Transition-State Analogues of Phenylethanolamine N-Methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT) is a critical enzyme in catecholamine synthesis. It transfers the methyl group of S-adenosylmethionine (SAM) to catalyze the synthesis of epinephrine from norepinephrine. Epinephrine has been associated with diverse human processes, including the regulation of blood pressure and respiration, as well as neurodegeneration found in Alzheimer's disease. Human PNMT (hPNMT) proceeds through an SN2 transition state (TS) in which the transfer of the methyl group is rate limiting. TS analogue enzyme inhibitors are specific for their target and bind orders of magnitude more tightly than their substrates. Molecules resembling the TS of hPNMT were designed, synthesized, and kinetically characterized. This new inhibitory scaffold was designed to mimic the geometry and electronic properties of the hPNMT TS. Synthetic efforts resulted in a tight-binding inhibitor with a Ki value of 12.0 nM. This is among the first of the TS analogue inhibitors of methyltransferase enzymes to show an affinity in the nanomolar range. Isothermal titration calorimetry (ITC) measurements indicated negative cooperative binding of inhibitor to the dimeric protein, driven by favorable entropic contributions. Structural analysis revealed that inhibitor 3 binds to hPNMT by filling the catalytic binding pockets for the cofactor (SAM) and the substrate (norepinephrine) binding sites.

Kinetic and pH studies on human phenylethanolamine N-methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-L-methionine (AdoMet) is converted to S-adenosyl-L-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the logV pH-profile was not well defined, plots of logV/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.

Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines as inhibitors of phenylethanolamine N-methyltransferase.

A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the alpha(2)-adrenoceptor. The THTP nucleus was suggested as an isosteric replacement for the 1,2,3,4-tetrahydroisoquinoline (THIQ) ring system on the basis that 3-thienylmethylamine (18) was more potent as an inhibitor of hPNMT and more selective toward the alpha(2)-adrenoceptor than benzylamine (15). Although the isosterism was confirmed, with similar influence of functional groups and chirality in both systems on hPNMT inhibitory potency and selectivity, the THTP compounds proved, in general, to be less potent as inhibitors of hPNMT than their THIQ counterparts, with the drop in potency being primarily attributed to the electronic properties of the thiophene ring. A hypothesis for the reduced hPNMT inhibitory potency of these compounds has been formed on the basis of molecular modeling and docking studies using the X-ray crystal structures of hPNMT co-crystallized with THIQ-type inhibitors and S-adenosyl-L-homocysteine as a template.

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase.

The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed with its product, S-adenosyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Val53, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased Km values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its kcat value. Both WT hPNMT and D267A had similar kcat values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The Ki values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenosyl-L-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.

Molecular recognition of sub-micromolar inhibitors by the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase.

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.

Getting the adrenaline going: crystal structure of the adrenaline-synthesizing enzyme PNMT.

BACKGROUND: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. RESULTS: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. CONCLUSIONS: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Modulatory role of the epinergic system in the neuroendocrine-immune system function.

