Phenoxybenzamine is neuroprotective in a rat model of severe traumatic brain injury.

Phenoxybenzamine (PBZ) is an FDA approved α-1 adrenergic receptor antagonist that is currently used to treat symptoms of pheochromocytoma. However, it has not been studied as a neuroprotective agent for traumatic brain injury (TBI). While screening neuroprotective candidates, we found that phenoxybenzamine reduced neuronal death in rat hippocampal slice cultures following exposure to oxygen glucose deprivation (OGD). Using this system, we found that phenoxybenzamine reduced neuronal death over a broad dose range (0.1 µM-1 mM) and provided efficacy when delivered up to 16 h post-OGD. We further tested phenoxybenzamine in the rat lateral fluid percussion model of TBI. When administered 8 h after TBI, phenoxybenzamine improved neurological severity scoring and foot fault assessments. At 25 days post injury, phenoxybenzamine treated TBI animals also showed a significant improvement in both learning and memory compared to saline treated controls. We further examined gene expression changes within the cortex following TBI. At 32 h post-TBI phenoxybenzamine treated animals had significantly lower expression of pro-inflammatory signaling proteins CCL2, IL1ß, and MyD88, suggesting that phenoxybenzamine may exert a neuroprotective effect by reducing neuroinflammation after TBI. These data suggest that phenonxybenzamine may have application in the treatment of TBI.

Antidiarrhoeal activity of DAS-77 (a herbal preparation).

BACKGROUND: DAS-77 is a traditional herbal preparation composed of the young callous bark of mango (Mangifera indica Linn., Anacardiaceae) and the dried root of pawpaw (Carica papaya Linn., Caricaceae). This phytomedicine is claimed to have beneficial effects in the treatment of gastrointestinal disorders, including diarrhoea. OBJECTIVE: To investigate the antidiarrhoeal effect of DAS-77 using standard pharmacological models. METHODS: Normal and castor oil-induced intestinal transit, and castor oil-induced diarrhoea tests wore carried out in mice while intestinal fluid accumulation and gastric emptying tests were carried out in rats. Acute toxicity test and preliminary phytochemical analysis were also conducted. RESULTS: The results obtained in this study revealed that DAS-77 had no significant inhibitory effect on normal intestinal transit, castor oil-induced diarrhoea, intestinal fluid accumulation and gastric emptying. However, the inhibitory effect of DAS-77 was significant (p<0.001) relative to control in the castor oil-induced intestinal transit test. Peak effect was produced at the dose of 100 mg/kg (p.o.). The effect of DAS-77 in this respect was reversed by pilocarpine and propranolol, but not by phenoxybenzamine. DAS-77 did not produce any mortality given p.o. up to 10 g/kg, indicating the relative safety of the preparation. The i.p. LD50 was estimated to be 1122 mg/kg. The remedy was found to contain saponins, tannins, phenols and alkaloids. CONCLUSION: Findings in this study suggest that DAS-77 possesses antidiarrhoeal activity due to the inhibition of intestinal motility possibly mediated by muscarinic and alpha adrenergic receptors.

Targeting the chromosome partitioning protein ParA in tuberculosis drug discovery.

OBJECTIVE: To identify inhibitors of the essential chromosome partitioning protein ParA that are active against Mycobacterium tuberculosis. METHODS: Antisense expression of the parA orthologue MSMEG_6939 was induced on the Mycobacterium smegmatis background. Screening of synthetic chemical libraries was performed to identify compounds with higher anti-mycobacterial activity in the presence of parA antisense. Differentially active compounds were validated for specific inhibition of purified ParA protein from M. tuberculosis (Rv3918c). ParA inhibitors were then characterized for their activity towards M. tuberculosis in vitro. RESULTS: Under a number of culture conditions, parA antisense expression in M. smegmatis resulted in reduced growth. This effect on growth provided a basis for the detection of compounds that increased susceptibility to expression of parA antisense. Two compounds identified from library screening, phenoxybenzamine and octoclothepin, also inhibited the in vitro ATPase activity of ParA from M. tuberculosis. Structural in silico analyses predict that phenoxybenzamine and octoclothepin undergo interactions compatible with the active site of ParA. Octoclothepin exhibited significant bacteriostatic activity towards M. tuberculosis. CONCLUSIONS: Our data support the use of whole-cell differential antisense screens for the discovery of inhibitors of specific anti-tubercular drug targets. Using this approach, we have identified an inhibitor of purified ParA and whole cells of M. tuberculosis.

