J. Mark Braughler

Glucocorticoid mechanisms in acute spinal cord injury: A review and therapeutic rationale

This review seeks to provide pharmacological evidence that intensive glucocorticoid dosing can enhance sensorimotor recovery after blunt spinal cord trauma. It is suggested that high doses of glucocorticoids can beneficially affect the injured cord through the influence of at least three mechanisms. These are: (1) a facilitation of neuronal excitability and impulse conduction; (2) an improved blood flow; and, perhaps most importantly, (3) the preservation of cord ultrastructure through a reduction of injury-induced, free radical--catalyzed lipid peroxidation. In the case of methylprednisolone, the minimal intravenous dosage required to initially achieve each of these effects is in the range of 15 to 30 mg per kilogram of body weight, which is beyond that used currently for neurosurgical purposes. In addition, based upon the hypothesized mechanism of action and the tissue pharmacokinetics, the earliest possible initiation of therapy is imperative and rigorous maintenance dosing for an as yet undetermined length of time is needed.

Novel membrane localized iron chelators as inhibitors of iron-dependent lipid peroxidation.

Attachment of various iron chelating moieties to hydrophobic steroids greatly enhanced their abilities to inhibit iron-dependent lipid peroxidation. Using whole rat brain homogenates, lipid peroxidation initiated by the addition of 200 microM Fe2+ was assessed by the formation of thiobarbituric acid reactive products (TBAR). Under these conditions, 50% inhibitory concentrations of Fe3+ chelators such as desferrioxamine or N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound II) were around 170 and 50 microM respectively. Coupling desferrioxamine or compound II to a steroid at the D ring increased their potency in lipid peroxidation assays by 5- to 10-fold. Evidence that inhibition of lipid peroxidation by the steroid-chelator adducts was due to iron chelation was suggested by the fact that methylation of the catechol oxygens of compound II, which are essential for chelation, completely eliminated activity of the steroid adduct. A series of 21-aminosteroids which complex Fe2+ iron and potently inhibit iron-dependent lipid peroxidation has also been synthesized. Coupling Fe2+ chelators to hydrophobic steroids increased their inhibitory potencies by as much as 10- to 100-fold. Some steroid-based Fe2+ chelators stimulated lipid peroxidation at low concentrations in the presence of Fe3+. The degree of stimulation was related to the affinity of a compound for Fe2+ with the stronger chelators causing greater stimulation. The most potent inhibitors of lipid peroxidation in the 21-aminosteroid series were found to be those compounds forming the weakest Fe2+ complexes. The findings suggest that it is iron at or near the membrane that is responsible for the catalysis of lipid peroxidation. The compounds described should provide useful tools for studies of the involvement of iron in the lipid peroxidation process.

Soluble guanylate cyclase activation by nitric oxide and its reversal: Involvement of sulfhydryl group oxidation and reduction☆

Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.

Acute enhancement of spinal cord synaptosomal (Na+ + K+)-ATPase activity in cats following intravenous methylprednisolone

The acute intravenous administration of high doses of methylprednisolone sodium succinate (15-90 mg/kg) to cats enhanced (Na+ + K+)-ATPase activity as much as 2-fold in synaptosomes prepared from lumbar spinal cord removed following drug administration. The effect was apparent within 5 min and was statistically significant at 1 h following a single injection of methylprednisolone 90 mg/kg. (Na+ + K+)-ATPase activity had returned to control levels by 24 h post-injection. The implication of these findings to the treatment of spinal cord trauma and the reported neurophysiological effects of glucocorticoids are discussed.

Acute Cardiovascular Response to a Single Large Intravenous Dose of Methylprednisolone and Its Effects on the Responses to Norepinephrine and Isoproterenol

Abstract The cardiovascular actions of single 10-μg/kg iv doses of norepinephrine (NE) and isoproterenol (ISO), before and after the administration of a single 30-mg/kg iv dose of the glucocorticoid methylprednisolone (MP) (sodium succinate ester), were compared in adult cats. Methylprednisolone increased both systolic and diastolic pressures as well as the pulse pressure by approximately 50%. These steroid effects persisted unabated for the duration of the experiment (40 min). Heart rate was unaffected by MP. MP treatment significantly reduced the increase in systolic and diastolic pressures caused by NE. This MP effect was unrelated to the higher baselines for these two parameters caused by the steroid. The systolic blood pressure and positive chronotropic effects of ISO were also significantly blunted by MP. These observations suggest that a single large dose of MP may cause a depression of cardiovascular α- and β-1-receptor sensitivity. The results are discussed in relation to the actions of massive dose glucocorticoid administration in certain shock states and central nervous system trauma and stroke.

Sulfhydryl group involvement in the modulation of guanosine 3′,5′-monophosphate metabolism by nitric oxide, norepinephrine, pyruvate and t-butyl hydroperoxide in minced rat lung

The cyclic GMP content of rat lung mice was increased nearly 50-fold within 4 sec following exposure to nitric oxide. This rapid increase in cyclic GMP accumulation was prevented by 10 mM, but not 1 mM, dithiothreitol which itself caused a slower yet massive (100-fold) increase in the cyclic GMP content of lung mince. Tissue cyclic GMP following nitric oxide exposure declined rapidly even in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. The decline in cyclic GMP was accelerated by the thiol oxidant diamide (1 mM). The cyclic GMP content of lung mince was also increased by norepinephrine, pyruvate and t-butyl hydroperoxide. Diamide blocked cyclic GMP accumulation in response to these other agents as well as that caused by nitric oxide or dithiothreitol. The results suggest that sulfhydryl group modification may be a common pathway for the enhancement of cyclic GMP synthesis in tissues by a variety of stimuli.

