Reserve of vasopressin-sensitive adenylate cyclase in toad urinary bladder

Studies have been performed on the effect of vasopressin on cyclic AMP content of toad bladders. A prompt increase in cyclic AMP content occurred after exposure to vasopressin, which reached maximal values within 8 min and remained elevated up to 30 min. By a comparison of the dose-response characteristics of vasopressin on cyclic AMP content, with those Na⁺ transport and osmotic water flow, it was shown that supramaximal concentrations of vasopressin with respect to physiological function generate more cyclic AMP than is required for maximal stimulation of Na⁺ transport and water flow. Thus, it would seem that a reverse of hormone-sensitive adenylate cyclase is present in this tissue.     

Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.     

The effects of vasopressin and catecholamines on cyclic AMP in homogenates of rat brain

Vasopressin is known to mediate its action on the kidney through increasing the concentrations of cyclic AMP. As vasopressin is widely distributed in many extra hypothalamic areas of the brain and can be shown to act centrally, we have investigated the effect of vasopressin on cyclic AMP levels in homogenates of the striatal and locus coeruleus areas. In contrast with the effect obtained on the kidney, vasopressin did not stimulate adenyl cyclase activity in rat brain homogenates in a dose-related manner. The stimulation of cyclic AMP observed with dopamine or noradrenaline in these brain areas and the hippocampus was not affected by the presence of vasopressin. These observations suggest that the action of vasopressin on the brain is not mediated through cyclic AMP.     

Effects of vasopressin on electrolyte transport in lizard intestine

Summary Vasopressin applied serosally had no effect on electrical parameters and unidirectional Na and Cl fluxes across anin vitro short circuited preparation of lizard ileum. Short circuit current (Isc) and transmural potential difference (PD) across colon were decreased by vasopressin and increased by cyclic AMP. Vasopressin increased the mucosal-to-serosal flux of sodium and chloride across short circuited colon. Cyclic AMP had no effect on the rate of Na absorption but reversed Cl absorption to secretion. Vasopressin enhanced the net absorption of water across the colon but had no effect on absorption across ileum. Cyclic AMP activity in homogenates of colon was not altered by vasopressin but was increased by theophylline. It is concluded that the colonic response of the lizard colon to vasopressin is mediated by a noncyclic AMP mechanism.     

The effect of turpentine-induced inflammation on rat liver glycosyltransferases and Golgi complex ultrastructure

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.     

Alterations in lactoperoxidase-catalyzed radio-iodination of membrane proteins associated with vasopressin-induced changes in tissue permeability to water.

Lactoperoxidase-catalyzed radio-iodination was used to study the effects of vasopressin upon the luminal membrane of the toad's urinary bladder. Iodination of several proteins of this membrane, which represents the primary barrier to the flux of water, was increased by exposing the tissue to vasopressin and cyclic AMP. This effect was inhibited by colchicine, which reduces the effects of vasopressin upon water but not sodium flux; aldosterone, which stimulates sodium transport but not water flux, had no effect on tissue labeling under these conditions. The results provide evidence of hormone-induced alterations in the molecular conformation of membrane proteins related to hormone-regulated membrane permability to water and indicate this technique may be used to identify and isolate those proteins mediating the hormone response.     

Membrane pathways for water and solutes in the toad bladder: I. Independent activation of water and urea transport

Summary Vasopressin activates a number of transport systems in the toad bladder, including the systems for water, urea, sodium, and other small solutes. Evidence from experiments with selective inhibitors indicates that these transport systems are to a large extent functionally independent. In the present study, we show that the transport systems can be separately activated. Low concentrations of vasopressin (1 mU/ml) activate urea transport with virtually no effect on water transport. This selective effect is due in part to the relatively greater inhibitory action of endogenous prostaglandins on water transport. Low concentrations of 8-bromoadenosine cyclic AMP, on the other hand, activate water, but not urea transport. In additional experiments, we found that varying the ratio of exogenous cyclic AMP to theophylline activated water or urea transport selectively. These studies support the concept of independently controlled systems for water and solute transport, and provide a basis for the study of individual luminal membrane pathways for water and solutes in the accompanying paper.     

