Extracellular acidification related to the stimulation of catecholamines release by strontium in the bovine adrenal medulla.

The possible modifications of extracellular pH associated with the secretion of catecholamines evoked by the introduction of 2.2 mM Sr2+ to a Ca(2+)-free, buffer-free, Locke solution were investigated in decorticated perfused bovine adrenal glands. A progressive and reversible decrease of external pH accompanied the catecholamine release promoted by Sr(2+)-introduction into the perfusion fluid. This extracellular acid shift was practically undetected when the chromaffin tissue was stimulated by the addition of Sr2+ to a buffered medium. Both the secretory response as well as the extracellular pH drop mediated by Sr(2+)-introduction to a Ca(2+)-free, buffer-free, Locke solution were markedly inhibited by methoxyverapamil (0.3 mM), Mg2+ (20 mM) and hyperosmolarity (750 mOsm). The exposure of the adrenal medulla to a Ca(2+)-free, buffer-free, high-K+ solution containing 2.2 mM Sr2+ for 6 min promoted a significant enhancement of both the secretory response and the acidification of the perfusates compared with the responses evoked by Sr2+ in a 5.6 mM K+ medium. These results are consistent with the existence of a close relationship between extracellular acidification and the release of catecholamines triggered by the introduction of Sr2+ to the perfusion fluid.

Effect of hyperosmolarity on extracellular acidification associated with catecholamine secretion evoked by barium in perfused bovine adrenal medulla.

1. The effects of the increase of osmotic strength on extracellular pH modifications produced during buffer-free, 5 mM Ba2+ stimulation was studied in decorticated perfused bovine adrenal glands. 2. A pronounced and reversible attenuation of the extracellular pH drop as well as of the release of catecholamines induced by Ba2+ was detected in glands exposed to a hyperosmotic (750 mOsm) perfusion fluid made up by the addition of NaCl. 3. Similar results were observed using sucrose as osmoticant instead of NaCl. 4. A progressive decline in both external acidification and secretory response to Ba2+ was found on gradually increasing the osmotic strength of the perfusion fluid. 5. These results provide additional evidence in favour of the existence of a close association between extracellular acidification and adrenomedullary secretion induced by buffer-free Ba2+.

Characterization of extracellular pH drop due to the activation of the secretory process by acetylcholine in the bovine adrenal medulla.

A progressive and reversible decrease of external pH accompanied the catecholamine release elicited by acetylcholine in decorticated bovine adrenal glands perfused with buffer-free Locke solution adjusted to an initial pH of 7.4. Both the secretory response as well as the extracellular acid shift promoted by the cholinergic agonist were antagonized by hexamethonium plus atropine, Mg2+ and verapamil. Experiments performed to assess the effects of the reduction of external pH on acetylcholine-induced release of catecholamines revealed that increasing the extracellular concentration of H+ significantly and reversibly reduced this secretory response. These findings are consistent with the idea that adrenomedullary activation of secretion by acetylcholine could be associated with a transient acidification of the extracellular fluid. This release of protons, arising mainly from the chromaffin granules, may be involved in a local automodulatory mechanism of the regulated secretory process.

In vitro evaluation of acute effects of mitoxantrone (Novantrone) in rat and guinea pig atria.

Since guinea pig and rat atria have been used as models to study acute anthracycline-induced cardiotoxicity, experiments were carried out in these preparations to evaluate possible acute cardiac effects mediated by mitoxantrone (MTX). After a latency period of approximately 90 min, MTX (10(-5)-10(-4) M) promoted a concentration-related and time-dependent decrease of spontaneous rate in guinea pig atria. A similar but less intense effect after a longer latency interval was observed in rat atria. In this preparation, MTX (10(-5)-10(-4) M) incubated up to 150 min., induced a gradual competitive beta-adrenergic blocking effect on the positive chronotropic action of isoproterenol. This was characterized by a progressive decline of pD2 values without altering Emax. A similar and stronger effect was found in isolated guinea pig atria incubated under same conditions with MTX, except that 10(-4) M exposed for 150 min. was able to depress the Emax to isoproterenol by 21.2%. In addition, MTX (10(-4) M) in this model promoted a non-competitive antagonistic effect on the chronotropic action of histamine. These data are compatible with the idea that MTX could induce cardiac acute effects qualitatively similar to but of lower potency than those produced by doxorubicin in these two models. In addition, guinea pig atria seemed to display higher sensitivity to MTX compared to rat atrial preparations.