Prevention of hypertensive crises in rats induced by acute and chronic norepinephrine excess.

Calcium Channel Blockers (CCBs), competitive α-adrenoceptor blockers, and phenoxybenzamine (POB) are used for preoperative treatment of pheochromocytomas. We analyzed the protection from hypertensive crisis provided by these drugs during acute and chronic norepinephrine excess. To ensure adaptive changes during chronic norepinephrine (NE) excess, we continuously exposed male Wistar rats to NE for 3 weeks (osmotic pumps). Afterwards, blood pressure (BP) was continuously measured while NE boli (0-1000 µg/kg, i. v.) were administered before and after antihypertensive treatment in anesthetized and catheterized rats. A single dose of urapidil (10 mg/kg), nitrendipine (600 µg/kg) and POB (10 mg/kg) lowered BP from 212 ± 12 mmHg by 52 ± 7%, 31 ± 9%, and 50 ± 6%, respectively. With NE boli a maximum BP of 235 ± 29, 240 ± 30 and 138 ± 3 mmHg was measured in urapidil, nitrendipine, and POB treated animals (p<0.05). The number of hypertensive episodes (delta BP >30 mmHg) was 3 (3), 1.5 (0-3), and 0 (0-1) (p<0.05). Because of inferiority, urapidil was excluded from further testing. Chronically NE exposed rats were treated with POB (10 mg/kg/d), nifedipine (10 mg/kg/d), or vehicle for 7 days. Marked BP elevations were observed at baseline (167 ± 7, 210 ± 7 , and 217 ± 7 mmHg, p<0.01) and maximum blood pressure was 220 ± 32, 282 ± 26, and 268 ± 40 mmHg (p<0.001) with NE boli. Further stabilization was achieved combining POB pretreatment with a continuous nifedipine infusion, which effectively prevented BP elevations during NE excess. POB was the most effective drug used in monotherapy, but BP stabilization was superior using a combination of POB pretreatment with a continuous nifedipine infusion in this model.

Potential of PSA-NCAM in neuron-glial plasticity in the adult hypothalamus: role of noradrenergic and GABAergic neurotransmitters.

The present study was designed to establish the dynamic regulation of polysialylated form of neural cell adhesion molecule (PSA-NCAM) expression by neurotransmitters controlling gonadotropin releasing hormone (GnRH) secretion. The expression of PSA-NCAM and glial fibrillary acidic protein (GFAP) on GnRH cell bodies and axon terminals in the medial preoptic area (mPOA) and median eminence-arcuate (ME-ARC) region of hypothalamus was studied in the proestrous phase of cycling rats treated with alpha-adrenergic receptor blocker phenoxybenzamine (PBZ) and gamma-aminobutyric acid (GABA) by using dual immunohistofluorescent staining and Western blot hybridization. To further elucidate whether activity mediated regulation of PSA-NCAM in GnRH neuron is via regulation of PSA biosynthesis by polysialytransferase (PST) enzyme, the expression of PST-1 enzyme was studied by using fluorescent in situ hybridization (FISH). Both GnRH and PSA-NCAM immunostaining was much higher in the mPOA and ME-ARC region from proestrous phase rats, whereas, PBZ and GABA treatments significantly reduced their expression, GFAP-ir and its content were increased in the PBZ and GABA treated proestrous rats. Taken together, our observations add to the growing evidence that PSA-NCAM plays permissive role for neuronal-glial remodeling and further suggest a functional link between activity dependent structural remodeling in GnRH neurons. Further, enhanced mRNA expression of PST suggests that the biosynthesis of PSA on NCAM is regulated at the transcriptional level.

Effect of dopamine on vascular reactivity in near-term lamb carotids: role of the endothelium.