It is well recognized that the reciprocal interaction established between the immune and neuroendocrine systems is crucial for the homeostatic adaptation of individuals during septicemia. In the present study, using an in vivo rat model, we investigated the degree of participation of central and peripheral epinergic systems in the modulation of the hypothalamic-pituitary-adrenal and immune axes' functions during endotoxemia. For this purpose, acute endotoxemia was induced in adult male rats pretreated intraperitoneally with either different inhibitors of phenylethanolamine-N-methyltransferase (PNMT) [which are active either peripherally (SKF 29661) or both peripherally and centrally (SKF 64139), thus lowering epinephrine (EPI) synthesis] or vehicle only (CTRL). Twelve hours after pretreatment, animals were intraperitoneally injected with vehicle alone (basal) or vehicle containing bacterial lipopolysaccharide (LPS) and sacrificed 2 h later. A significant (p < 0.05 vs. the respective basal value) hypoglycemia was found in all groups studied. No pretreatment modified basal plasma adrenocorticotropic hormone (ACTH), glucocorticoid and cytokine concentrations. Endotoxin-stimulated ACTH secretion was severalfold (p < 0.05) higher than the respective basal value in CTRL and in SKFs-pretreated rats; however, the plasma ACTH levels after LPS were significantly (p < 0.05 vs. CTRL and SKF-29661 values) reduced in SKF-64139-pretreated rats. All groups studied showed an appropriate adrenal response to endotoxin challenge. Although no differences were found in basal anterior pituitary (AP) ACTH content among groups, LPS treatment significantly (p < 0.05 versus the respective basal value) decreased AP ACTH in CTRL and SKF 29661 groups. No pretreatment modified the basal medial basal hypothalamus (MBH) corticotropin-releasing hormone (CRH) content. Conversely, SKF 64139 pretreatment significantly (p < 0.05 vs. CTRL and SKF 29661 values) reduced basal median eminence (ME) CRH content, and LPS administration significantly (p < 0.05) decreased ME CRH in CTRL and SKF-29661-pretreated rats. SKF 64139 pretreatment significantly (p < 0.05) enhanced basal MBH and ME arginine vasopressin (AVP) contents. LPS administration significantly (p < 0.05) decreased MBH AVP in CTRL and SKF-29661-pretreated rats and diminished (p < 0.05 vs. basal values) ME AVP in all groups. The plasma tumor necrosis factor alpha (TNFalpha) concentrations were enhanced severalfold (p < 0.05 vs. basal values) after LPS treatment in CTRL rats, but not in SKFs-treated animals. In order to explore the reduced cytokine release after LPS in PNMT-inhibited rats, additional ex vivo experiments were performed by using peripheral mononuclear cells (PMNC) from both CTRL and SKF-29661-pretreated rats. The results of these experiments confirmed an immune dysfunction after inhibition of peripheral EPI synthesis; in fact, basal and concanavalin-A-stimulated TNFalpha secretions were significantly (p < 0.05) lower in SKF-29661-treated than in CTRL PMNC, while, interestingly, addition of EPI (10(-7) M) to the medium fully restored these effects. These data demonstrate that: (1) the mechanism whereby LPS-induced hypoglycemia was independent of epinergic activity; (2) selective central inhibition of epinergic function reduced endotoxin-stimulated ACTH secretion, an effect that could mainly be due to a decrease in CRH-ergic activity; (3) the central epinergic system positively and negatively modulates CRH- and AVPergic functions, respectively, and (4) inhibition of peripheral PNMT activity reduced immune system function in vivo and ex vivo. It is further suggested that low peripheral levels of EPI could be beneficial for the body's defense mechanisms during endotoxic shock.

Changes in hypothalamic catecholamines, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase in the catfish Heteropneustes fossilis in relation to season, raised photoperiod and temperature, ovariectomy, and estradiol-17 beta replacement.

In Heteropneustes fossilis, contents and turnovers of hypothalamic catecholamines (CA) and activities of dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) showed significant seasonal variations with significantly high day values. The seasonal pattern of dopamine (DA) on one hand and that of noradrenaline (NA) and adrenaline (A) on the other hand showed an inverse relationship, the former decreasing and the latter increasing during the progress of gonadal recrudescence. The DBH and PNMT levels were low in the resting phase and increased to the peak in the prespawning (DBH) and spawning (PNMT) phases. Maintenance of the fish under long photoperiods (16L:8D) and high temperature (28 +/- 2 degrees) stimulated the NA and A, and DBH and PNMT activities, and suppressed the DA mechanism, the changes being maximal in the raised temperature groups. In the resting phase (December), ovariectomy (OVX) or estradiol-17 beta (E2) replacement in 4-week ovariectomized fish did not produce any significant effects on the CA and enzyme activities. On the contrary, in the prespawning phase (May), OVX produced differential and biphasic responses on CA and the enzymes. The contents and turnovers of both NA and A increased significantly at 2-5 weeks and decreased in the sixth week. However, the reverse was true for DA. The DBH and PNMT activities (assayed only 3, 4, and 6 weeks after OVX) were elevated significantly in the third and fourth weeks but decreased in the sixth week. Plasma levels of gonadotropin (GTH) increased significantly at all durations of OVX in a bimodal pattern while the E2 levels decreased consistently. Supplementation with a low dose (0.1 microgram/g BW) of E2 restored the NA and A and enzyme activities while the higher doses (0.5, 1.0, and 5.0 micrograms/g BW) depleted them. The reverse was true for DA. The low dose of E2 restored the GTH level while the higher ones inhibited it significantly. These results indicate that both environmental photoperiod and temperature and E2-negative feedback act on the CA to modulate GTH secretion.

