Nephrogenic diabetes insipidus, which can be inherited or acquired, is characterized by an inability to concentrate urine despite normal or elevated plasma concentrations of the antidiuretic hormone arginine vasopressin. Polyuria, with hyposthenuria, and polydipsia are the cardinal clinical manifestations of the disease. About 90% of patients with congenital nephrogenic diabetes insipidus are males with the X-linked recessive form of the disease (OMIM 304800) who have mutations in the arginine vasopressin receptor 2 gene (AVPR2), which codes for the vasopressin V2 receptor. The gene is located in chromosomal region Xq28. In <10% of the families studied, congenital nephrogenic diabetes insipidus has an autosomal-recessive or autosomal-dominant (OMIM 222000 and 125800, respectively) mode of inheritance. Mutations have been identified in the aquaporin-2 gene (AQP2), which is located in chromosome region 12q13 and codes for the vasopressin-sensitive water channel. When studied in vitro, most AVPR2 mutations result in receptors that are trapped intracellularly and are unable to reach the plasma membrane. A few mutant receptors reach the cell surface but are unable to bind arginine vasopressin or to properly trigger an intracellular cyclic AMP signal. Similarly, aquaporin-2 mutant proteins are misrouted and cannot be expressed at the luminal membrane. Chemical or pharmacological chaperones have been found to reverse the intracellular retention of aquaporin-2 and arginine vasopressin receptor 2 mutant proteins. Because many hereditary diseases stem from the intracellular retention of otherwise functional proteins, this mechanism may offer a new therapeutic approach to the treatment of those diseases that result from errors in protein kinesis.

Nephrogenic diabetes insipidus.

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible. The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTA-opened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

https://rupress.org/jcb/article-pdf/87/3/746/1389175/746.pdf

Occluding junctions and cytoskeletal components in a cultured transporting epithelium

Cytoplasmic microtubules and their functions

It is now well established that the action of antidiuretic hormone (ADH) is mediated by cAMP and that ADH induces physiological changes in the luminal cell Less certain at this stage are the events that link an elevation of intracellular cAMP to an increase in luminal membrane permeability to water and solutes. In recent years, three observations on the toad bladder have provided important insights into these post-CAMP events: (1) The demonstration that by a variety of maneuvers the changes in water permeability can be dissociated from changes in solute Permeability and therefore that separate pathways are involved.5-7 (2) The observations demonstrating that microtubules and microfilaments are involved in the water permeability response.8-10 (3 ) The finding that intramembrane particle aggregates visualized by freeze-fracture electron microscopy are associated with the modulation of luminal-membrane water pem1eabi1ity.ll-l~ Observations reported by many laboratories clearly demonstrate that these organized membrane structures perform an important role in the action of ADH.ll-la Most workers in the field now strongly suspect that these structures are the site of transmembrane water channels. The evidence that aggregates play a role in the ADH response has been discussed in several review papers.l73 Here we will describe how freeze-fracture observations have led to a new proposal for the mechanism of action of ADH and discuss to what extent this proposal is supported by the available evidence.

ADH Action: Evidence for a Membrane Shuttle Mechanism

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothi...

Actin filaments regulate epithelial Na+ channel activity.

Colchicine, vinblastine, podophyllotoxin, and cytochalasin B inhibit the action of vasopressin and cyclic adenosine monophosphate on osmotic water movement across the toad bladder The findings suggest that microtubules, and possibly microfilaments, play a role in the action of vasopressin, perhaps through involvement in the mechanism of release of secretory material from the bladder epithelial cells