Regulation of Cyclic AMP Accumulation by Peptide Hormone Receptors in Immunocytochemically: Defined Astroglial Cells

Primary cultures of neonatal murine brain have been reported to express multiple receptors that regulate adenylate cyclase activity. Since for the most part these results were obtained with mixed cell cultures, it has been difficult to define receptor profiles for specific cell types. With this concern in mind a series of studies has been initiated designed to identify specific receptors present on highly purified, immunocytochemically defined astroglia derived from the cerebral cortices of neonatal rats. In this study the capacity of a variety of peptide hormones to regulate cyclic AMP metabolism in these cells was examined. Fibroblasts derived from the meninges represent a predictable source of contamination in primary CNS culture. Thus, to assign more clearly specific receptors to the astroglial cell population, receptor-mediated regulation of cyclic AMP accumulation was also examined in fibroblasts. Cyclic AMP accumulation in astroglia was stimulated by catecholamines (acting at beta 1-adrenergic receptors), prostaglandin E1, vasoactive intestinal polypeptide, alpha-melanocyte-stimulating hormone, and adrenocorticotropin. Bombesin, luteinizing hormone-releasing hormone, neurotensin, thyrotropin-releasing hormone, somatostatin, secretin, and vasopressin did not significantly increase cyclic AMP levels in these cultures. Catecholamines, acting at alpha 2-adrenergic receptors, and somatostatin inhibited agonist-stimulated cyclic AMP accumulation. In meningeal cell cultures catecholamines (acting at beta 2- and alpha 2-adrenergic receptors) and prostaglandin E1 regulated cyclic AMP levels. However, vasoactive intestinal peptide did not stimulate and somatostatin did not inhibit cyclic AMP accumulation in these cells.     

Anterior falcate artery in adults: histology of the relation of superior sagittal sinus, dural vein and the arachnoid granulations]

The anterior falcate artery, which is the continuation of the anterior ethmoidal, supplies the dura mater in the region of the superior sagittal sinus as far almost as the coronal suture. Graphic reconstructions show its relationships to the veins, the sinus, and the arachnoid granulations. Histological studies of the artery emphasize its adaptation to longitudinal stretching. The complexity of its relationships suggests a functional significance beyond that of nourishing the dura.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Activation of cyclic AMP-dependent protein kinase A common step in vasopressin- and hypertonicity-induced water flow

Water flow across the amphibian urinary bladder can be induced by either vasopressin or serosal hypertonicity. In an effort to determine the common intracellular steps mediating both responses, we determined the in situ activation of cyclic AMP-dependent protein kinase in bladders stimulated by vasopressin or hypertonicity. Treatment of bladders with vasopressin (1 mU/ml) caused in situ activation of cytosolic cyclic AMP-dependent protein kinase of epithelial cells, with a rise in the kinase ratio and cyclic AMP content. Similarly, hyperonicity increased the kinase ratio, but this occured without a measurable increase in cyclic AMP content per mg protein. Because of the hypertonicity-induced cell shrinkage, epithelial cell water decreased by 20%, which may result in a proportionate increase in cyclic AMP concentration (per ml cell water). Furthermore, cell shrinkage also increases intracellular electrolyte concentration, which, in turn, should delay reassociation and consequent inactivation of the predominant Type II cyclic AMP-dependent protein kinase of the epithelial cells. Thus activation of cyclic AMP-dependent protein kinase during hypetonicity may be the result of cell shrinkage, with an associated increase in cyclic AMP and electrolyte concentrations. Studies with prostaglandin synthesis inhibitors and colchicine, a microtubule disrupting agent, also indicated common pathways for vasopressin and hypertonicity. Both naproxen and meclofenamate significantly enhanced the hypertonicity response. Colchicine pretreatment, on the other hand, caused a small (18%) but significant inhibition of the hypertnicity response, similar to its effect on the vasopressine response (25% inhibition). Thus, the increased water permeability of the toad bladder in response to both vasopressin and hypertonicity follows a similar pathway. Activation of cyclic AMP-dependent protein kinase represents the first common step yet identified.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi.

The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.     

The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by vasopressin and the inhibition of this mitogenic action by thyrocalcitonin

Lysine vasopressin (antidiuretic hormone), like cyclic adenosine 3',5'-monophosphate (cyclic AMP), rapidly (in less than 1 hour) stimulates the initiation of deoxyribonucleic acid synthesis and thereby increases the flow of cells into mitosis in rat thymic lymphocyte populations in vitro. This mitogenic action of vasopressin, again like that of cyclic AMP, is potentiated by caffeine, an inhibitor of the intracellular phosphodiesterase which catalyzes the degradation of cyclic AMP. On the other hand, vasopressin's mitogenic action (also like that of cyclic AMP) is blocked by imidazole, an activator of cyclic nucleotide phosphodiesterase activity. The hormone, thyrocalcitonin (calcitonin) which is known to block the cyclic AMP-mediated mitogenic effect of parathyroid hormone by interfering with cyclic AMP action, also blocks the mitogenic action of vasopressin. The inhibitory effects of imidazole and thyrocalcitonin on vasopressin's mitogenic action are both overcome by the phosphodiesterase inhibitor, caffeine. It is concluded from these observations that the mitogenic action of vasopressin is mediated by cyclic AMP.     

