Effect of phenylethanolamine N-methyltransferase and dopamine-beta-hydroxylase inhibition on epinephrine levels in the brain.

The effects of phenylethanolamine N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DβH) inhibition on the epinephrine content in specific regions of the brain were studied. SKF 64139, a potent PNMT inhibitor, is effective in lowering brain epinephrine levels. The time course of PNMT inhibition by SKF 64139 parallels the lowering of epinephrine levels in the brain. Diethyldithiocarbamate (DDC), a potent inhibitor of DβH, is effective in lowering norepinephrine and epinephrine levels and in elevating dopamine levels in the analyzed regions of the brain. The epinephrine levels in the brain appear to be under similar biosynthetic control as in the adrenal glands.     

Lowering of blood pressure in hypertensive rats by SKF 64139 and SKF 72223

The effects of a centrally acting phenylethanolamine N-methyl-transferase (PNMT) inhibitor, SKF 64139, and of its analog, SKF 72223, which is devoid of PNMT inhibitory activity on blood pressure and heart rate, were investigated in spontaneously hypertensive rats (SHR) and in DOCA-salt hypertensive rats. SKF 64139 lowers blood pressure and decreases pulse rate, while SKF 72223 lowers blood pressure and transiently increases pulse rate in SH-rats and in DOCA-salt hypertensive rats. SKF 72223 has no effect on blood pressure or heart rate in normotensive Wister-Kyoto rats. These results suggest that the antihypertensive action elicited by these two tetrahydroisoquinoline (TIQ) derivatives is not due to lowering of central epinephrine (E) levels. To determine whether the cardiovascular response elicited by SKF 72223 is due to stimulation of presynaptic alpha 2-adrenoreceptors, or to blockade of alpha 1-adrenoreceptors, we have examined its effect in combination with the partial alpha 2-agonist clonidine, or with the alpha 1-antagonist prazosin. The administration of clonidine slightly decreases the antihypertensive action of SKF 72223. The clonidine induced reduction in pulse rate is reversed by SKF 72223. In animals pretreated with prazosin, SKF 72223 elicits an additional decrease in blood pressure. Since SKF 64139 and SKF 72223 interact with alpha 2-adrenoreceptors, it is suggested that blockade of peripheral vascular alpha 2-adrenoreceptors might be in part responsible for their antihypertensive action. However, the antihypertensive action of these two drugs might also be due to some central mechanisms.     

Facilitation of lordosis in guinea pigs by an alpha-noradrenergic agonist is independent of progestin receptor stimulation

Because manipulations of the noradrenergic system affect both lordosis behavior and progestin receptor levels in female guinea pigs, the present study attempted to determine if the noradrenergic (NE) system affects lordosis solely because of its impact on progestin receptors. Although the progestin receptor antagonist RU486 significantly reduced progesterone-facilitated lordosis, it had no effect on lordosis induced by the alpha-NE agonist clonidine in estrogen-primed female guinea pigs. This indicates that although progesterone may facilitate lordosis in female guinea pigs via activation of progestin receptors, the alpha-noradrenergic agonist clonidine does not mediate lordosis through the same mechanism.     

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.     

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.     

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.     

The nature of adrenoceptors in the guinea pig cerebral cortex: a microiontophoretic study

1. Noradrenaline, isoprenaline, and phenylephrine have been applied my microiontophoresis to neurones in the guinea pig cerebral cortex. All three compounds produced depression of neuronal firing, and all could be antagonized to some extent by phentolamine or propranolol. 2. The responses to isoprenaline were substantially reduced in size after a few applications. Noradrenaline and phenylephrine responses were partially reduced at the time of isoprenaline insensitivity, and the responses could now be blocked completely by phentolamine. 3. The results suggest that two kinds of receptors are present in the guinea pig cerebral cortex, with properties similar to alpha and beta receptors in the periphery. A single receptor with intermediate properties would not readily explain the present results. 4. The results are not consistent with the proposal that alpha receptors mediate neuronal excitation, and beta receptors inhibition in the cerebral cortex. 5. It is also suggested that the failure of some previous studies on guinea pig cortex in vitro to demonstrate the presence of beta receptors may be due to the particularly rapid desensitization of these receptors.     