Many neonates are diagnosed with hypotension in the first 24 hr of life. Those with severe hypotension are often given high doses of dopamine at 10 to 20 microg/kg/min. This study examined the hypothesis that dopamine, a vasoactive drug commonly used in the neonatal intensive care unit, alters vascular reactivity. Vascular reactivity was measured by comparing 5HT dose-response characteristics in untreated near-term lamb common carotid arteries and arteries treated with 15 microg/kg/min of dopamine. The authors found that dopamine pretreatment for 60 min significantly potentiated 5HT-induced contractile tone by approximately 100% ( p < .05). This observed increase in tone was accompanied by a significant decrease in the affinity of 5HT to its receptor ( p < .05), suggesting an activation of a separate contractile pathway, or a mechanism downstream from agonist-receptor binding. Interestingly, an increase in contractility was observed only in endothelium-intact arteries. In arteries with denuded endothelium, dopamine pretreatment resulted in a small but significant decrease in tone compared to control arteries ( p < .05), suggesting a vasodilatory mechanism unmasked by endothelium removal. Although multiple mechanisms can increase vascular resistance, these data described the in vitro effects of high doses of dopamine on vascular tone as well as the role of the endothelium in dopaminemediated vasoconstriction.

Effects of electroacupuncture stimulation applied to limb and back on mesenteric microvascular hemodynamics.

The effects of electroacupuncture stimulation (EAS) of the hind paw and the back on the mesenteric microhemodynamics in anesthetized rats were investigated using an intravital microscope system. Red blood cell (RBC) velocity in the mesenteric arterioles was measured by the dual-sensor method developed by the authors. Electrical stimulation was applied using two acupuncture needles inserted into the skin and the underlying muscles of the hind paw and the dorsal Th13-L1 level area. The hind-paw EAS evoked intensity-dependent pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles, while the back EAS evoked depressor responses and decrease responses in RBC velocity. Heart rate showed increase responses accompanying EAS either on the hind paw or the back. The pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles accompanying the hind paw EAS were abolished by an intravenous administration of alpha-adrenergic receptor antagonist (phenoxybenzamine; POB), and the tachycardiac responses were abolished by administration of beta-adrenergic receptor antagonist (propranolol). Occasional but notable reflex vasoconstrictions in the mesenteric terminal arteriole were induced by EAS either on the hind paw or the back. These vasoconstrictive responses were not affected by the administration of POB. The present study directly demonstrated that hemodynamic changes at the level of precapillary arterioles accompanying EAS either on the hind paw or the back mainly depend on the changes of systemic arterial pressure regardless of stimulus current intensities. Moreover, the results in the present study suggest some receptors other than alpha-adrenergic receptor might be involved in the mechanism of EAS-induced vasoconstriction in the mesenteric arteriole.

Modulation of ET(B) receptor-induced arginine-vasopressin secretion by N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA)-dependent mechanisms in hypothalamo-neurohypophysial explants.

The endothelins (ETs) stimulate the secretion of arginine-vasopressin (AVP) in vivo and in vitro. The activation of hypothalamic ET(B) receptors increases AVP release, but the neurotransmitters mediating these responses are not known. In the compartmentalized hypothalamo-neurohypophysial explant model, the overall basal release of AVP was 53+/-17 pg x h(-1) x PP(-1) (where PP is posterior pituitary). ET(B) receptor activation in hypothalamic sites by 1 nM IRL1620 dose-dependently increased AVP secretion, with a maximal response of 340+/-70% of basal x h(-1) x PP(-1), whereas 1 nM ET-1, the ET(A) receptor-selective agonist, inhibited AVP release to 44+/-8% x h(-1) x PP(-1). Addition of MK801 along with IRL1620 inhibited AVP release to a value no different from basal (122+/-41% x h(-1) x PP(-1)). In contrast, 10 microM DNQX [6,7-dinitroquinozaline-2,3-(1H,4H)-dione] did not block ET(B) receptor-induced AVP release (326+/-73% x h(-1) x PP(-1)), and nor did non-selective alpha-adrenergic receptor antagonism. The GABA(A) (where GABA is gamma-aminobutyric acid) receptor agonist muscimol (10 microM) inhibited AVP release in response to IRL1620 (127+/-30% x h(-1) x explant(-1)). These data suggest that AVP release induced by activation of hypothalamic ET(B) receptors is mediated by a hypothalamic N-methyl-D-aspartate (NMDA) receptor-mediated mechanism. In turn, the local release of GABA associated with NMDA activation may exert an inhibitory influence and dampen the AVP secretory response.

