Mammalian urinary bladder permeability is altered by cationic proteins: modulation by divalent cations.

It was previously demonstrated that protamine sulfate (PS, a cationic polypeptide) as well as synthetic cationic polypeptides (CpP, e.g., polylysine and polyarginine) caused an increase in the apical membrane conductance of the mammalian urinary bladder epithelium that was voltage dependent. The membrane conductance induced by these CpP was mediated by a saturable binding site and was partially blocked by CpP (self-inhibition). The PS-induced membrane conductance can be modified by polyvalent cations at three sites. The first site was to competitively inhibit the interaction of PS with an apical membrane binding site. The second site was to reversibly block the conductance induced by PS. The relative binding affinity (block of PS-induced conductance) sequence was as follows: UO2(2+) > La3+ > Mn2+ > Ba2+ > or = Ca2+ > Sr2+. Although La3+, Mn2+, Ba2+, Ca2+, and Sr2+ inhibited > or = 81% of the PS-induced conductance, UO2(2+) inhibited only 51% and Mg2+ was without effect. The third site was to increase the rate of loss of the PS-induced conductance from the apical membrane. Although neither carbodiimides (carboxyl group reactive reagents) nor neuraminidase (cleaves sialic acid residues) altered the effect of PS on the urinary bladder conductance, PS increased the conductance of lipid bilayers composed of negatively charged phospholipids. A candidate for the binding site might be the negatively charged phosphate groups of membrane lipids.

Modification of epithelial permeability by cationic polypeptides.

It has been demonstrated that protamine sulfate (PS; a cationic polypeptide composed of 70% arginine) increases the apical membrane conductance of the mammalian urinary bladder. In this report, synthetic cationic polypeptides (CpP; e.g., polyarginine) were used to determine whether the response of the bladder to PS was due to its cationic nature (i.e., its arginine content). We demonstrate that CpP induce a large increase in the cation and anion conductance of the apical membrane of the rabbit urinary bladder epithelium. The modulation of the membrane conductance by CpP is dependent upon a number of parameters. 1) The magnitude of the conductance change was voltage dependent. 2) An increase in the total charge per molecule increased the rate of conductance change. 3) An increase in the charge density (ratio of charged amino acids to total amino acids) increased the rate of change of conductance. 4) La3+ inhibited the ability of CpP to alter the membrane conductance. 5) The rate of reversal of the CpP-induced conductance was dependent upon the total charge per molecule as well as the charge density. 6) The level of self-inhibition (ability of solution CpP to inhibit the CpP-induced membrane conductance) was inversely correlated with the charge density and was also concentration dependent, with less inhibition occurring at low mucosal CpP concentrations. These data are consistent with a model developed to describe the effect of PS on the conductive properties of the urinary bladder epithelium.

Effect of protamine sulfate on the permeability properties of the mammalian urinary bladder.

Protamine sulfate (PS, an arginine-rich protein of molecular weight 5,000) has been reported to affect the ionic permeability of gallbladder epithelium, the permeability of cultured epithelial cells to mannitol, and the permeability of endothelial cell layers to albumin. Although the effect of PS has been widely investigated, the mechanism of its action on membrane permeability is presently unknown. The effect of PS on the rabbit urinary bladder epithelium was studied using both transepithelial and intracellular microelectrode techniques in conjunction with equivalent circuit analysis. The addition of 100 micrograms/ml of PS to a NaCl-containing mucosal solution caused (over a 40-min period) a large increase in the transepithelial conductance (Gt) and a transient hyperpolarization of the transepithelial voltage (Vt) followed by a depolarization of Vt. This secondary depolarization of Vt was not present if the mucosal solution was a KCl or a K-gluconate Ringer. The PS-induced increase in Gt was due to an increase in the apical membrane permeability to both cations (Na+ or K+) and anions (Cl- or gluconate). Further studies revealed the following features of the PS-induced conductance. (i) Trypsin inhibits the PS effect; however, this was due to PS hydrolysis by trypsin and not a membrane effect. (ii) Mucosal PS partially inhibited the PS-induced apical membrane conductance. (iii) The ability of PS to increase the membrane conductance was enhanced when the apical membrane potential was cell interior negative. (iv) The rate of conductance change (at any given membrane potential) was a saturating function of the PS concentration. This finding suggests that PS must interact with a membrane binding site before it can induce a change in the membrane conductance. (v) Lanthanum inhibited the PS-dependent conductance by two different mechanisms. One was as a reversible blocker of the PS-induced conductance. The other was by inhibiting the interaction between PS and a membrane binding site. A kinetic model is developed to describe the steps involved in the increase in membrane conductance.