Testosterone and erectile function in hypogonadal men unresponsive to tadalafil: results from an open-label uncontrolled study.

The study was aimed at investigating the efficacy of tadalafil (Cialis) in combination with transdermal testosterone (Testogel) for the treatment of tadalafil-refractory erectile dysfunction in hypogonadal patients. In an open-label, retrospective trial, 69 hypogonadal nonresponders to tadalafil monotherapy (mean age: 59 years, total testosterone < or =3.4 ng ml(-1)) were randomly divided into two homogeneous groups. Group I (n = 35) received Testogel (5 g containing 50 mg testosterone, daily) for 4 weeks, followed by concurrent therapy with tadalafil (20 mg, twice a week). Group II (n = 34) was assigned to treatment with Testogel (5 g containing 50 mg testosterone, daily) for a duration of 10 weeks before adjunctive therapy with tadalafil was initiated. Total testosterone levels were measured at baseline, week 4 and week 10. Sexual function was assessed employing the International Index of Erectile Function (IIEF). As an additional measure of efficacy, a questionnaire completed by the patients' partner was used. Mean testosterone levels were observed to increase from baseline to study end. Following 4 weeks of therapy, an improvement in Erectile Function (EF) from baseline was observed, which was greater in group I than in group II. The assessment after week 10 showed that EF had further increased and was quite similar now in both groups. Partners found that erectile capacity had greatly improved from baseline to study end. No adverse effects have been observed. These data suggest that combination therapy with testosterone and tadalafil is an effective means in a subset of hypogonadal patients who did not respond to tadalafil alone. We assume that testosterone-induced remodelling of penile tissue structure is one underlying reason for the observed improvement of erectile function. The results imply that this process may require a longer period of testosterone administration than 4 weeks.

The capillary transport system for free fatty acids in the heart.

The nature of the process by which free fatty acids, which are tightly bound to albumin, traverse the endothelium of cardiac capillaries to reach the cardiac muscle cells, so that they are extracted to a net extent of approximately 40%, needs clarification. Previous studies have indicated that a membrane fatty acid-binding protein provides for carrier-mediated uptake of free fatty acids by isolated hepatocytes, cardiomyocytes, and jejunal mucosal cells. A monoclonal monospecific antibody was prepared against purified membrane fatty acid-binding protein from rat liver. Multiple-indicator dilution experiments were carried out in the isolated rat heart with labeled albumin, sucrose, and palmitate in the presence of control perfusate or perfusate containing either specific antibody or comparable nonspecific myeloma cell supernatant (each of the latter containing additional albumin, in identical concentrations). Analysis of the labeled-sucrose curves provided a permeability-surface area product for sucrose to which that for palmitate could be compared. In comparison with control supernatants, myeloma supernatant produced a minor inhibition of palmitate uptake, as a result of the increase in albumin concentration. The specific antibody, which contained identical albumin concentrations, produced a major inhibition of palmitate uptake, significantly greater than with the myeloma supernatant. The data indicate that the membrane fatty acid-binding protein mediates the transfer of free fatty acid across the endothelial cells of cardiac capillaries for presentation to heart muscle. Passive intramembrane lateral diffusion of palmitate could not provide an explanation for the findings.

Identification and characterization of a monoclonal antibody to the membrane fatty acid binding protein.

A monoclonal antibody to the rat liver membrane fatty acid binding protein (MFABP) was prepared by immunizing mice with purified MFABP isolated from solubilized rat liver plasma membrane proteins by oleate-agarose affinity chromatography technique. The monoclonal antibody K15/6 identified a single 40 kDa protein in rat liver plasma membranes with pI values of 8.5, 8.8 and 9.0, which is identical to the authentic MFABP, but clearly distinct from rat mitochondrial GOT. The antibody K15/6 selectively inhibited cellular influx as well as membrane binding of fatty acids, but not of cholesterol or vitamin E. The same antibody was used in immunofluorescence, ELISA and Western blot analysis to determine the subcellular and organ distribution pattern of MFABP. The protein was identified in rat liver plasma membranes and mitochondria, but in no other cell compartment. It was detectable in homogenates of rat liver but not in homogenates of other organs. Therefore, the monoclonal antibody K15/6 represents an organ specific antibody to MFABP which reveals inhibitory action on membrane binding/transport of fatty acids.

The membrane fatty acid-binding protein is not identical to mitochondrial glutamic oxaloacetic transaminase (mGOT).

For evaluation whether the membrane fatty acid-binding protein is related to mGOT, studies on the structure and function of both purified proteins were performed. Physicochemical characterization revealed that both proteins are different: the membrane fatty acid-binding protein has a molecular weight of 40 kD and a pI of 8.5-9.0, whereas rat mGOT has a molecular weight of 44 kD and a pI of 9.5-10.0. According to this distinct differences, they migrated separately on 2-dimensional electrophoresis. Furthermore, monospecific antibodies against the membrane fatty acid binding protein did not react with rat mGOT. In co-chromatography studies only the membrane fatty acid-binding protein showed affinity for long chain fatty acids, but not mGOT. Moreover, membrane binding studies were performed with the monospecific antibody to the membrane fatty acid binding protein. The inhibitory effect of this antibody on plasma membrane binding of oleate was reversed after preabsorption of the antibody with the membrane fatty acid binding protein, but was not affected after preabsorption with mGOT. These results indicate that the membrane fatty acid binding protein and mGOT are structurally and functionally not related. The data also support the significance of this membrane protein in the plasma membrane binding process of long chain fatty acids.

Cellular uptake of conjugated bilirubin and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 is mediated by a membrane BSP/bilirubin binding protein.

Cellular influx kinetics of 4-50 microM bilirubin diglucuronide and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 was examined at 37 degrees C. In confluent monolayer cultures, cellular influx of increasing concentrations of conjugated bilirubin and BSP revealed similar saturation kinetics with Km values of 9.9 and 12.1 microM, and Vmax values of 0.512 and 0.473 nmol.mg cell protein-1.min-1, respectively. Uptake of [3H]bilirubin diglucuronide was competitively inhibited by unlabeled BSP, and was temperature dependent with maximal cellular influx rates at 37 degrees C. When the confluent monolayer cultures were pretreated with a monospecific antibody to the rat liver BSP/bilirubin binding membrane protein, initial uptake rates of conjugated and unconjugated bilirubin as well as of BSP were significantly inhibited, whereas uptake of oleate was not affected. Furthermore, immunoblot analysis of the homogenate of Hep G2 cells with the same antibody revealed predominant reactivity with a 55 kDa protein. These data suggest that cellular uptake of bilirubin and related cholephilic organic anions by the human hepatoma cell line Hep G2 is mediated by a specific 55 kDa membrane BSP/bilirubin binding protein.

Cellular influx of sulfobromophthalein by the biliary epithelium carcinoma cell line Sk-Cha-1 reveals kinetic criteria of a carrier-mediated uptake mechanism.

Cellular uptake of the cholephilic organic anion sulphobromophthalein (BSP) by the human biliary epithelium carcinoma cell line Sk-Cha-1 was examined at 37 degrees C. In confluent monolayer cultures the cellular influx rate of increasing concentrations of [35S]BSP followed saturation kinetics with a Km value of 18 microM and a Vmax value of 243 pmol.min-1.mg protein-1. Uptake of [35S]BSP was competitively inhibited by the presence of bilirubin diglucuronide, but not by taurocholate or cholate. Furthermore, uptake was temperature dependent with maximal cellular influx rates at 37 degrees C.

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process.

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.