Circadian regulation of Limulus visual functions: A role for octopamine and cAMP

The purpose of this contribution is to review our current understanding of the source and biochemistry of the circadian efferent input to the eyes of the American horseshoe crab Limulus polyphemus and the impact of this input on the structure, physiology and biochemistry of Limulus eyes. Special emphasis is given to the role of the biogenic amine octopamine and biochemical cascades it activates in the eyes. In addition to reviewing published data, we present new data showing that octopamine elevates cAMP levels in Limulus lateral eyes, and we partially characterize the pharmacology of the receptors involved in this response. We also present new data showing that octopamine regulates gene expression in Limulus lateral eyes by activating a cAMP cascade.

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca2+ current ( ICa) and intracellular Ca2+ concentration ([Ca2+]i) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after Gs activation by α,β-tubulin. Colchicine depolymerizes microtubules into α,β-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca2+ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 μM) induced ACh-activated K+ current [ IK(ACh)], which happens via βγ-subunits from the activation of Gi. Carbachol also reduced [Ca2+]i transients and contractions. Once Gi is activated by muscarinic agonist, the αi-subunit is released from the βγ-subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by ICa reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca2+]i transients and contractions than in untreated cells. The αi-subunit became effective in signaling through the AC/cAMP cascade and reduced ICa without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed ICa inhibition by CCh. Stimulation of ICa by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on IK(ACh) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase IK(ACh) by βγ-subunits released from Gs to mask this effect of CCh. Microtubules suppress ICa regulation by αi; their disruption releases restraints that unmask muscarinic inhibition of ICa. Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.

Sphingolipid-Cholesterol Domains (Lipid Rafts) in Normal Human and Dog Thyroid Follicular Cells Are Not Involved in Thyrotropin Receptor Signaling

Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the α- subunit of the heterotrimeric Gs and Gq proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mm methyl-β-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.