The Involvement of Enkephalin System in Analgesia Induced by Brain Iron Deficiency.

Iron deficiency (ID) induces in rats marked reduction of brain iron and dopamine D2 receptor. The resultant effects are a wide variety of changes in dopamine-mediated behaviors including thermo-regulation, motor activity, stereotyped behavior and diminished learning. Another behavioral change resulting from ID rats is an increase in the pain threshold, which is dependent on the duration and severity of ID. The results showed that peripheral administration of enkaphalines (0.1-3.0 mg/kg I.P.) potentiates ID-induced analgesia and causes a higher pain threshold, a phenomenon not observed in control rats.This effect may be associated with the known functional alteration in blood brain barrier resulting from ID. The opiate antagonists, naloxone (2 mg/kg and MIF-I (1 mg/kg), blocked the opiate-induced pain threshold potentiation in ID. Furthermore, the levels of dynorphin B and met-enkephalin are significantly increased in dopamine opiate-rich brain areas (caudate nucleus, substantia nigra, nucleus accumbens and globus pallidus) during ID. These phenomena can be reversed by placing rats on iron plus (control) diet for 24 days. It is concluded that a decrease in brain dopamine neurotransmission is associated with an increased functional level of the opiate system and that this mechanism mediates not only the analgesic effect but may also be associated with learning deficit observed in ID rats.

Angiotensin II induces acrosomal exocytosis in bovine spermatozoa.

Ejaculated mammalian spermatozoa must reside in the female genital tract for some time before gaining the ability to fertilize the egg. During this time, spermatozoa undergo some physiological changes that collectively are called capacitation. Capacitation of mammalian spermatozoa is a prerequisite for acrosome reaction, which is an exocytotic event occurring before fertilization. The specific biophysical and biochemical changes that accompany sperm capacitation and the agonists inducing acrosome reaction are not fully understood. Using SDS-gel electrophoresis and immunoblotting, we demonstrate the existence of a class of angiotensin receptors (AT1) in bovine spermatozoa. In capacitated sperm, we show that angiotensin II (ANG II) AT1 receptors are localized in the head and tail, whereas in noncapacitated cells the receptors are localized in the tail only. We find that ANG II markedly stimulates acrosomal exocytosis of capacitated bovine spermatozoa in vitro in a concentration range of 0.1-10 nM. No effect of ANG II was found in noncapacitated cells. The ability of ANG II to stimulate the acrosome reaction depends on the presence of calcium ions in the incubation medium. The ANG II-induced acrosome reaction was markedly inhibited by a selective AT1 receptor antagonist, losartan (DUP 753). PD-123319, a selective antagonist of the ANG II AT2 receptor, had no effect on the ANG II-induced acrosome reaction. Thus ANG II via activation of AT1 receptors may play a regulatory role in the induction of the acrosome reaction.

Atrial natriuretic peptide induces acrosomal exocytosis of human spermatozoa.

Acrosomal exocytosis in mammalian spermatozoa is a process essential for fertilization. We report here that atrial natriuretic peptide (ANP) markedly stimulates acrosomal exocytosis of capacitated human spermatozoa. Typically, ANP exerts some of its actions via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that ANP-stimulated acrosome reaction was inhibited by the competitive ANPR-A antagonist anantin, indicating a receptor-mediated process. A linear fragment of ANP, ANP-(13-28), and another ANP-like compound, brain natriuretic peptide, were inactive. The stimulatory effect of ANP on acrosome reaction was mimicked by the permeable cGMP analog, 8-bromo-cGMP (8-BrcGMP). Addition of the protein kinase C (PKC) inhibitors, staurosporine and GF-109203X, resulted in a dose-related inhibition of ANP-induced acrosome reaction. Also, downregulation of endogeneous PKC activity resulted in inhibition of ANP- but not 8-BrcGMP-induced acrosome reaction. Removal of extracellular Ca2+ abolished ANP-induced acrosome reaction. Thus ANP via Ca2+ influx, PKC activation, and stimulation of particulate guanylyl cyclase may play a role in the induction of acrosome reaction of human spermatozoa.

Thrombin stimulates atrial natriuretic peptide secretion from rat cardiac atrium.

