Modulation of polymyxin B effects on mammalian urinary bladder.

This report demonstrates that Ca2+, Mg2+, and protons alter the ability of polymyxin B (PX, a cationic antibiotic used clinically as a bactericidal agent) to increase the apical membrane conductance of the rabbit urinary bladder. Using electrophysiological methods, we determine that these alterations occur by two mechanisms. First, they blocked the PX-induced conductance in a rapid and reversible manner; second, they competed with PX for a membrane binding site. In addition, Ca2+ (but not Mg2+ or protons) altered the rate at which the induced conductance could be reversed. When solution pH was greater than 8.8, PX was not able to induce a conductance. This ability of high pH to inhibit the action of PX was due to a decrease in the number of positive charges on PX. Further studies demonstrated that for maximal activity, PX required its fatty acid tail. These data were used to develop a model describing the mechanism by which PX can induce a conductance in the apical membrane of the rabbit urinary bladder.

Effects of polymyxin B on mammalian urinary bladder.

Previous reports have demonstrated that large cationic polypeptides (of molecular mass 5,000 daltons or greater) cause an increase in the apical membrane conductance of the rabbit urinary bladder epithelium. This report investigates the effects of the small cationic molecule polymyxin B (PX: a 1,400 dalton antibiotic) on the permeability of the rabbit urinary bladder. The addition of micromolar concentrations of polymyxin B to the luminal solution of the rabbit urinary bladder resulted in an increase in the transepithelial conductance of the bladder. The magnitude of the increase in the conductance was dependent upon the concentration of PX, and the polarity and magnitude of the apical membrane potential. As the apical membrane potential was made more cell interior negative, the larger was the increase in the membrane conductance. This voltage-dependent increase in conductance was an exponential function of the applied voltage, with a negligible increase in conductance occurring when the membrane potential was cell interior positive. Upon changing the membrane voltage from cell interior positive to negative, there was a delay before there was a measurable change in the membrane conductance. The longer the apical membrane was exposed to PX, the more poorly reversible was its effect on the transepithelial conductance, suggesting a toxic effect of PX on this epithelium.

Modulation of epithelial permeability by extracellular macromolecules.

Epithelia are sheets of cells joined together by tight junctions. This geometry allows an epithelium to act as a barrier, i.e., restrict the movement of substances between two compartments that it separates (typically 1 compartment is the blood) and also to actively and selectively transport substances between the two compartments. It has been known for a number of years that both the barrier and transport functions of epithelia can be regulated by hormones and neurotransmitters, and this regulation is a central component of plasma electrolyte and nonelectrolyte homeostasis. Less appreciated is that these epithelial functions can be modified by macromolecules other than neurotransmitters and hormones. These macromolecules have been divided into the following categories: proteases, cytokines, cellular constituents, nonbacterial xenobiotics, and bacterial xenobiotics. Such macromolecules can alter epithelial transport and barrier function by a number of different mechanisms. These include proteolysis of epithelial ion channels and tight junctional complexes, conversion of an ion pump into a nonselective cation channel, increase in epithelial membrane permeability resulting in cell swelling and lysis, and up- or downregulation of cellular second messenger systems that can alter ion transport capabilities or prove cytotoxic to the cells. Finally, these modifications can be either transient or chronic in nature and in many circumstances result in a perturbation of the electrolyte and nonelectrolyte status of the host organism.

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.

Role of intracellular Ca2+ in modulation of tight junction resistance in A6 cells.

The role of intracellular Ca2+ in the development and maintenance of epithelial tight junctional integrity is poorly understood. We assessed tight junctional resistance (Rj) in confluent monolayers of A6 cells that were treated with mucosal amiloride such that the transepithelial resistance (Rt) reflects Rj. Solution Ca2+ concentration [Ca2+] was reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) addition to the bathing solutions. Reduction of mucosal [Ca2+] to 1 microM or reduction of serosal Ca2+ to 100 microM did not significantly alter Rt. However, a further decrease of serosal Ca2+ to 40 microM caused the resistance to fall to < 12% of the control value. Following restoration of serosal [Ca2+], Rt increased to a new steady-state value within approximately 15 min. The magnitude of recovery of Rt was inversely correlated with the length of time the epithelium was exposed to low serosal [Ca2+]. To further test the effects of asymmetric Ca2+ removal, the serosal [Ca2+] was chelated using EGTA to reduce Rt. When the Ca2+ ionophore A-23187 was subsequently added to the mucosal solution, Rt increased from 20% to 60% of the control level. In addition, cells were loaded with the fluorescent Ca2+ indicator, Calcium Green, and the temporal relationship between changes in Rt and intracellular Ca2+ was determined. Following removal of serosal Ca2+, cell Ca2+ decreased, followed by a decrease in Rt. In contrast, returning Ca2+ to the serosal bathing solution resulted in a parallel increase of both Rt and cell [Ca2+]. These data strongly suggest that changes in intracellular [Ca2+] play an important role in the regulation of Rj.

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.

Mycoplasma gallisepticum (MG)--laboratory and field studies evaluating the safety and efficacy of an inactivated MG bacterin.

A highly antigenic isolate of Mycoplasma gallisepticum (MG) was utilized in the production of an inactivated, oil-emulsified MG bacterin (MGB). Laboratory tests indicated that the bacterin was capable of protecting chickens from clinical signs of MG caused by intrasinus challenge with the R, S-6, PG-31, or 1150 strain of MG. Vaccinated turkeys also were protected from clinical signs of disease when challenged with MG. Use of the MGB in chickens under laboratory conditions resulted in a reduction in airsacculitis from 44% in nonvaccinates to 10% in vaccinates and further reduced the number of organisms present in the trachea post-challenge. Commercial chickens vaccinated subcutaneously midway or lower in the nape of the neck showed no untoward effects due to the bacterin. Those improperly vaccinated at the base of the skull developed a transient edema around the eye(s). This swelling did not appear to affect the performance of the chickens and had been reabsorbed by the next observation period. Subcutaneous inoculation should be at the mid or lower neck region. Field trials at a commercial egg operation comparing production efficiency showed that chickens vaccinated with the MGB had higher egg production, a greater percentage of eggs graded large and over, a smaller percentage of undergrades, and better feed conversion than chickens vaccinated with a live-culture, low-virulence Conn-F strain vaccine. The results of these studies indicate that the oil-emulsified MG bacterin is safe and highly efficacious.