https://science.sciencemag.org/content/sci/181/4097/347.full.pdf

Vasopressin: Possible Role of Microtubules and Microfilaments in Its Action

Vasopressin (antidiuretic hormone) has two major physiological actions. Vasopressin induces the contraction or relaxation of certain types of smooth muscle and, in addition, promotes the movement of water (and in some instances of sodium and urea) across responsive epithelial tissues, most notably, the distal tubule of the mammalian kidney and amphibian urinary bladder and skin.’ The action of vasopressin on water movement in epithelial tissues has been extensively investigated; however, the cellular mechanisms involved in this action of the hormone are still to a large extent unknown.z The hormone appears to promote the osmotic movement of water across responsive cells by inducing an increase in the permeability of the membrane at their apical surface 3-6 (FIGURE 1); the permeability change is thought to result from alteration in the size 3, or number of aqueous channels in this rate-limiting barrier.? Under physiological conditions, the effect on the apical membrane is elicited only when vasopressin is present at the basal (i.e. the opposite) surface of the epithelial It is generally accepted that the interaction of the hormone with specific receptors in the basal cell membrane leads to activation of adenyl cyclase and generation of cyclic 3’,5’-adenosine monophosphate (CAMP). Vasopressin has been shown to stimulate specific membrane-bound adenyl cyclase systems 9, l o and to induce an increase in intracellular cAMP levels in target tissues,11* and cAMP mimics the effects of the hormone in these tissues.*? l4 The cellular events that intervene between the generation of cAMP at the level of the basal cell membrane and the increase in permeability of the apical membrane have not been defined. Cyclic AMP-dependent protein kinases have been described in amphibian bladder15 and renal medulla,l6 and a vasopressinand CAMP-dependent phosphoprotein phosphatase has been reported in toad bladder; l7 however the functional role of these enzymes in the response to the hormone is not known. Economy of mechanism at the cellular and molecular level appears, increasingly, to be a characteristic of biological systems. The possibility that the apparently discrete actions of vasopressin on smooth muscle contraction and transcellular water movement involve some common or analogous mechanism( s ) 5 ,

Part XII. Michotubules and the Secretory Pocess: EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE ACTION OF VASOPRESSIN*

A conductometric method for measuring micromolar quantities of carbon dioxide.

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 X 10(-6) M; podophyllotoxin, 5 X 10(-7)M; and vinblastine, 1 X 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 tubulin in 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.

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder. I. Functional studies on the effects of antimitotic agents on the response to vasopressin.

SummaryColchicine and other antimitotic agents have been found to inhibit the action of vasopressin and cyclic AMP on transcellular water movement in the toad bladder; functional and biochemical studies suggest that the effect of these agents is due to interference with microtubule function. To further assess this hypothesis, a quantitative ultrastructural analysis of the content and distribution of microtubules was performed on epithelial cells of bladders exposed to colchicine, vasopressin, and cyclic AMP. The content (volume density) of microtubules was estimated by a point-counting stereological technique. The results indicate that the content of assembled microtubules in the granular epithelial cells is reduced in hemibladders exposed to colchicine; this effect is dose-dependent and is estimated to be half-maximal at a colchicine concentration of 1.4×10−6m. In contrast, the content of assembled microtubules in the granular cells is slightly (∼30%) but significantly increased after exposure of hemibladders to vasopressin (100 mU/ml) or cyclic AMP (10mm). The content of microtubules in mitochondria-rich cells was not found to be significantly altered after exposure to vasopressin. The combined results of functional, biochemical, and morphological studies provide evidence that cytoplasmic microtubules in the granular epithelial cells play a critical role in the action of vasopressin on transcellular water movement in the toad bladder. Precisely how microtubules are involved in the action of the hormone remains to be determined.

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder. III. Morphological studies on the content and distribution of microtubules in bladder epithelial cells.

Roy H. Maffly

Papers

Vasopressin: Possible Role of Microtubules and Microfilaments in Its Action

Part XII. Michotubules and the Secretory Pocess: EVIDENCE FOR INVOLVEMENT OF MICROTUBULES IN THE ACTION OF VASOPRESSIN*

A conductometric method for measuring micromolar quantities of carbon dioxide.

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder. I. Functional studies on the effects of antimitotic agents on the response to vasopressin.

Evidence for involvement of microtubules in the action of vasopressin in toad urinary bladder. III. Morphological studies on the content and distribution of microtubules in bladder epithelial cells.