Catecholamine synthesis inhibitors increase pineal adrenaline content by stimulating adrenal medullary activity.

Estimation of noradrenaline and adrenaline utilization in the pineal gland of female rats was attempted using inhibitors of the enzymes that catalyse the catecholamine biosynthetic pathway. Treatment with FLA63, an inhibitor of dopamine beta-hydroxylase (10 mg/kg, 2 h before killing), induced depletion of noradrenaline and adrenaline in the preoptic area and median eminence (sites, respectively, inside and outside the blood-brain barrier) but, paradoxically, resulted in a significant increase (+77%) in the pineal content of adrenaline without affecting that of noradrenaline. Treatment with LY134046, an inhibitor of phenylethanolamine N-methyltransferase (40 mg/kg, 5 and 2 h before killing), induced depletion of adrenaline in the preoptic area and median eminence but, again, resulted in a paradoxical and large increase in pineal adrenaline (+224%); this increase was prevented by prior adrenalectomy. Blood samples taken from free-moving rats fitted with intravenous and intraperitoneal cannulae revealed a marked increase in plasma levels of adrenaline after each injection of LY134046. These results suggest that the adrenal medulla is the primary source for the increase in pineal adrenaline seen after administration of the enzyme inhibitors. The precise site of uptake and the biological implications of this phenomenon remain to be elucidated. Nevertheless, interpretation of in vivo experiments involving these catecholamine synthesis inhibitors should take this adrenal response into account.

Inhibition of guinea pig lordosis behavior by the phenylethanolamine N-methyltransferase (PNMT) inhibitor SKF-64139: mediation by alpha noradrenergic receptors.

Experiments were undertaken to determine whether the steroid-dependent lordosis response of female guinea pigs is under adrenergic control. In initial experiments, treatment with the centrally active phenylethanolomine N-methyltransferase (PNMT; the enzyme catalyzing methylation of norepinephrine to epinephrine) inhibitor SKF-64139 inhibited lordosis behavior induced by estradiol-17 beta benzoate plus progesterone. SKF-29661, a PNMT inhibitor that does not cross the blood-brain barrier, did not affect lordosis. However, no detectable epinephrine was found in brain or spinal cord of drug- or vehicle-treated guinea pigs. This suggests that epinephrine neuronal systems do not exist in the guinea pig CNS. In agreement with this idea, the inhibitory effects of SKF-64139 on lordosis were found to be primarily attributable to the blockade of alpha noradrenergic receptors rather than to PNMT inhibition. Two lines of evidence support this conclusion. First, using in vitro receptor binding techniques, SKF-64139 was found to have a relatively high affinity for alpha 1 and particularly alpha 2 receptors in guinea pig forebrain. Second, presumably through competitive inhibition of SKF-64139 binding to alpha receptors, treatment with clonidine (an alpha receptor agonist) overrode the inhibitory effects of SKF-64139 on lordosis. Taken together, these findings indicate the possible absence of epinephrine neuronal systems in guinea pig brain and reemphasize the importance of alpha receptors in regulating steroid-dependent lordosis behavior in this species.

5'-thioadenosine derivatives as potent and selective inhibitors of histamine N-methyltransferase.