Decrease of extracellular pH associated with the secretion of catecholamines induced by barium in perfused bovine adrenal medulla.

1. The possible modifications of extracellular pH produced during buffer-free 5 mM Ba2+ stimulation was studied in decorticated perfused bovine adrenal glands. 2. A significant and reversible drop of pH accompanied the release of catecholamines each time the tissue was exposed to a buffer-free 5 mM Ba2+ solution. 3. A progressive declination of the magnitude of this acidification associated with a gradual attenuation of the secretory response was observed consecutive to successive periods of Ba(2+)-stimulation. 4. D-600 (methoxyverapamil), in a concentration of 3 x 10(-4) M, markedly antagonized both Ba(2+)-induced secretory response and extracellular pH drop. 5. Perfusion of adrenal medulla for 4 min period with Locke solution buffered at pH 6.9, significantly and reversibly reduced the secretory response to 5 mM Ba2+ (pH 6.9) compared to a first control response obtained 35 min before at pH 7.4. 6. These results are compatible with the view that Ba(2+)-induced secretory activity is accompanied by the release of protons which could be involved in a local negative automodulatory mechanism of adrenomedullary secretion.

Comparison of acute effects of mitoxantrone and doxorubicin in guinea-pig atria.

1. The acute effects of doxorubicin (DOX) and mitoxantrone (MTX) on basal rate and on positive chronotropic activity induced by 1-noradrenaline (1-NA) were investigated in isolated guinea-pig atria. 2. DOX (10(-5)-10(-4)M) progressively depressed atrial rate after a short latency period. Only 10(-4) M MTX reduced the spontaneous frequency after 120 and 180 min incubation. This effect was significantly lower to that elicited by DOX (10(-4)M). 3. Atropine (1.5 x 10(-6) M) and reserpine pretreatment did not affect the negative chronotropic action induced by DOX or MTX. 4. DOX (10(-5)-10(-4) M) produced a significant reduction of the maximal chronotropic response (Emax) to 1-noradrenaline (1-NA) after 60, 120 and 180 min of exposure. 5. MTX (10(-5)-10(-4) M) after 60 and 120 min incubation induced a beta-adrenergic, concentration- and time-dependent, competitive blocking effect. After 180 min of exposure, MTX (10(-4) M) reduced the Emax to 1-NA which was of less magnitude to that produced by DOX (10(-4) M). 6. Although both DOX and MTX depressed spontaneous and 1-NA induced chronotropic activity, MTX effects were of a slower onset and development compared to those exerted by DOX.

Estrogens regulate angiotensin-converting enzyme and angiotensin receptors in female rat anterior pituitary.

We studied the effects of the estrous cycle, ovariectomy and estrogen replacement on angiotensin-converting enzyme (ACE) (kininase II, EC 3.4.15.1) and angiotensin II (AT) receptors in the pituitary gland of the female rat. Quantitative autoradiography, with the use of consecutive pituitary sections, allowed for simultaneous determination of changes in binding and in the potential AT synthetic ability of individual pituitaries, and for a correlation between these two phenomena. In the anterior pituitary, ACE activity and binding of the ACE inhibitor [125I]-351A were not changed during the estrous cycle. Ovariectomy produced a significant increase in ACE activity and binding, and both of these parameters returned to normal after estrogen replacement. There were no changes in ACE activity or binding in the posterior pituitary during the estrous cycle or after ovariectomy or hormone replacement. AT receptors were characterized as of the AT1 type, since they were displaced by the selective AT1 antagonist DuP 753 and not by the AT2 competitor PD 123177. There were marked changes in the concentration of AT1 receptors during the estrous cycle, with highest numbers in metestrus, lower in estrus and diestrus, and lowest during proestrus. Estrogen replacement in ovariectomized rats decreased AT1 receptor number in the anterior pituitary. Our results indicate a dual effect of estrogen on anterior pituitary AT, physiologically on AT receptor expression and pharmacologically on ACE activity.

Increased beta 2-adrenoceptors in the superior cervical ganglia of genetically hypertensive rats.