Atrial natriuretic peptide (ANP) is a hormone secreted predominantly by atrial myocytes. Although atrial distension is the primary stimulus of ANP secretion, several hormones have also been implicated in the regulation of ANP secretion. alpha-Thrombin, a serine protease participating in the blood coagulation system, has additional hormone-like effects in several cell types, apparently via interaction with specific cell surface receptors. Here we report that alpha-thrombin enhanced ANP secretion from isolated rat atrium within 10 min, in a concentration-dependent manner. The protease also significantly increased ANP release from cultured atrial myocytes, in a concentration-dependent manner. The alpha-thrombin-induced release of ANP from cultured atrial myocytes was completely abolished by hirudin, a specific alpha-thrombin protease inhibitor. Furthermore, synthetic peptides, identical in their amino acid sequence to the N-terminal segment of the proteolytically cleaved thrombin receptor, enhanced ANP release from adult rat cultured atrial myocytes. Our data suggest that thrombin may regulate ANP release from the cardiac atrium. This action involves activation of thrombin receptors in atrial myocytes.

Erythropoietin stimulates atrial natriuretic peptide secretion from adult rat cardiac atrium.

Hypoxia is a powerful stimulus for erythropoietin (EPO) secretion from the kidney and for atrial natriuretic peptide (ANP) secretion from atrial myocytes. EPO is involved in the long term defense mechanism against hypoxia via stimulation of erythropoiesis. ANP is involved in the short-term defense mechanism against hypoxia via improved pulmonary and heart functions. We investigated a possible interaction between these two hormones. We tested the hypothesis that EPO may stimulate ANP secretion from the cardiac atrium. This hypothesis was tested in two in vitro models; isolated rate atrium and cultured adult atria rat myocytes. Recombinant human EPO (5-10 units/ml) enhanced ANP secretion from the isolated atrium (by approximately 2-fold) within 10 min in a concentration-dependent manner. To define whether the action of EPO on ANP secretion is direct, we examined the effect of EPO on ANP release from adult rat cultured atrial myocytes. EPO failed to stimulate ANP secretion from cultured atrial myocytes, suggesting that EPO-induced ANP secretion is an indirect effect. Cyclooxygenase products (e.g.,prostaglandins) and endothelin 1 were shown to be potent secretagogues of ANP from cardiac atrium. To test whether EPO-induced ANP secretion from isolated perfused atrium is mediated by cyclooxygenase products and/or endothelin, we used inhibitors of the enzyme cyclooxygenase (indomethacin or aspirin) and the endothelin receptor ETA subtype antagonist BQ123. EPO-stimulated ANP secretion was not affected by indomethacin (10(-4) M) or aspirin (10(-4) M), whereas BQ123 (10(-6) M) completely abolished EPO-stimulated ANP secretion from cardiac atrium. Our results expand our knowledge on the interaction between EPO and ANP hormonal systems and the possible role in the acute defense mechanism against hypoxia.

Epinephrine dose-response of the isolated working heart in O2-exposed rats.

BACKGROUND: Heart energy efficiency, which is affected by catecholamines, has previously been shown to decline in rats with prolonged normobaric O2 exposure. HYPOTHESIS: Oxygen exposure affects dose response of the heart to catecholamines. METHODS: Epinephrine dose-response (10(-10) - 5 x 10(-6) mol.L-1) was measured in the isolated working heart excised from control rats breathing air, and rats exposed to normobaric 100% oxygen for either 24 h or 49 h. The variables measured were input (oxygen consumption (VO2) and output power, cardiac contractility (Emax and maximal dP/dT), coronary resistance, heart frequency (fH) and left ventricular pressure. Variable (Y*) dose response to epinephrine concentration (C) was fitted to the equation: Y* = Ymax/(1 + (C/C50)n), Ymax--maximal Y*, C50--C for half Ymax and n--an empirical power. RESULTS: Oxygen exposure of the intact rat had little influence on baseline cardiac variables, but did affect sensitivity to catecholamines. A general effect of the O2 exposure was a left shift of the dose-response curve for example, C50 was reduced by 72, 41 and 43 x 10-8 mol.L-1 for VO2, fH and Emax, respectively, after the 24 h exposure. CONCLUSIONS: There was a pronounced change in the dose-response in hearts from 24 h O2-exposed rats, a change partially reversed in hearts from 49 h O2-exposed rats. The high dose, which had a stimulatory effect on hearts from control rats, failed to stimulate hearts from hyperoxic rats.

Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa.

The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.

Effects of ANP receptor antagonists on ANP secretion from adult rat cultured atrial myocytes.