Several new analogues of adenosine bearing a lipophilic side chain at the 5'-position have been synthesized and investigated for their ability to inhibit histamine N-methyltransferase (HNMT). The 5'-deoxy-5'-[4-(3-indolyl)but-1-yl]thio]adenosine (2e), exhibited a pI50 of 5.00 against guinea pig brain HNMT. Interestingly, the polar methyl sulphonium analogue (1c) was a more potent inhibitor of this enzyme (pI50 = 5.26). Both compounds were relatively ineffective inhibitors of rabbit adrenal phenylethanolamine N-methyltransferase (PNMT), rabbit lung indoleamine N-methyltransferase (INMT), and rat brain catechol O-methyltransferase (COMT). 5'-[N(4-phenylbutyl)]-amino-5'deoxyadenosine (2a) and 5'-[N-methyl,N-(4-phenylbutyl]-amino-5'deoxyadenosine (2b) also exhibited potent and selective inhibition against guinea pig brain HNMT. Results from kinetic studies indicate that the above compounds are inhibitors that compete for both the histamine and the S-adenosylmethionine (SAM) binding sites of HNMT. Compound 1c is one of the most potent adenosine analogue inhibitors of HNMT known.

Epinephrine mediates the growth hormone-releasing effect of galanin in infant rats.

The mechanism underlying the GH-releasing effect of galanin (GAL), a novel 29-amino acid peptide, was investigated in the neonatal rat. The effect of galanin was compared to that of clonidine (CLO), a drug known to release GH via endogenous GHRF. GAL administration (5-25 micrograms/kg BW, sc) induced in 10-day-old pups a clear-cut and dose-related rise in plasma GH 15 min postinjection. CLO (50-450 micrograms/kg BW, sc) induced a marked rise in plasma GH, but no dose-related effect was evident. Inhibition of hypothalamic norepinephrine and epinephrine biosynthesis by DU-18288 (6 mg/kg BW, ip) or selective inhibition of epinephrine biosynthesis by SKF-64139 (50 mg/kg BW, ip) completely abolished the GH-releasing effect of GAL (25 micrograms/kg, sc), but left unaltered the GH rise induced by CLO (150 micrograms/kg, sc). Passive immunization with an anti-GHRF serum decreased basal GH levels and prevented the GH-releasing effect of either GAL or CLO, whereas in pups pretreated with an antisomatostatin serum, CLO, but not GAL, increased the already elevated plasma GH titers. In all these data indicate that in the infant rat 1) GAL is a potent GH secretagogue; 2) the action of GAL is not exerted directly on GHRF- or somatostatin-secreting structures, but requires the intervention of catecholaminergic neurons; 3) the GH-releasing effect of GAL is ultimately exerted via GHRF release, although a mechanism operating to inhibit hypothalamic somatostatin release cannot be ruled out; and 4) differently from GAL, CLO releases GH via postsynaptic stimulation of GHRF-secreting neurons.

Effects of catecholamine-depleting agents and receptor blockers on basal and angiotensin II- or norepinephrine-stimulated luteinizing hormone release in female rats.

Depletion of hypothalamic norepinephrine (NE) and epinephrine by administration of diethyldithiocarbamate abolished the stimulatory effects of intraventricular (IVT) angiotensin II (AII) on LH release in ovariectomized rats pretreated with estradiol and progesterone. The increase in blood LH produced by IVT NE or iv LHRH was unaffected in these drug-treated animals. Selective depletion of hypothalamic epinephrine by the administration of LY134046 (8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride) potentiated the effect of AII on LH secretion. Blockade of alpha 1-, alpha 2-, or beta-adrenergic receptors resulted in a transient increase in basal LH levels. The stimulation of LH secretion induced by IVT AII or NE was unaffected by alpha 1-receptor blockade with prazosin, but was abolished by alpha 2-receptor blockade with yohimbine. beta-Receptor blockade with propranolol potentiated both NE- and AII-induced LH release. AII receptor blockade with IVT saralasin prevented the LH rise due to AII without modifying that due to NE. Taken together, these data suggest that IVT AII stimulates LH release in ovariectomized rats treated with estradiol and progesterone by releasing endogenous NE, which, in turn, acts on facilitatory alpha 2-receptors to affect LH secretion, presumably by increasing the secretion of LHRH. Exogenous NE also acts at this receptor. beta-Receptors provide inhibitory tone to this facilitatory system, and blockade of this receptor subtype results in potentiated LH responses to both AII and NE.

Epinephrine synthesis in the PC12 pheochromocytoma cell line.