[125I]Iodocyanopindolol binding sites were characterized by autoradiography in the superior cervical ganglia of Wistar-Kyoto (WKY) rats. A high concentration of (-)-[125I]iodocyanopindolol binding sites, characterized as beta-adrenoceptors by (-)-propranolol displacement, was distributed throughout the ganglia and in the postganglionic (internal carotid) nerve. ICI 118,551, a beta 2-selective antagonist, displaced more than 85% of the binding sites, whereas CGP 20712A, a beta 1-selective antagonist, displaced less than 10% of the binding sites, indicating that the beta-adrenoceptors were primarily of the beta 2-subtype. Emulsion autoradiography demonstrated that at least part of the binding sites were associated with principal ganglion cells. Unilateral deafferentation did not modify the number of binding sites in the superior cervical ganglia of WKY or spontaneously hypertensive rat (SHR). These results suggest that at least part of these receptors may correspond to prejunctional beta 2-adrenoceptors originated in principal ganglion cells. The concentration of beta 2-receptors was increased in the superior cervical ganglia of young and adult SHR when compared to age-matched WKY rats (49% and 39%, respectively). There were no differences in beta 2-adrenoceptor number in the stellate ganglia of young and adult WKY and SHR. These results suggest that beta 2-adrenoceptor stimulation may be selectively enhanced in some peripheral sympathetic ganglia in SHR and this could play a role in the development and maintenance of the increased sympathetic activity in this strain.

Inhibitory effect of hyperosmolality on catecholamine secretion from the bovine adrenal medulla.

We examined the effect of hyperosmolality on exocytotic catecholamine secretion in isolated-perfused bovine adrenals, in both the absence and presence of Ca2+ in the extracellular medium. Exposure of adrenal glands to hyperosmotic media (600 mOsm/Kg H2O) for 20 minutes significantly decreased spontaneous catecholamine output, and was independent of extracellular Ca2+ concentration. Adrenal glands were exposed to a hyperosmotic Locke solution (600 mOsm/Kg H2O) for 10 minutes and then stimulated with acetylcholine. Hyperosmolality produced a significant, reversible inhibition of acetylcholine-evoked catecholamine secretion. As the osmotic strength of the perfusion fluid was increased, either by addition of sucrose or NaCl, there was a progressive reduction in the acetylcholine response. Hyperosmolality also decreased catecholamine release induced by 56 mM K+ and by removal of NaCl from the perfusion medium. Similar results were seen in glands exposed to a NaCl-free solution in the absence of extracellular Ca2+. These results suggest that inhibitory effects of hyperosmolality on stimulus-secretion coupling in the adrenal medulla are exerted after Ca2+ enters the chromaffin cells, and support one of the primary criteria of the chemiosmotic hypothesis.

Different inhibitory sites of action for magnesium and verapamil in the bovine adrenal medulla.

The effects of verapamil and Mg2+ on catecholamine release induced by the substitution of NaCl by sucrose in the extracellular medium were studied in the perfused bovine adrenal medulla. Verapamil (3 X 10(-4) M) totally blocked the acetylcholine-evoked release of amines, but failed to antagonize the secretory response promoted by NaCl-omission. Perfusing glands with a Locke solution containing 10 mM Mg2+, produced a 61.4 and 62.8% inhibition of catecholamine release induced by acetylcholine and by NaCl-deprivation respectively. A similar inhibitory effect of Mg2+ (10-20 mM) was obtained in glands exposed to a NaCl-free solution in the absence of extracellular Ca2+. In adrenal glands previously perfused with verapamil (3 X 10(-4) M) and then exposed to a Locke solution containing verapamil (3 X 10(-4) M) and Mg2+ (10 or 20 mM) the inhibition of the secretory responses promoted by NaCl-omission was of the same magnitude as that obtained when the glands were perfused with Mg2+ (10 or 20 mM) in the absence of verapamil. A similar lacking effect of verapamil on the blocking action of Mg2+ on catecholamine release evoked by NaCl-deprivation was observed in the absence of extracellular Ca2+. These results are compatible with the idea that in the bovine adrenal medulla verapamil and Mg2+ could have different inhibitory sites of action. Verapamil would exert its blocking effect at an extracellular site, while the inhibitory action of Mg2+ may be mediated by an intracellular site. An additional extracellular locus of action for Mg2+ at the external surface of the chromaffin cell cannot be discarded. These results also suggest that calcium channels may not be involved in the inward transport mechanism of Mg2+ to its intracellular locus of action.