Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.     

EVIDENCE FOR EXTRANORADRENERGIC DOPAMINE-β-HYDROXYLASE ACTIVITY IN RAT SALIVARY GLAND

—Three days after superior cervical ganglionectomy of adult Sprague-Dawley rats, the levels of endogenous norepinephrine, the uptake process for [³H]norepinephrine and the activity of tyrosine hydroxylase decreased 99 per cent in the ipsilateral salivary gland. In contrast, the activity of dopamine-β-hydroxylase and DOPA decarboxylase fell to 30 per cent of the activity of the contralateral innervated gland. Examination of the cofactor requirements, the characteristics of activation by cupric ion and the immunologic identity of this residual hydroxylase activity indicated that it was authentic dopamine-β-hydroxylase. The residual dopamine-β-hydroxylase in the denervated gland had the same subcellular distribution as the enzyme in the innervated salivary gland. Procedures that caused atrophy or hypertrophy of the acinar cells did not affect the total content of dopamine-β-hydroxylase in the denervated salivary gland. Chemical sympathectomy with 6-hydroxy-dopamine caused a 40 per cent decrement in the serum levels of dopamine-β-hydroxylase but a 30 per cent increase in its activity in the denervated salivary gland. Although denervation caused a complete loss of endogenous norepinephrine in the salivary gland, it resulted in only a 15 per cent decrement in the levels of endogenous octopamine and β-phenylethanolamine, two other products of dopamine-β-hydroxylase.     

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.     

In vivo tracer studies of glucose metabolism,cerebral blood flow,and protein synthesis in naloxone precipitated morphine withdrawal

Quantitative autoradiography of [¹⁴C]deoxyglucose, [¹⁴C]iodoantipyrine, and [¹⁴C]leucine was used to estimate regional cerebral glucose metabolism, cerebral blood flow, and cerebral protein synthesis, respectively, in rats during morphine dependence and withdrawal. Glucose metabolism was elevated in 19 of 26 selected brain regions; the elevations in glucose metabolism were similar when data were expressed as either optical density ratios or as calculated rate values of mol/100 gm/min. Restraining the rats produced heterogeneous effects on glucose metabolism during morphine withdrawal (MW). Neither estimated cerebral blood flow nor cerebral protein synthesis were affected by morphine and/or naloxone treatments in either naive or morphine-dependent rats. The data demonstrate that changes in regional cerebral glucose utilization occur independently of blood flow changes and exclude the possibility that regional changes in glucose utilization occur as a consequence of large regional changes in protein synthesis rates in brain. These data confirm the utility of 2-deoxyglucose measures of MW as objective biochemical indices of opiate agonist and antagonist effects in vivo.     

Venus flytrap biomechanics: Forces in the Dionaea muscipula trap

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the “escaping” force which is very strong and can reach up to 4 N.     

Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

Regional cerebral glucose utilization in withdrawal following systemic and intracerebroventricular sufentanil administration

Regional cerebral glucose utilization (RCGU) and behavior were studied during naloxone-precipitated withdrawal in rats after chronic intravenous (IV) or intracerebroventricular (ICV) administration of sufentanil citrate, a potent, highly selective mu opiate agonist. Changes in RCGU were indistinguishable between the two groups (p<0.05) in 21 of 24 anatomically related limbic and brainstem structures known to be activated during withdrawal. Rats made dependent by ICV infusions of sufentanil had smaller RCGU changes in the lateral septal areas, lateral habenular nuclei and paratenial nuclei than rats made dependent by IV infusions of sufentanil. These observations are consistent with infusion artifact, given the proximity of these structures to the site of IVC infusion. All 24 structures had increased RCGU in experimental groups compared with controls (p<0.05). Although linear regression analysis suggests slightly greater RCGU changes in rats after IV sufentanil than in rats after ICV sufentanil (m=0.81), the changes in corresponding structures are highly correlated (r=0.96) indicating qualitatively almost identical RCGU changes. Behavioral changes paralleled RCGU changes and revealed slightly greater withdrawal in rats after IV sufentanil but no clear qualitative differences. Taken together, these results suggest that cerebral metabolic changes in withdrawal following chronic sufentanil administration result exclusively from effects at CNS opiate receptors and not from peripheral receptors. Additionally, the current study provides a model for the production of opiate dependence, by the ICV administration of a specific mu opiate receptor agonist that is relatively free of infusion artifact.