Atrial natriuretic peptide (ANP) is a hormone-secreted predominantly by atrial myocytes. ANP exerts many of its actions via activation of the particulate guanylyl cyclase receptor ANPR-A and the formation of guanosine 3',5'-cyclic monophosphate (cGMP), which serves as a second messenger in the target cells. Using membrane-permeable cGMP analogues (8-bromo-cGMP and dibutyryl- cGMP), we first tested the hypothesis that ANP secretion by adult rat cultured atrial myocytes can be modulated through the second messenger cGMP. Second, we examined the effects of two competitive ANPR-A receptor antagonists, namely HS-142-1 and anantin, on cGMP formation and ANP secretion from cultured atrial myocytes. Cultured atrial myocytes secreted large quantities of immunoreactive (ir) ANP under basal conditions. We found that cGMP analogues inhibited basal irANP secretion from cultured atrial myocytes, whereas HS-142-1 and anantin had stimulating effects. HS-142-1 and anantin reduced cGMP formation in cultured atrial myocytes at basal conditions. These results suggest an autoregulatory mechanism of ANP secretion by atrial myocytes in an autocrine/paracrine fashion.

Release of atrial natriuretic peptide in brief ischemia-reperfusion in isolated rat hearts.

We studied the effects of short-term global ischemia and reperfusion on ANP secretion from Langendorff-perfused rat hearts compared with isolated ventricles. Effects of regional ischemia, with or without increased atrial pressure, were examined in Langendorff-perfused and working heart models. Five minutes of global ischemia were associated with elevated levels of atrial natriuretic peptide (ANP) in the coronary effluent immediately and for approximately 10 min after resumption of reperfusion, resulting in a net hormone excess of 23 +/- 5 ng/g wet wt. The ventricles produced on the average 11% ANP compared with the whole heart, and their contribution of to postischemic ANP overflow was approximately proportional to their basal production. In Langendorff-perfused hearts, regional ischemia increased the concentration of ANP in the coronary effluent 51 +/- 11%, whereas the secretion rate (per minute) decreased 18 +/- 5%. In the presence of atrial distension in the working heart model, a trend for increase in ANP secretion was apparent. We conclude that global ischemia, even of brief duration, has an independent stimulatory effect on ANP release, the ischemic atrium being responsible for most of the excess. Regional ischemia, when not accompanied by atrial distention, reduces the ANP secretion rate during the ischemic period. Heart failure secondary to ischemia stimulates ANP secretion, but this response seems to be both delayed and attenuated compared with atrial stretch alone.

Atrial natriuretic peptide attracts human spermatozoa in vitro.

Here we report that atrial natriuretic peptide (ANP), a known activator of particulate guanylate cyclase, induces attraction and swimming speed enhancement of human spermatozoa in vitro. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of ANP, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that spermatozoa followed the gradient of ANP and accumulated in it. Speed enhancement was detected in the presence of ANP without a gradient. These observations suggest either that an ANP-like substance is the physiological attractant for human spermatozoa, or, more likely, that ANP directly affects guanylate cyclase in a manner similar to that caused by the physiological attractant.

Thyroid hormone restores atrial stretch-induced secretion of atrial natriuretic peptide in hypophysectomized rats.

Acute blood volume expansion caused a rapid and profound release of atrial natriuretic peptides (ANP) from cardiac atria. The stimulated release of ANP was markedly blunted (more than 50%) in hypophysectomized rats (8 days after surgery). Daily subcutaneous injections of thyroxine (30 micrograms/kg) to hypophysectomized rats for 7 days, completely restored acute volume expansion induced release of ANP. Our data suggest that thyroid hormone is required for ANP secretion in response to acute volume expansion.

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.

The hypertensinogenic activity of 18,19-dihydroxycorticosterone in adrenalectomized rats.

Adrenalectomized, spontaneously hypertensive rats (SHR) were used to test the hypertensinogenic property of recently synthesized 18,19-dihydroxycorticosterone (18,19-Di-OH-B). The steroid was given via Alzet miniosmotic pump, with and without aldosterone. Neither 18,19-Di-OH-B (5 micrograms) nor aldosterone (5 micrograms) increased blood pressure in SHR when given alone; when administered together they resulted in a significant rise in blood pressure. Results indicate a high probability that certain mineralocorticoids, which are inactive by themselves, might play a role in the etiology of hypertension when acting together under physiological conditions.

Physiologic regulation and distribution of the renal vasopressin receptor.

