructive pulmonary disease and acute respiratory failure

PURPOSE To investigate the effects of hypoxemia, hypercapnia, and cardiovascular hormones (norepinephrine, endothelin-1, and atrial natriuretic factor) on blood pressure during acute respiratory failure. PATIENTS AND METHODS Patients with chronic obstructive pulmonary disease and acute respiratory failure were divided into four groups of 10 patients each: hypoxemia-normocapnia, hypoxemia-hypercapnia, hypoxemia-hypocapnia, and normoxemia-hypercapnia. Plasma norepinephrine levels were determined by high-performance liquid chromatography with electrochemical detection. Plasma endothelin-1 and atrial natriuretic factor levels were radioimmunoassayed after chromatographic preextraction. RESULTS Systolic blood pressure and cardiovascular hormone levels were greater in patients with hypercapnia (whether or not they also had hypoxemia) than in those with normocapnia and hypoxemia. For example, in patients with hypercapnia and normoxemia, the mean (+/- SD) systolic blood pressure was 183+/-31 mm Hg and the mean norepinephrine level was 494+/-107 pg/mL, as compared with 150+/- 6 mm Hg and 243+/-58 pg/mL in those with normocapnia and hypoxemia (both P<0.05). Similar results were seen for endothelin-1 and atrial natriuretic factor levels, and for the comparisons of hypoxemic patients who were hypercapnic with those who were normocapnic. CONCLUSIONS These results suggest that blood carbon dioxide levels, rather than oxygen levels, are responsible for hypertension during acute respiratory failure, perhaps as a result of enhanced sympatho-adrenergic activity.

Inhibitory action of opioid peptides on ouabain-sensitive Na+-K+ and Ca2+-dependent ATPase activities in bovine cardiac sarcolemma

The present study demonstrates that morphine (10(-6) and 10(-5) M), methionine-enkephalin or leucine-enkephalin (10(-10), 10(-8), and 10(-6) M) were able to inhibit significantly, in a dose-dependent manner, both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. The inhibitory action of these opioids on the two ATPases was not antagonized by preincubation with naloxone (10(-6) M). Naloxone alone (10(-8), 10(-6) and 10(-5) M) did not affect both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. Heat-denatured methionine-enkephalin (10(-6) M) or leucine-enkephalin (10(-6) M) also unaffected both the ATPases. The possibility is also discussed that opioid peptides may regulate myocardial contractility by modulating the movement of ions across the heart sarcolemma.