PC12 is a rat pheochromocytoma cell line which has been increasingly used as a model system for both neural differentiation and chromaffin cell function. PC12 cells have been reported to synthesize dopamine and norepinephrine, but not epinephrine. We have found that PC12 cells do synthesize small amounts of epinephrine and that dexamethasone increases both epinephrine content as well as phenylethanolamine-N-methyltransferase (PNMT) activity. These results suggest that the PC12 cell line may be useful in the investigation of the regulation of PNMT.

Reduction of central epinephrine concentrations is consistent with the continued occurrence of ovulation in rats treated with an inhibitor (LY 134046) of phenylethanolamine N-methyltransferase.

Groups of 4-day cyclic rats were injected (i.p.) with LY 134046 (50 mg/kg), a central inhibitor of phenylethanolamine N-methyltransferase, or saline at 09.00, 13.00 and 19.00 h on the day of proestrus. The incidence of ovulation was examined the following estrous morning. There was no difference in the number of ova in drug-treated animals compared to saline-treated controls. In other groups of 4-day cyclic rats, LY 134046 or saline was injected daily at 10.00 h for 5 consecutive days from proestrus to proestrus inclusive. The animals were decapitated the following day and ova were counted. Epinephrine concentrations were determined by radioenzymatic assay in the mediobasal hypothalamus (MBH) and the medial preoptic area (MPOA). All saline-treated controls and 10/14 of the drug-treated animals had ovulated, while epinephrine concentrations in the MBH and MPOA had been reduced by 95.8 and 94.7%, respectively, compared to saline-treated controls. These experiments suggest that a significant surge of luteinizing hormone occurs to initiate ovulation even after a severe reduction in central epinephrine concentration has taken place.

Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 10. Base- and amino acid modified analogues of S-aristeromycinyl-L-homocysteine.

A series of base- and amino acid modified analogues of S-aristeromycinyl-L-homocysteine, a carbocyclic nucleoside, were synthesized and evaluated as inhibitors of S-adenosyl-L-methionine-dependent methyltransferases, including catechol O-methyltransferase, phenylethanolamine N-methyltransferase, and histamine N-methyltransferase. The base-modified analogues (8-azaadenine, 3-deazaadenine, and N6-methyladenine) were prepared by reaction of the corresponding carbocyclic 5'-chloro-5'-deoxynucleosides with the anion of homocysteine generated in situ either from L-homocystine or S-benzyl-L-homocysteine in Na/liquid NH3 or with DL-homocysteine thiolactone in alkaline solution. S-Aristeromycinyl-D-homocysteine was prepared with use of D-homocystine in the Na/liquid NH3 reaction. The sulfoxide and sulfone analogues were prepared by oxidation of S-aristeromycinyl-L-homocysteine. The various base- and amino acid modified analogues of S-aristeromycinyl-L-homocysteine were inactive as inhibitors of catechol O-methyltransferase. In contrast, the 3-deaza analogue was a good inhibitor (Ki = 20.5 +/- 1 microM) of phenylethanolamine N-methyltransferase whereas S-aristeromycinyl-D-homocysteine was an excellent inhibitor (Ki = 10.4 +/- 2.4 microM) of histamine N-methyltransferase. On the basis of these results, it would appear that the structural requirements for the binding S-aristeromycinyl-L-homocysteine are similar to those for binding S-adenosyl-L-homocysteine. Therefore, these carbocyclic analogues have the potential of being better inhibitors in vivo, because they should be more stable to metabolism than the ribosyl analogues.

Effects of brain epinephrine depletion on thermoregulation, reflex bradycardia, and motor activity in rats.

Two hours after i.p. administration of 2-cyclooctyl-2-hydroxyethylamine (CONH), 1-aminomethylcycloundecanol (CUNH), 2,3-dichloro-alpha-methylbenzylamine (DCMB), or 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (SKF64139), the hypothalamic and brain stem epinephrine (EPI) contents of rat brain were decreased. Depletions of brain EPI with these phenylethanolamine N-methyltransferase (PNMT) inhibitors reduced the rectal temperatures of rats at ambient temperatures of 8 and 22 degrees C. The hypothermia in response to these PNMT inhibitors was due to decreased metabolism and cutaneous vasodilatation. The locomotor stimulant responses induced by thyrotropin-releasing hormone were also reduced by administration of any one of these PNMT inhibitors. On the other hand, acute administration of any of these PNMT inhibitors enhanced the reflex bradycardia induced by i.v. infusion of EPI. The data suggest that brain (particularly the hypothalamus and brain stem) EPI-containing neurons are involved in the regulation of body temperature, reflex bradycardia, and motor performance in the rat.