Characterization of dopamine-mediated relaxation in experimental vein bypass grafts.

BACKGROUND: Dopamine is an endogenous inotropic agent commonly used during coronary artery surgery and in the medical therapy of a revascularized patient. In this study the responses of intimal hyperplastic vein grafts to dopamine are examined. METHODS: The in vitro isometric tension responses to dopamine of common carotid jugular vein bypass grafts in New Zealand White rabbits were determined. The responses were compared to those obtained in the jugular vein and in the common carotid artery. Both endothelialized and denuded vessels were precontracted with prostaglandin F(2alpha) and the responses to dopamine were assessed. The contributions of nitric oxide and prostanoids to the response were also determined. RESULTS: Each vessel showed a biphasic dose response to dopamine with relaxation at low concentrations followed by contraction at high concentrations. Dopamine relaxation in the jugular vein was endothelial independent while in the carotid artery it was endothelial dependent and decreased. The sensitivity of both vessels was significantly greater than the vein graft (6.62 +/- 0.12; P < 0. 05); however, after endothelial denudation, the sensitivity of dopamine-mediated relaxation of the vein graft (8.91 +/- 0.09) was significantly enhanced. Preincubation with L-NMMA (to block NO synthesis) inhibited vein graft relaxation to dopamine and preincubation with indomethacin (to block cyclooxygenase activity) inhibited carotid artery relaxation to dopamine. Addition of phenoxybenzamine, a broad alpha-adrenergic antagonist, enhanced dopamine relaxation in the jugular vein and depressed the relaxation in the carotid artery. There was no effect on the dopamine response in the vein graft. Jugular vein and carotid artery responded to dopamine with cholera toxin-sensitive (Galpha(s)) responses. In contrast, dopamine relaxation in the vein graft was enhanced by inhibition of Galpha(s). CONCLUSION: Dopamine relaxation in vein grafts is mediated in part by NO but not by either prostanoids or alpha-adrenergic receptor activation. It is diminished compared to native vessels due to an endothelium-dependent, Galpha(s)-mediated pathway.

Adenosine formation during hypoxia in isolated hearts: effect of adrenergic blockade.

Adrenergic receptor blockade has been reported to decrease cardiac adenosine formation and release during hypoxia. We wished to determine whether this occurs by an improvement in the energy supply/demand ratio. Isolated guinea pig hearts were perfused at a constant pressure of 50 mm Hg. Hypoxia (30% O2) was maintained for 20 min while adenosine release and venous PO2 were measured in the coronary venous effluent. beta-adrenergic blockade with 5 microM atenolol did not change hypoxic adenosine release (Control: 15.6 +/- 2.7, Atenolol: 23.6 +/- 5.7 nmol/g/20 min). Addition of 6 microM phentolamine with atenolol significantly reduced hypoxic adenosine release (4.4 +/- 1.4 nmol/g/20 min, P < 0.05). Atenolol was without hemodynamic effects, but addition of phentolamine reduced left ventricular pressure development, heart rate, and oxygen consumption prior to hypoxia. Atenolol plus phentolamine did not change venous PO2 during hypoxia. Treatment with phenoxybenzamine (1 microM) plus atenolol also reduced adenosine release (7.4 +/- 0.8 nmol/g/20 min). Control experiments and atenolol plus phentolamine experiments were repeated using 31P-NMR to measure high energy phosphates. Adrenergic blockade had no effect on phosphate concentrations during normoxia, but resulted in higher [PCr], lower [P(i)] and higher phosphorylation potentials during hypoxia. Adrenergic blockade also prevented the hypoxia-induced rise in intracellular [H+], [AMP] and [ADP] seen in control hearts. The changes in phosphorylation potential are correlated with similar changes in adenosine release in adrenergically intact hearts. We conclude that the primary effect of adrenergic blockade during hypoxia is a reduction in ATP use due to alpha-receptor blockade. This leads to improved high energy phosphate concentrations during hypoxia and a reduction in adenosine formation.