Species Differences in the Brain Regional Distribution of Receptor Binding for Thyrotropin-Releasing Hormone

Abstract: A survey of the regional distribution of binding of 1 nM [³H](3-MeHis²)thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in the brains of eight mammalian species revealed major species differences in both the absolute and relative values of TRH receptor binding in different brain regions. Several brain regions exhibited binding equal to or exceeding that in the anterior pituitary gland of the same species, including the amygdaia in the guinea pig and rat, the hypothalamus in the guinea pig, the nucleus accumbens in the rabbit, and all these and other regions in the cat and dog, for which pituitary binding was exceptionally low. Species could be divided into two groups according to which brain region appeared highest in binding: rabbits, sheep, and cattle had highest binding in the nucleus accumbens/septal area, whereas guinea pigs, rats, dogs, cats, and pigs had highest binding in the amygdala/temporal cortex area. The nucleus accumbens consistently exceeded the caudate-putamen in receptor binding. For most brain regions, rabbits, rodents, and sheep tended to be higher than carnivores, cattle, or pigs. Further regions that exhibited appreciable binding in most species included the olfactory bulb and tubercle, hippocampus, and various cortical and brain stem areas. In fact, essentially all brain regions appeared to have detectable levels of TRH receptors in at least some species, but no rat peripheral tissues have yet shown detectable receptor binding. The species differences appeared to reflect largely if not entirely differences in receptor density, although this was not tested in every species.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Effects of drugs interfering with the metabolism of octopamine on blood pressure of rats

1. p-Octopamine injected in lateral ventricle of conscious spontaneously hypertensive rats decreased systolic blood pressure (SBP). 2. Precursors of octopamine--tyrosine, tyramine and phenylethanolamine (PEA)--had the same effect. The administration of pargyline, a MAO inhibitor, which increased brain octopamine, resulted in a reduction of systolic blood pressure; and this decrease was greater after administration of octopamine precursors and PEA. 3. Similarly, drugs known to inhibit activity of phenylethanolamine N-methyl-transferase (PNMT) and to increase brain octopamine level such as SKF 64139 and DCMB decreased SBP. 4. p-Octopamine hypotension was not antagonized by piperoxan, yohimbine and prazosin, a relatively selective antagonist of post-synaptic alpha adrenoceptors. 5. These results suggest that octopamine may be involved in central blood pressure regulation, and the receptors sensitive to octopamine appeared to be distinct from those receptive to the catecholamines.     

Morphine suppresses the ovarian steroid hormone-dependent lordosis response of female guinea pigs: reversal by naloxone but not clonidine.

The opiate agonist morphine caused a dose- and time-dependent suppression of lordosis responding in ovariectomized guinea pigs treated with estradiol-17 beta and progesterone. The suppression of lordosis by morphine appears to be mediated by opiate receptors since the opiate antagonist naloxone blocked its effects both in terms of the percentage of animals showing lordosis and the duration of individual responses. Naloxone, when given alone, did not affect lordosis responding in estradiol-17 beta + progesterone-primed animals and did not induce lordosis in animals primed with estradiol-17 beta alone. Thus, endogenous opioids might not tonically inhibit lordosis under the physiological conditions examined. The alpha-noradrenergic agonist clonidine did not reverse the effects of morphine on lordosis. Thus, the inhibitory effects of morphine on this behavior might be independent of its presynaptic effects on norepinephrine release in brain.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Reduction in opiate receptor reserve in morphine tolerant guinea pig ilea

Spare opiate receptors in the guinea pig ileum have been detected by the use of the opiate receptor alkylating agent beta-chlornaltrexamine (CNA). Treatment of the guinea pig ileum longitudinal muscle in vitro with low concentrations (less than 10nM) of CNA resulted in an irreversible parallel shift to the right of the normorphine log concentration response curve. With increasing concentration of the reagent, the agonist EC50 becomes progressively greater. Finally a point is reached at which the maximal agonist effect decreases, so that parallelism is no longer seen. The maximal parallel shift provides a measure from which one can estimate the spare receptor fraction that is present in untreated tissue. In ilea from normal guinea pigs, roughly 80-90% of the opiate receptors for normorphine were found to be spare. Even after the largest parallel shifts that could be achieved, the naloxone Ke value for antagonism was unchanged, indicating that normorphine acts through spare mu receptors. Ilea from guinea pigs made tolerant by chronic morphine pellet implantation were found to be more sensitive to the effects of CNA treatment; there was a reduction in the number of spare receptors for normorphine. It is suggested that the opiate spare receptor fraction is physiologically modulated to control neuronal sensitivity to opioid effect.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.     

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α₂-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N -methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μ M . Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I₁-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.     

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.     

Substance P-induced airway hyperreactivity is mediated by neuronal M(2) receptor dysfunction

Neuronal muscarinic (M(2)) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M(2) autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M(2) receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar(9),Met(O(2))(11)]- substance P, guinea pigs were hyperreactive to vagal stimulation and M(2) receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) all prevented substance P-induced M(2) dysfunction and hyperreactivity. Intravenous heparin acutely reversed M(2) receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK(1) receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M(2) receptors by MBP, resulting in increased release of acetylcholine.