Evidence that norepinephrine and epinephrine systems mediate the stimulatory effects of ovarian hormones on luteinizing hormone and luteinizing hormone-releasing hormone.

Results from previous investigations have suggested an important role for central epinephrine (EPI) systems in mediating the stimulatory effects of ovarian hormones on LH release in ovariectomized female rats. The purpose of these experiments was 1) to test whether selective inhibition of EPI synthesis blocks the sequential accumulation and decline of LHRH concentrations in the median eminence that precedes the ovarian hormone-induced LH surge and 2) to test whether the stimulatory ovarian hormone regimen enhances the activity of EPI systems in the hypothalamus. Ovariectomized rats were treated with estradiol, followed 2 days later by progesterone. Animals were treated before progesterone administration with saline, one of the EPI synthesis inhibitors [SK&F 64139 (2,3-dichloro-tetrahydroisoquinoline HCl) or LY 78335 (dichloro-alpha-methylbenzylamine)], or the dopamine-beta-hydroxylase inhibitor FLA-63 (bis-4-methyl-1-homopiperazinyl thiocarbonyl disulfide), which inhibits NE and EPI synthesis. The catecholamine synthesis inhibitors blocked or delayed the afternoon LH surge. FLA-63 completely prevented the accumulation of LHRH in the median eminence that preceded the rise in LH release. However, selective EPI synthesis inhibition with SK&F 64139 only partially prevented this increase in LHRH. A second EPI synthesis inhibitor, LY 78335, delayed both the LH surge and the rise in LHRH. In a second experiment, the administration of estradiol and progesterone to ovariectomized rats increased the alpha-methyltyrosine-induced depletion of hypothalamic EPI, suggesting increased activity in this system during the LH surge. Further experiments localized this effect to the medial basal hypothalamus. The depletion of both NE and EPI after synthesis inhibition was also enhanced during an earlier period, approximating the time of LHRH accumulation. These results suggest that the ovarian hormones activate both NE and EPI systems to stimulate the early afternoon rise of LHRH in the median eminence and to induce the subsequent LH surge.

Effects of L-dopa on epinephrine concentration in rat brain: possible role of inhibition of norepinephrine N-methyltransferase by S-adenosylhomocysteine.

L-Dopa injected at 200 mg/kg i.p. into rats caused a slight reduction in hypothalamic concentration of epinephrine and completely prevented the accumulation of epinephrine after monoamine oxidase inhibition. The lowering of epinephrine concentration was greater with L-dopa than with D-dopa, was dose-related over a dosage range of 50 to 200 mg/kg of L-dopa and was not prevented by a dopamine receptor antagonist. Hypothalamic norepinephrine N-methyltransferase activity measured in vitro was not altered in rats treated with L-dopa. L-Dopa injection decreased S-adenosylmethionine (SAMe) concentration and increased S-adenosylhomocysteine (SAH) concentration in hypothalamus, probably a result of extensive O-methylation of L-dopa and its metabolites. The decrease in SAMe and epinephrine concentration and the increase in SAH concentration occurred at lower doses of L-dopa in carbidopa-pretreated rats than in control rats. Norepinephrine N-Methyl-transferase activity assayed in vitro was markedly inhibited by SAH; the inhibition was competitive with SAMe as the variable substrate and the Ki for SAH was 1.9 x 10(-5) M. Increasing the SAH/SAMe ratio in in vitro experiments sharply reduced norepinephrine N-methyltransferase activity. The apparent inhibition of hypothalamic epinephrine synthesis in vivo after L-dopa injection is suggested to be a consequence of the increased SAH/SAMe ratio.