Activation-dependent regulation of galanin gene expression in gonadotropin-releasing hormone neurons in the female rat.

In rats, galanin is colocalized in GnRH neurons, and galanin mRNA in GnRH neurons is increased coincidentally with the preovulatory gonadotropin surge. Whether the induction of galanin mRNA in GnRH neurons at proestrus reflects the action of sex steroids is unknown. We tested this hypothesis by challenging ovariectomized rats (n = 7) with estrogen and progesterone (E/P) to induce a LH surge and measuring galanin mRNA in GnRH neurons to determine whether there was an associated induction of galanin message in these cells. We used single and double label in situ hybridization and image analysis to compare among groups the levels of both galanin mRNA and GnRH mRNA in GnRH neurons. We found that steroid-primed animals showed an approximately 400% induction of galanin mRNA signal in GnRH neurons over that in vehicle-treated animals. Second, we hypothesized that steroid-dependent events which induce the expression of galanin mRNA in GnRH neurons depend on transsynaptic input to GnRH neurons. We tested this hypothesis by examining the effect of a pharmacological blockade of the steroid-induced activation of GnRH neurons on levels of galanin mRNA in these cells. We killed groups of ovariectomized adult female rats at the peak of a E/P-primed LH surge (n = 7) and after steroid priming followed by blockade of the LH surge with either the general anesthetic pentobarbital (n = 7) or the specific alpha-adrenergic receptor blocker phenoxybenzamine (n = 7). When we examined signal levels representing galanin mRNA content in GnRH neurons, we observed a 4-fold increase in signal for galanin mRNA in the GnRH neurons of steroid-primed (E/P surge) animals compared with that in oil-treated controls (P < 0.0004). This increase in galanin mRNA was prevented when the LH surge was blocked by treatment with either pentobarbital or phenoxybenzamine (P < 0.03 and P < 0.0001 vs. E/P surge controls, respectively). Cellular levels of GnRH mRNA were not different among control, E/P, and E/P plus pentobarbital groups (P > 0.2). These observations suggest that an increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a LH surge. By inference, induction of galanin mRNA in GnRH neurons reflects their activation, possibly via afferent neurons that transduce the steroid signal to GnRH neurons.

Sexual differences in hypothalamic adenosine 3',5'-cyclic monophosphate content.

Hypothalamic adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured in the morning (10:00) and afternoon (16:00) in castrated and intact rats of both sexes to evaluate a possible sexual difference. Castration produced different effects on the hypothalamic cAMP concentration. In females, ovariectomy significantly reduced total hypothalamic cAMP concentration, whereas in males, orchidectomy elevated hypothalamic cAMP content. This observation was independent of the time of day at which the animals were killed. On the other hand, administration of the alpha-adrenoreceptor blocking agent phenoxybenzamine or the beta-adrenoreceptor blocker propranolol lowered the concentration of cAMP in the hypothalamus. However, this effect was selective and related to the time of day when the animals were killed. Phenoxybenzamine reduced the hypothalamic cAMP content in males killed in the afternoon, whereas the effect of propranolol was significant in males killed in the morning. These results indicate a response of hypothalamic cAMP to adrenoreceptor blockers in male rats opposite to that reported previously from our laboratory in female rats, suggesting that the functional behavior of the hypothalamic adrenergic response is strongly sex related.

Release of corticotropin-releasing factor from superfused rat hypothalami induced by interleukin-1 is not dependent on adrenergic mechanism.