Previous studies suggest that circulating levels of vasopressin (AVP) influence the responsiveness of the kidney to AVP. To determine how changes in renal AVP receptors and adenylate cyclase (AC) contribute to such altered responsiveness, we analyzed AVP receptors and AVP-sensitive AC in kidneys from Sprague-Dawley, Long-Evans and homozygous Brattleboro rats. In autoradiographic studies, the distribution of [3H]AVP binding sites was similar in all groups, corresponding to the location of AVP-sensitive AC: collecting ducts greater than outer medullary collecting ducts greater than medullary thick ascending limb greater than cortical collecting ducts. No differences in AVP receptor affinity or content were observed in kidney medullary membranes from Sprague-Dawley, Long-Evans or Brattleboro rats (KD = 0.8, 0.9, and 0.7 nM; Bmax = 116 +/- 9, 95 +/- 11 and 98 +/- 6 fmol/mg). Basal and AVP-stimulated AC activities were lower in kidney membranes from Brattleboro rats compared with Sprague-Dawley and Long-Evans animals (basal = 28 +/- 4, 40 +/- 4 and 38 +/- 3 pmol cAMP/mg/min; EDmax = 57 +/- 5, 80 +/- 7 and 71 +/- 2 pmol cAMP/mg/min) with no change in ED50. In 48-hour water-deprived Sprague-Dawley rats, AVP receptors were decreased from 116 +/- 9 to 58 +/- 2 fmol/mg, suggesting that AVP receptors are down-regulated by elevated AVP blood levels. The absence of changes in basal or AVP-stimulated AC in dehydrated rats indicates that receptor-AC coupling is normal and that maximum AC activation can occur with partial receptor occupancy. The data also indicate that impaired renal responsiveness to AVP in Brattleboro rats is not due to down-regulation of AVP receptors.

Ouabain induces secretion of proatrial natriuretic factor by rat atrial cardiocytes.

Neonatal rat atrial and ventricular cardiocytes in primary culture secrete proatrial natriuretic factor (proANF), the 126-amino acid precursor of atrial natriuretic factor (ANF). The cellular mechanisms of proANF secretion differ in the two cell types: atrial cardiocytes store proANF in abundant secretory granules before secretion, whereas ventricular cardiocytes secrete the precursor rapidly after synthesis. In this study, we used the Na+-K+-adenosinetriphosphatase (ATPase) inhibitor ouabain to investigate the functional significance of these differing secretory mechanisms. Ouabain increased immunoreactive ANF (iANF) secretion by atrial cardiocytes 1.5- to 2.4-fold. Metabolic labeling studies demonstrated that proANF was the form of iANF released in response to ouabain. Ventricular secretion of iANF was unaffected by the Na+-K+-ATPase inhibitor. These observations are consistent with a model in which atrial cardiocytes are able to release proANF via a regulated secretory pathway, whereas ventricular cardiocytes utilize a constitutive secretory pathway. The ability of ouabain to stimulate proANF secretion suggests that an increase in intracellular calcium concentration may promote fusion of secretory granules with atrial cell membranes thereby mediating exocytosis of stored proANF.

The distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of rat, guinea-pig, pig and man.

The distribution of melanin-concentrating hormone-like immunoreactivity was investigated by radioimmunoassay in the CNS of rat, guinea-pig, pig and man. Highest concentrations of melanin-concentrating hormone-like immunoreactivity were found in the hypothalamus of all the species: rat 204.4 +/- 14.9; guinea-pig 159.5 +/- 23.3; pig 10.9 +/- 4.5 and man 80.1 +/- 19.1 pmol/g. Gel chromatographic analysis of hypothalamic extracts showed five immunoreactive peaks of melanin-concentrating hormone-like immunoreactivity in the rat and pig and six in the guinea-pig and man. High-performance liquid chromatography analysis of hypothalamic extracts showed five immunoreactive peaks in rat, guinea-pig, pig and four in man. However, these peaks appeared at different retention times from that of the single peak of salmon melanin-concentrating hormone. Examination of subcellular fractions of whole rat brain showed that most of the melanin-concentrating hormone-like immunoreactivity is found in the synaptosome fraction. Stimulation of melanin-concentrating hormone-like immunoreactivity release from rat hypothalamic slices revealed that potassium in the presence of calcium stimulated melanin-concentrating hormone-like immunoreactivity release. These findings suggest that mammalian melanin-concentrating hormone-like immunoreactivity has a different amino acid sequence from salmon melanin-concentrating hormone and may exist in multiple molecular forms. It is possible that melanin-concentrating hormone may play a role as a neurotransmitter or modulator in the mammalian CNS.

Role of autonomic nervous system and vasoactive hormones in the release of atrial natriuretic peptides in conscious rats.

