Joseph R. Sherwin

Iodide Induced Suppression of Thyrotropin-Stimulated Adenosine 3´,5´-Monophosphate Production in Cat Thyroid Slices

Cat thyroid slices were studied to investigate their responsiveness to thyrotropin stimulation of cyclic AMP accumulation. Ovine and bovine thyrotropin, in the presence of 2.5 mM aminophylline, induced a dose-dependent increase in the cyclic AMP content of cat thyroid tissue. Half-maximal stimulation of cyclic AMP accumulation was obtained at a thyrotropin concentration of 1-2 mU/ml. The maximal effect of thyrotropin was observed at 10 mU/ml, and was associated with a mean 77 +/- 19-fold increase in thyroidal cyclic AMP. Preincubation of cat thyroid tissue for 2 h with 50 micron NaI resulted in an impairment in the subsequent ability of thyrotropin to enhance cyclic AMP accumulation, without altering the level of cyclic AMP in tissues not exposed to the hormone. Preincubation alone was without effect on thyrotropin stimulation of cyclic AMP, and the inhibitory effect of iodide was prevented by addition of 3 mM methimazole to the preincubation medium. In addition, the time course of thytrotropin stimulation of cyclic AMP accumulation in cat thyroid slices was not significantly altered by the preincubation with excess iodide. These studies provide additional evidence that excess iodide inhibits the adenylate cyclase-cyclic AMP system in thyroid tissue.

Effects of prostaglandins on distribution of blood flow in the cat.

Abstract Part of the variability of the vascular effect of prostaglandins may be due to differences in techniques of measuring flow in only one organ at a time in addition to not measuring flow under steady state hemodynamic conditions. Using anesthetized cats, PGE 1 , PGE 2 , PGF 2α or PGI 2 (prostacyclin) or their vehicle was infused (i.e., μg/kg/min for PGE l , PGE 2 and PGF 2α ; 0.175 μg/kg/min for PGI 2 ) for 30 minutes. Following the 30 min infusion period, blood flow measurements as determined by radioactive labeled microspheres (15 ±3 μ diameter) were simultaneously measured for heart, lung, kidney, adrenal gland, skeletal muscle, stomach, jejunum, pancreas, spleen and liver. PGE 1 significantly increased gastric blood flow, PGE 2 increased cardiac output, lung, and gastric blood flow, and PGF2a increased skeletal muscle and gastric blood flow. Prostacyclin increased cardiac output, skeletal muscle, gastric, jejunal and pancreatic blood flow. These results indicate that these prostaglandins appear to exert vasodilator effects in vascular beds in which they have vasoactivity. Moreover, prostacyclin is more potent and exerts a broader spectrum vasodilator action than either PGE 1 , PGE 2 or PGF 2α . These properties of PGI 2 may ultimately prove beneficial in the therapeutics of hemorrhagic shock where splanchnic flow is severely compromised.

Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells

Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma x glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10(-8) M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10(-10) M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10(-8) or 10(-10) M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10(-7)-10(-10) M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.

Tetanus toxin inhibits neurotensin-induced mobilization of cytosolic protein kinase C activity in NG-108 cells

There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma x glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10(-10)-10(-13) M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 microM neurotensin. Pretreatment with 10(-10) M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10(-11) M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.

Cellular responses to acute injury: The role of the plasma membrane in cell response to two clostridial toxins

The plasma membrane plays an important role in the pathogenesis of acute cell injury. This brief review outlines the role of the plasma membrane in the cellular response to two clostridial toxins, the botulinum C2 toxin and the tetanus toxin. These two toxins belong to the same family of toxins as botulinum toxin type A and type F, those used clinically for the treatment of facial spasm. The actions of C2 toxin on cultured cells give rise to an acute injury characterized by a dissociation of the actin filaments of the cell cytoskeleton. While this toxin can be lethal to intact organisms, the acute cellular response need not necessarily result in cell death. In the case of tetanus toxin, the toxin appears to perturb the plasma membrane so that the function of one important cell second messenger system, protein kinase C, is altered.

Tetanus Toxin and Protein Kinase C

The pathogenesis of tetanus toxin poisoning is characterized by an inhibition of neurotransmitter release in the central nervous system; however, the exact mechanism through which the toxin inhibits exocytosis is not yet understood. A suitable neuronal model in which to study the biochemical effects of tetanus toxin on secretory processes is the differentiated neuroblastoma x glioma hybrid cell line NG-108.1 We have recently reported that tetanus toxin pretreatment of differentiated NG-108 cells attenuated the ability of phorbol myristate acetate (PMA) and neurotensin to mobilize cytosolic protein kinase C (PKC) to the plasma membrane.2,3 One implication of these observations is that the ability of tetanus toxin to alter PKC metabolism is related to its ability to inhibit exocytosis. In this study we have used a permeabilized cell/synthetic peptide assay to directly measure membrane PKC activity following tetanus toxin pretreatment. Further, we have used this assay to determine the efficacy of four putative inhibitors of membrane PKC activity; staurosporine and H-7 inhibit the catalytic site of the kinase, calphostin and sphingosine block the regulatory site. Finally, experiments were performed on neuromuscular preparations to test the hypotheses that PKC is required for short-term neuromuscular transmission and that the kinase could be the direct target of tetanus toxin.