Oxidative modulation of soluble guanylate cyclase by manganese

Homogeneous or partially purified soluble guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from rat liver exhibited variable sensitivity to assay pH that was dependent upon buffer composition and the cation cofactor. Enzyme activity with 3 mM Mn2+ in excess of Mn2+-GTP was considerably less in Tris buffers above pH 8.0 than in glycine buffer. In the pH range of 6.0-7.6, however, manganese-supported activity was greater in Tris buffers than in imidazole or cacodylate buffers of corresponding pH. The differences in activity seen with various buffers were not apparent when Mg2+ was the sole cation cofactor but were dependent upon Mn2+ concentrations in excess of Mn2+-GTP. The effects of excess Mn2+ on guanylate cyclase varied with assay pH and buffer composition. At pH 7.6 in Tris-HCl buffer, excess Mn2+ increased guanylate cyclase activity with an apparent Ka of 0.25 mM and concentrations above 3 mM were slightly inhibitory. At pH 9.0 in Tris-HCl buffer, however, concentrations of excess Mn2+ above 0.1 mM were strongly inhibitory. By comparison, in cacodylate (pH 7.6) or glycine (pH 9.0) buffers, high concentrations of excess Mn2+ were considerably less inhibitory and the apparent Ka values for excess Mn2+ were greater than in Tris-HCl buffer at equivalent pH. The variable effects of Mn2+ on enzyme activity as a function of buffer pH and composition were qualitatively similar to its effects on catecholamine oxidation. Furthermore, the inhibition of guanylate cyclase by excess Mn2+ was partially prevented by dithiothreitol and the stimulation of enzyme activity by excess cation was completely blocked by the antioxidant hydroquinone. The studies suggest that the apparent requirement and preference of soluble guanylate cyclase for excess Mn2+ as cation cofactor, as well as the inhibition of enzyme activity by excess Mn2+ may be mediated by oxidative events associated with changes in the oxidation state of the free cation.

Dissociation of increases in cyclic GMP from relaxation of arterial smooth muscle.

Glyceryl trinitrate increased the cyclic GMP content of epinephrine-contracted aortic strips from glyceryl trinitrate-tolerant rats in a dose-dependent manner. Glyceryl trinitrate (.05 millimicron/ml) at a concentration that caused to relaxation of aortas from tolerant animals but a 42.4 3.7% relaxation of control aortas, elevated cyclic GMP levels in both groups approximately 3-fold. The results are not consistent with the hypothesis that cyclic GMP is involved in the relaxation of arterial smooth muscle.

Alterations in the subcellular distribution of Guanylate cyclase and its responsiveness to nitric oxide in diethylstilbestrol-induced renal tumors.

The cyclic GMP content of diethylstilbestrol‐induced renal tumors in the male golden hamster was increased nearly 130‐fold over that in kidney from control animals. Cyclic GMP in tumors was 91.80 ± 19.18 pmoles cyclic GMP/mg protein compared to 0.72 ± 0.07 in control kidneys. Cyclic AMP in tumors was also increased over control, however, to a much lesser degree (2.7‐fold). In control kidneys, 84.6% of homogenate guanylate cyclase activity was recovered in the 100,000 × g supernatant fraction. Total homogenate guanylate cyclase activity from diethylstilbestrol‐induced renal tumors was increased 5.5‐fold over that in control kidneys and only 8.1% was associated with the 100,000 × g supernatant fraction. Neither the soluble or particulate guanylate cyclase from renal tumors could be activated by nitric oxide. The unresponsiveness of tumor guanylate cyclase to nitric oxide was independent of the cation cofactor, and not due to a shift in the dose response curve for nitric oxide. Responsiveness to nitric oxide was not restored by thiols, sugars, other proteins, or hemoglobin. Basal cyclic AMP formation by soluble guanylate cyclase from renal tumors was dramatically increased over that observed in control kidneys, and could not be increased further by nitric oxide. This is the first study of cyclic GMP and guanylate cyclase in a primary estrogen‐induced tumor. The possibility that the changes observed in guanylate cyclase from diethylstilbestrol‐induced renal tumors are related to in vivo activation of the enzyme by epoxide metabolites of diethylstilbestrol is discussed. Cancer 50:78–84, 1982.

An isolated perfused dog lung preparation for the study of cyclic GMP metabolism: Effects of sodium nitroprusside and oxygen

The intact, isolated perfused dog lung was evaluated as a model for studies directed at defining the role of oxidative modulation of lung cyclic GMP metabolism in pulmonary function. Sodium nitroprusside added to the perfusion blood increased the cyclic GMP content of lung over 4-fold in a dose-dependent manner. Although sodium nitroprusside administration caused changes in lung vascular resistance, these occurred independently of the changes in cyclic GMP. Ventilation of lungs with a high oxygen gas mixture containing 95% O2. 5% CO2 acutely increased the cyclic GMP content of lungs after 15 min from 1.3 +/- 0.06 (mean +/- SE) to 3.4 +/- 0.12 pmol cyclic GMP/mg protein. Cyclic GMP levels returned toward control during continued ventilation with the high oxygen concentration. The oxygen-induced elevation of lung cyclic GMP content was not accompanied by changes in lung vascular resistance. The results indicate that the isolated perfused lung may be useful in studies of cyclic GMP, tissue oxidation and pulmonary function.