Atrial natriuretic peptides (ANPs) have been implicated in volume homeostasis and blood pressure regulation; however, little is known about factors regulating the release of ANPs. We therefore examined the roles of the sympathetic and parasympathetic nervous systems, the renin-angiotensin system, and vasopressin in the release of ANPs, under basal conditions and after acute blood volume expansion or acute salt loading in conscious rats. Plasma concentrations of immunoreactive ANPs (ir-ANPs) were measured by specific radioimmunoassay. Both acute blood volume expansion and acute salt loading increased basal plasma levels of ir-ANPs (90.4 +/- 5.2 pg/ml) four- to fivefold within 2 min. Basal and stimulated plasma levels of ir-ANPs were unaltered by pretreatment with methylatropine or with propranolol or by ganglionic blockade with hexamethonium. Total adrenergic blockade (combined treatment with yohimbine, prazosin, and propranolol) had no effect on basal or salt loading-stimulated plasma levels of ir-ANPs and slightly attenuated blood volume expansion-induced release of ANPs. Ganglionic blockade with chlorisondamine tripled basal plasma concentrations of ir-ANPs but had no effect on stimulation-induced release of ANPs. Acute blockade of the renin-angiotensin system with captopril or infusion of saralasin or pretreatment with a vasopressin pressor antagonist had no effect on basal or stimulated release of ANPs. Following intravenous bolus injections of graded doses of l-norepinephrine, angiotensin II, and vasopressin, mean arterial pressure and plasma levels of ir-ANPs increased in a dose-dependent manner. In conclusion, plasma concentrations of ir-ANPs can be acutely elevated by several physiological stimuli: volume expansion; salt loading; and pressor hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of heroin on opioid levels in the rat brain.

Dependence on the potent narcotic agent heroin in rats was produced by the implication of double-coated pellets. Chronic administration of this drug induced significant alterations in dynorphin B and [Met5]enkephalin-Arg6-Gly7-Leu8 (ME-RGL) levels in discrete areas of the rat brain. The level of the latter increased by 53.6% in the substantia nigra while the levels of dynorphin B increased in the globus pallidus and ventral tegmental area (188.0 and 41.8%, respectively).

Environmentally-induced modification of the benzodiazepine/GABA receptor coupled chloride ionophore.

The benzodiazepine/GABA receptor coupled chloride ionophore was examined in brain membranes of rats maintained in either a conventional animal facility or a "protected" (low-stress) environment. Following a 10 min ambient temperature swim, animals maintained in both environments had qualitatively similar increases in the number (Bmax) of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, the apparent affinity of this radioligand, and the efficacy and potency of Cl- to enhance [3H]flunitrazepam binding. Nonetheless, the Bmax of [35S]TBPS and efficacy of Cl- to enhance [3H]flunitrazepam binding were significantly lower in animals housed in the protected environment compared to those maintained in a conventional facility both before and after swim stress. Furthermore, in rats housed in a protected environment, sequential removal of animals from a common cage (cohort removal), produced a very rapid increase (less than or equal to 15 s) in Cl(-)-enhanced [3H]flunitrazepam binding in cortical and hippocampal but not cerebellar membranes. Cohort removal also produced a sequential increase in the number of [35S]TBPS binding sites and apparent affinity of this radioligand in cerebral cortical membranes. The effects of cohort removal were not observed in animals subjected to ambient temperature swim or if animals were removed from different cages. Changes in the benzodiazepine/GABA receptor coupled chloride ionophore produced by cohort removal from a common cage preceded any statistically significant changes in circulating levels of alpha-MSH, beta-endorphin, ACTH or corticosterone. These findings suggest that the benzodiazepine/GABA receptor chloride ionophore complex (supramolecular complex) is under both tonic and acute regulation by the environment, and may subserve a physiologically relevant function in the response to stressful or anxiety producing stimuli.

Anterior pituitary gland modulates the release of atrial natriuretic peptides from cardiac atria.

Myocytes in the atria contain a prohormone that gives rise to atrial natriuretic peptides (ANP), which have intrinsic hemodynamic regulatory activity. Little is known about mechanisms regulating ANP release. In rats with indwelling catheters, acute blood volume expansion with 5% (wt/vol) dextrose increases the amount of circulating immunoreactive ANP by a factor of 2-3, as determined by radioimmunoassay. Pithing, which both removes neurogenic influences and interrupts humoral influences of the brain and pituitary gland on the heart, completely blocked stimulus-induced release of ANP. Because our studies using pharmacological blockade of the autonomic nervous system had suggested that neurogenic mechanisms do not play a major role in ANP release, we sought a humoral mechanism involved in ANP secretion. Basal and stimulated release of ANP were significantly blunted in hypophysectomized rats (8 days after operation) but were completely restored when the resected anterior pituitary was reimplanted under the kidney capsule. This suggests that hormones of anterior pituitary origin are required for ANP secretion in response to acute volume loading.