Aromatic L-amino acid decarboxylase: histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution.

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Interactions of amantadine with the cardiac muscarinic receptor.

The antiviral drug amantadine has anticholinergic effects in the guinea-pig atrium at concentrations greater than 1 X 10(-4)M. It is a competitive inhibitor of [3H]-quinuclidinyl benzilate binding to the muscarinic receptor, but antagonizes the negative inotropic effect of acetylcholine in a non-competitive manner. It increases the duration of the atrial action potential and also increases the force of atrial contraction. These effects are evident at approximately 10 times lower concentrations than the antimuscarinic effects. The increase in contractility can be reversed by propranolol (5 X 10(-7)M) but the increase in action potential duration is potentiated by propranolol. Shortening of the action potential duration by acetylcholine was reversed by amantadine, but at approximately ten times lower levels than were needed to reduce the negative inotropic effect. Interactions between beta adrenoceptor binding of [3H]-dihydroalprenolol and amantadine could not be demonstrated. Similarly, binding of [3H]-nitrendipine to the calcium channel is not influenced. It is suggested that amantadine may exert its positive inotropic effect by interaction with the potassium channel, causing a delay in outward current.

Effect of dexamethasone on biopterin levels and tyrosine hydroxylase activity in PC-12 cells.

Culture of PC-12 cells in 1 microM dexamethasone for 24, 48 or 72 hr did not alter significantly PC-12 cell total biopterin levels, although tyrosine hydroxylase activity in extracts of cell homogenates was increased 2- to 3-fold. Increasing the concentration of dexamethasone to 10 microM did not change biopterin levels or result in further increases in tyrosine hydroxylase activity measured after 72 hr. Culture of cells in dexamethasone markedly decreased the ratio of biopterin concentration to tyrosine hydroxylase activity. The molar ratio of tyrosine hydroxylase subunits to biopterin in control cells can be estimated to be approximately one. Therefore, following induction of the enzyme by dexamethasone, there appears to be an excess of enzyme molecules relative to cofactor molecules in these cells.

Effects of methotrexate on biopterin levels and synthesis in rat cultured pineal glands.

Culture of rat pineal glands in methotrexate (0.5, 5, or 10 microM) for 6 or 24 h did not alter pineal tetrahydrobiopterin (85-90% of total biopterin in cultured glands), except for a decrease of 30% after 24 h culture in 10 microM methotrexate. However, pineal dihydrobiopterin and/or biopterin (10-15% of total biopterin) was increased by methotrexate up to 2.5-fold. Biopterin detected in the culture medium following pineal culture was also increased to a similar extent after methotrexate treatment and appeared to represent leakage of pineal dihydrobiopterin and/or biopterin. Culture of glands in 5 microM methotrexate did not alter the conversion of [U-14C]-guanosine to [14C]biopterin, suggesting that pineal tetrahydrobiopterin synthesis was not altered by methotrexate. Complete inhibition of dihydrofolate reductase activity measured in pineal homogenates was obtained following culture of glands in all concentrations of methotrexate studied. Therefore, dihydrofolate reductase and dihydrobiopterin do not appear to be involved in a major biosynthetic pathway for pineal tetrahydrobiopterin from GTP, although they may have a minor role in tetrahydrobiopterin synthesis.

Comparison of histamine-H(1) receptors in bovine intracerebral microvessels with cerebral grey matter by [(3)H]mepyramine binding.

Small intracerebral blood vessels (microvessels) of bovine brain are known to contain the vasoactive amine histamine, and the presence of histamine-H(1) receptors in microvessels was examined using the radioligand, [(3)H]mepyramine. Microvessels were isolated from cerebral cortex grey matter, striatum and hippocampus by a sieving technique and membranes prepared for binding studies. [(3)H]Mepyramine bound to a single, high affinity site, which displayed stereoselectivity for (+) chlorpheniramine relative to its (?) isomer and was consistent with binding to H(1)-receptors. The density of binding sites (B(max)), in microvessel membranes from cortical grey matter, was approximately one-third of that seen in membranes prepared from cortical grey matter. Microvessels isolated from striata and hippocampi had a similar density of H(1)-receptor sites to that seen in cortical microvessels. These results demonstrate that bovine intracerebral microvessels contain significant numbers of histamine-H(1) receptors and strengthen the hypothesis that histamine could regulate the calibre of intracerebral blood vessels.

Characterization of alpha-adrenoceptors in rat and guinea pig tissues using radiolabeled agonists and antagonists.

Tritium-labeled preparations of the selective alpha 2-adrenoceptor agonist, clonidine, and the selective alpha 1-adrenoceptor antagonists, prazosin and WB 4101, were examined for their suitability as ligands for receptor assay. In the rat brain, 3H-clonidine bound specifically with high affinity to membrane sites, and drug displacement studies indicated that these were alpha 2-adrenoceptors. The structural requirements for this receptor were defined by examining a range of clonidine analogues. 3H-Clonidine binding to peripheral tissues of the rat was very low, although peripheral tissues from guinea pig exhibited higher binding. Specific 3H-clinidine binding was not reduced after chemical sympathectomy, suggesting that the binding site, although having characteristics of an alpha 2-adrenoceptor, was not located presynaptically. Studies with 3H-WB 4101 in rat cerebral cortex indicated that this ligand bound to an alpha 1-adrenoceptor which was independent of the 3H-clonidine-binding site. 3H-Prazosin was a ligand for alpha 1-adrenoceptors in guinea pig brain and also in membranes from seminal vesicle. However, in the latter tissue, characterization of the binding site was hampered by a high degree of nonspecific binding (50% of total binding).

alpha- and beta-Adrenoceptor activities of norepinephrine analogues in isolated tissues of the rat.

A range of norepinephrine analogues has been studied in rat tracheal, atrial, and aortic preparations. Isopropyl appeared to be the most favorable N-substituent for agonist action on tracheal adrenoceptors, whereas hydroxyphenylisopropyl gave highest activity on atrial adrenoceptors. Selectivity for the beta 2-adrenoceptor was improved with tertiary branching at the gamma-carbon and further increased by aromatic substitution with resorcinol or saligenin. Alpha-Adrenoceptor properties were substantially modified by N-substitution, whereas aromatic substitution was an important factor in determining beta-adrenoceptor agonist or antagonist activity at the beta-adrenoceptor. The data were consistent with norepinephrine analogues having multiple sites of attachment to the receptor membrane influencing receptor potency and selectivity. The data also suggest that compounds of this type can have actions on both alpha- and beta-adrenoceptors.