Effect of amiloride on conductance of toad urinary bladder

Summary The transepithelial conductance of toad bladder epithelia and the amplitude of the fluctuations of this conductance caused by the action of the underlying smooth muscle have been further investigated. In particular, amiloride was found to reduce both tissue conductance and its fluctuating component to the same extent. Analysis suggests that the steady-state conductance of the toad urinary bladder may be associated only with the paracellular pathway for ions.     

Mode of action of amiloride in toad urinary bladder

Summary The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (V _t ) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10^–4 m markedly reduced the mucosal membrane potential (V _m ) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, theV _t could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16mm, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation, Na permeability was calculated from the data ofV _t 's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10^–4 m) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride.     

Effects of lidocaine, mepivacaine and prilocaine on the detrusor muscle of the rat urinary bladder

Lidocaine, mepivacaine and prilocaine cause in rats an increase of the frequency of the spontaneous contractions and an increase of the tone on the detrusor muscle of the urinary bladder. These effects are not antagonized by atropine and by fentola mine. On the contrary verapamil and papaverine antagonize these above effects.     

Effects of 2-deoxy-d-glucose,amiloride, vasopressin,and ouabain on active conductance andE Na in the toad bladder

Summary The effects of various agents on active sodium transport were studied in the toad bladder in terms of the equivalent circuit comprising an active conductanceK ^a, an electromotive forceE _Na, and a parallel passive conductanceK ^p. For agents which affectK ^a, but notE _Na orK ^p, the inverse slope of the plot of total conductance against short-circuit currentI ₀ evaluatesE _Na, and the intercept representsK ^p. Studies employing 5×10^–7 m amiloride to depressK ^a indicate a changingE _Na, invalidating the use of the slope technique with this agent. An alternative suitable technique employs 10^–5 m amiloride, which reducesI ₀ reversibly to near zero without effect onK ^p. Despite curvilinearity of the -I₀ plot under these conditions,K ^p may therefore be estimated fairly precisely from the residual conductance. It then becomes possible to follow the dynamic behavior ofK ^a andE _Na (in the absence of 10^–5 m amiloride) by frequent measurements of andI ₀, utilizing the relationshipsK ^a=K-K ^p, andK _Na=I _O/(K-K ^p). 2-deoxy-d-glucose (7.5×10^–3 m) depressedK ^a without affectingE _Na. Amiloride (5×10^–7 m) depressedK ^a and enhancedE _Na. Vasopressin (100 mU/ml) enhancedK ^a markedly and depressedE _Na slightly. Ouabain (10^–4 m) depressed bothK ^a andE _Na. All of the above effects were noted promptly;K ^p was unaffected. The electromotive force of Na transportE _Na appears not to be a pure energetic parameter, but to reflect kinetic factors as well, in accordance with thermodynamic considerations.     

Effects of caffeine on the intracellular distribution of calcium in frog sartorius muscle

The influence of caffeine on the intracellular distribution of calcium in the frog sartorius muscle was studied by differential centrifugation in an attempt to identify the locus of action of this alkaloid. The problem was approached in two ways. In the first, the locus of action was sought by relating the kinetic functions of ⁴⁵Ca washout curves of muscles to changes in the distribution of ⁴⁵Ca in the isolated fractions from the same muscles. It was not possible to make any correlation of the ⁴⁵Ca-washout curves to the activity in the fractions; the relative distribution of this nuclide remained essentially unchanged at 1-, 2-, and 3-hour intervals along the curve. The washout curves appear to be the net effect of a complex interaction of the calcium in pools containing both readily exchangeable calcium and calcium which has a slow exchange or turnover rate. The second approach centered upon the examination of the effect of caffeine on the intracellular distribution of ⁴⁵Ca and of calcium among the cellular fractions. Caffeine treatment resulted in a distinct increase in the calcium content of the mitochondrial fraction and a decrease in the calcium of the microsomal fraction. Electron micrographic studies revealed significant morphological changes in the whole muscle and in the isolated mitochondrial fraction after the muscle had been exposed to caffeine in a concentration producing irreversible contracture or rigor (10 mM). The increase in calcium content of the mitochondrial fraction after caffeine treatment may be due to an actual accumulation of calcium by the mitochondria or may be the consequence of the appearance of granular vesicles in the fraction.     

Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder

Summary A simple model of the action of amiloride to block apical Na channels in the toad urinary bladder was tested. According to the model, the positively charged form of the drug binds to a site in the lumen of the channel within the electric field of the membrane. In agreement with the predictions of the model: (1) The voltage dependence of amiloride block was consistent with the assumption of a single amiloride binding site, at which about 15% of the transmembrane voltage is sensed, over a voltage range of ±160 mV. (2) The time course of the development of voltage dependence was consistent with that predicted from the rate constants for amiloride binding previously determined. (3) The ability of organic cations to mimic the action of amiloride showed a size dependence implying a restriction of access to the binding site, with an effective diameter of about 5 angstroms. In a fourth test, divalent cations (Ca, Mg, Ba and Sr) were found to block Na channels with a complex voltage dependence, suggesting that these ions interact with two or more sites. at least one of which may be within the lumen of the pore.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

Effects of melittin on a model renal epithelium, the toad urinary bladder

The bee venom melittin, 10(-6) M, on the mucosal (urinary) side of the toad urinary bladder (in vitro), markedly decreased transepithelial potential difference, short-circuit current (Isc, sodium-dependent) and resistance. However, these effects were not seen when the toxin was placed on the opposite (serosal) side of the membrane preparation. The electrical effects were accompanied by a large increase in the transepithelial permeability to 22Na. The response was not changed by meclofenamic acid (which blocks formation of prostaglandins) but it was inhibited by La3+. In the presence of amiloride, which usually inhibits active Na transport and Isc, melittin, on the mucosal side, increased the Isc. The action of melittin appears to involve an interaction with anionic sites, which mediate its effects. Such sites appear to be present on the apical plasma membranes of the toad bladder epithelial cells, but they are not as abundant or they are inaccessible on the basal plasma membrane.     

Effects of step changes in pH on isometric tetanic tension of toad sartorius muscle

The effect of a rapid change in pHe (pH of bathing solution) on the isometric tetanic tension developed by sartorius muscles of toads acclimated to 5 and 25 degrees C was measured at 5 and 25 degrees C. The pH was altered by changing the carbon dioxide concentration of a bicarbonate buffered physiological solution. Acclimation temperature did not modify the response to a rapid change in pH, but test temperature did. Following a pH decrease from 9.0 to 6.0, tetanic tension decreased at a faster rate at 5 degrees C than at 25 degrees C. A new steady state was reached in 15 min at 5 degrees C but in 40 min at 25 degrees C. Following a pH increase from 6.0 to 8.5, tetanic tension increased at a faster rate at 25 degrees C than at 5 degrees C. A new steady state was reached in 60 min at 5 degrees C but in 10 min at 25 degrees C. We conclude that the rate of carbon dioxide diffusion through the sartorius muscle is only one factor that determines how rapidly tetanic tension changes following the step change in pH, and that muscle resists pH change more effectively at higher temperatures.     

Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle

Bladder smooth muscle contraction is mediated by both direct calcium entry through the cell membrane, and by calcium induced calcium release (CICR) from the sarcoplasmic reticulum (SR) storage sites. Ryanodine is a neutral plant alkaloid which binds to an ion channel located on the SR membrane. Its effects in cardiac skeletal muscle are well characterized where it inibits the efflux of intracellular calcium stores, and thus it serves as a negative inotrope. It has also been shown that in the develpping rabbit myocardium, there is a gradual increase in the expression of this ion channel. Little has been written about the expression and function of the ryanodine sensitive ion channel in smooth muscle. Recently we have shown that neonatal rabbit bladder smooth muscle is not very sensitive to ryanodine, while that from mature rabbits is extremely sensitive. This leads us to quantify the expression of the ryanodine sensitive ion channel. In this paper we demonstrate that the Kd values do not change to any significant degree with normal rabbit bladder development. However the Bmax values for 3 day, 2, 4, 6, and 8 week rabbit bladder smooth muscle are 7, 10, 15, 29, and 44 fmol specifically bound ryanodine/mg protein. The differences between the neonatal groups and the mature groups are significant (P<0.5). This increase in ryanodine sensitive ion channel expression with normal growth would suggest that with normal maturation, the bladder smooth muscle cell acquires an increased pool of sequestrered intracellular calcium. This would follow a similar pattern of development that has already been described in rabbit myocardium.