Interleukin-1 (IL-1) is a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis. The hypothalamus seems to be the most important site of action of IL-1 on the HPA axis, inducing corticotropin-releasing factor (CRF) secretion. Catecholamines are important modulators of CRF secretion. In turn, IL-1 stimulates catecholamine release from the hypothalamus. In the present study, we examined the possible involvement of hypothalamic catecholamines in the effect of IL-1 beta on hypothalamic CRF secretion, by using an in vitro rat hypothalami continuous perifusion system. Neither in vivo pretreatment with an inhibitor of catecholamine synthesis nor in vitro exposure to alpha- or beta-adrenoceptor antagonists (phenoxybenzamine or propranolol, respectively) nor combination of both treatments altered the effect of IL-1 on CRF secretion from superfused hypothalami. These data indicate that catecholamines are not involved in the in vitro stimulatory action of IL-1 on hypothalamic CRF secretion.

Aminergic control of vasopressin secretion in the conscious rat.

The influence of aminergic pathways on basal and stimulated vasopressin (AVP) release was studied in conscious rats, the stimulus for hormone release being an intracerebroventricular (ICV) injection of 5 microliters 0.85M sodium chloride. The animals were treated with either phenoxybenzamine, propranolol or haloperidol prior to administration of the central hypertonic stimulus. Phenoxybenzamine elevated basal plasma vasopressin concentrations, while propranolol and haloperidol had no effect. The secretion of AVP in response to the hypertonic stimulus was potentiated by phenoxybenzamine and haloperidol, but the effect of propranolol was equivocal. The antagonists had no effect on basal arterial pressure at the time of hypertonic saline administration or the pressor response to ICV sodium chloride.

Norepinephrine--stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat.

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3',5' cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle.

Norepinephrine: stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3,5 cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle (Au)

Norepinephrine: stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3',5' cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle

Adrenergic mediation of the naloxone-induced GnRH release from hypothalami of ovariectomized, steroid-treated immature rats.

The present study examines the effect of naloxone on GnRH release in vitro under different steroid milieus. Naloxone (6.1 mumol/kg) administered 30 min before decapitation was highly effective in evoking GnRH release from superfused hypothalamic tissues derived from ovariectomized, estradiol- and progesterone-treated immature rats, while ineffective in altering GnRH release from intact, ovariectomized and vehicle- or estradiol-treated rats. To further explore the possible involvement of catecholamines in the naloxone-stimulated GnRH release, diethyldithiocarbamic acid (2.9 mmol/kg), an inhibitor of noradrenalin synthesis, was administered ip 30 min before naloxone injection into ovariectomized, estradiol- and progesterone-treated rats. Diethyldithiocarbamic acid markedly reduced the naloxone-evoked GnRH release, although it was ineffective in modifying the spontaneous release of GnRH. A blockade of alpha-adrenergic receptor with phenoxybenzamine significantly suppressed the naloxone-stimulated GnRH release, whereas treatment with propranolol, a beta-adrenergic receptor blocker, failed to alter GnRH release. The present data suggest that the endogenous opioid peptide may participate in the regulation of GnRH release under a particular steroid milieu, and the inhibitory action of endogenous opioid peptide seems to require the mediation of adrenergic neurotransmission, presumably through alpha-adrenergic receptor.

Stress- and endotoxin-induced increases in brain tryptophan and serotonin metabolism depend on sympathetic nervous system activity.

Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan. The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects. Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min. The footshock- and restraint-related increases in 5-hydroxyindoleacetic acid (5-HIAA) were also prevented by chlorisondamine. The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin-1 (IL-1) were also prevented by chlorisondamine pretreatment. The footshock-induced increases in brain tryptophan and 5-HIAA were attenuated by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine. Thus the autonomic nervous system appears to be involved in the stress-related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system. Moreover, the main effect of the sympathetic nervous system is exerted on beta- as opposed to alpha-adrenergic receptors. We conclude that activation of the sympathetic nervous system is responsible for the stress-related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake. Endotoxin and IL-1 also elevate brain tryptophan, presumably by a similar mechanism. The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to 5-HIAA that occurs in stressed animals, and which may reflect increased serotonin release.

Alpha 1-adrenergic receptor coupling with phospholipase-C is negatively regulated by protein kinase-C in primary cultures of hypothalamic neurons and glial cells.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha 1-receptor (alpha 1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha 1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha 1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha 1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.