Inhibition of stimulated osmotic water flow by fluphenazine, a calmodulin inhibitor, in the isolated toad skin

The effects of fluphenazine, a phenothiazine calmodulin inhibitor, on the osmotic water flow (Posm) across isolated skins of Bufo arenarum toads were tested. Fluphenazine inhibited the increase in Posm induced by agents known to act through the cyclic AMP system (oxytocin, db-cAMP, theophylline, KCl and isoproterenol). The inhibitory effect was faster and more intense when the drug was present in the epidermal bath, and persisted after rinsing the preparation for 100 min. Our results indicate that phenothiazine's action may be primarily exerted at a site distal to cyclic AMP generation.     

Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production.

Isolated rat hepatocytes in primary monolayer culture were maintained for 18-24 h in the presence of 10% (v/v) serum and [3H]inositol. Vasopressin (100 nM) stimulated the production of inositol mono-, bis- and tris-phosphates (IP1, IP2, and IP3). Prior exposure of hepatocytes to 8-bromo cyclic AMP (8Br-cAMP; 100 microM), but not 8-bromo cyclic GMP, enhanced the vasopressin-mediated stimulation of inositol phosphate accumulation, but had no significant effect on their formation in the absence of vasopressin. The effect of the cyclic AMP analogue was mimicked by glucagon (10 nM), and was seen whether cyclic AMP or glucagon was added 5 min or 12 h before the addition of vasopressin. An 8 h incubation with dexamethasone (100 nM) enhanced the accumulation of IP3, but not that of IP2 or IP1, in the presence of 8Br-cAMP and vasopressin. Cycloheximide or actinomycin D had little effect on the vasopressin stimulation of inositol phosphate accumulation, after an 8 h incubation in the presence or absence of 8Br-cAMP.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.     

The superficial cerebral veins

Estimated were the number, the course, and the width of the superficial cerebral veins. The veins on the superolateral surface of the brain are the prefrontal superficial lateral superior, the precentral superficial lateral superior, the central superficial lateral superior, the parietal superficial lateral superior, and the occipital superficial lateral superior veins which drain to the superior sagittal sinus, to bridging veins, and to the falx cerebri. The veins which drain the lateral surface of the brain downwards are the middle superficial cerebral veins, the temporal inferior, occipital inferior, and anastomotic veins. The diameters of these veins were measured at the perforation of the arachnoid membrane and the diameters of the anastomotic veins on their narrowest area. On the medial side of the hemispheres, we divided in precentral superficial superior medial, central superficial medial, parietal superficial medial, occipital superficial medial dorsal veins of the corpus callosum, and internal occipital veins. On the basal surface of the hemispheres, we studied and described the uncal veins and the inferior hemispheric veins. Studied and discussed are also the bridging veins in the course of the inferior cerebral veins, the paracavernous sinuses, and the last course of the veins and their connections with the dura mater or the course inside the dura. Given are besides the numbers of these veins, the area of perforation of the arachnoid membrane, and their width and medical importance.     

Effect of H-7 on the modulation of glucagon actions by activators of protein kinase-C

The stimulations of ureagenesis and cyclic AMP accumulation induced by glucagon were inhibited by 10 nM vasopressin or 100 nM phorbol 12-myristate 13-acetate (PMA). The maximal accumulation of cyclic AMP induced by glucagon was clearly diminished by these agents without change in the EC50 for the peptide hormone suggesting a non-competitive type of inhibition. H-7 blocked the inhibition of glucagon-stimulated ureagenesis induced by PMA and vasopressin and diminished their effect on the accumulation of cyclic AMP induced by glucagon. It is concluded that activation of protein kinase C inhibits the stimulation of ureagenesis and the accumulation of cyclic AMP induced by glucagon in liver cells from hypothyroid rats; H-7 inhibits the effects of protein kinase C activation.     

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder

Summary The antimitotic agents colchicine, podophyllotoxin, and vinblastine inhibit the action of vasopressin and cyclic AMP on osmotic water movement in the toad urinary bladder. The alkaloids have no effect on either basal or vasopressin-stimulated sodium transport or urea flux across the tissue. Inhibition of vasopressin-induced water movement is half-maximal at the following alkaloid concentrations: colchicine, 1.8×10^–6 m; podophyllotoxin, 5×10^–7 m; and vinblastine, 1×10^–7 m. The characteristics of the specificity, time-dependence and temperature-dependence of the inhibitory effect of colchicine are similar to the characteristics of the interaction of this drug with tubulinin vitro, and they differ from those of its effect on nucleoside transport. Inhibition of the vasopressin response by colchicine, podophyllotoxin, and vinblastine is not readily reversed. The findings support the view that the inhibition of vasopressin-induced water movement by the antimitotic agents is due to the interaction of these agents with tubulin and consequent interference with microtubule integrity and function. Taken together with the results of biochemical and morphological studies, the findings provide evidence that cytoplasmic microtubules play a critical role in the action of vasopressin on transcellular water movement in the toad bladder.