A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.     

Protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice.

Neutralizing and nonneutralizing monoclonal antibodies to the peplomer glycoprotein and nucleocapsid protein of a mouse hepatitis virus (MHV), MHV-NuU, protected mice against lethal MHV-2 challenge. Histopathologically, livers of mice receiving protective antibodies showed some focal necrotic lesions with remarkable cellular infiltration instead of fulminant hepatitis caused by MHV-2.     

ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit

Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca²⁺uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent K_m for Ca²⁺ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa²⁺ transport by protoplast and tonoplast vesicles, respectively.Ca²⁺ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca²⁺ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca²⁺ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent. ¹Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986)     

Microtubules and the endoplasmic reticulum are highly interdependent structures

The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

The binding of 3H-acetylcholine to cholinergic receptors in bovine cerebral arteries

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and alpha-bungarotoxin exceeded 50 microM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries.     

delta 9-Tetrahydrocannabinol elicited ipsilateral circling behavior in rats with unilateral nigral lesion

The present study was designed to examine the influence of delta 9-tetrahydrocannabinol (THC) on the central dopaminergic system using circling behavior. THC 5 mg/kg i.p. produced ipsilateral circling in rats with unilateral nigral lesion by 6-hydroxy-dopamine. THC-induced ipsilateral circling was completely antagonized by 0.2 mg/kg of haloperidol. These findings suggest that THC may cause a presynaptic stimulation of nigrostriatal dopaminergic neurons.     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis

The membrane potential and the ionic activity gradients of K⁺and Cl^– across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K⁺ and Cl^–.Our results indicate that K⁺ is close to electrochemical equilibriumwhereas Cl^– is not. ¹ Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.