Expression of the androgen receptor gene in rat penile tissue and cells during sexual maturation.

The cessation of rat and human penile growth at completion of sexual maturation appears to be related to a tissue-specific decrease in the number of androgen receptors (AR) in the penis. To find out whether this is due to either transcriptional or translational regulation of the AR gene, we determined the levels of AR mRNA by Northern blots in the corpora cavernosa from groups of 16-, 19-, 22-, 27-, 52-, and 90-day-old rats. The AR mRNA rapidly decreases with age and is nearly undetectable in the 52- and 90-day-old rats, paralleling the decline in AR number. The persistence of a low amount of AR mRNA in the adult penis was confirmed by reverse transcription/polymerase chain reaction amplification of total RNA, and its level was estimated by a semiquantitative modification of this procedure at less than 1/25th of that found in the youngest rats. Smooth muscle cells derived from the 16- and 90-day-old corpora cavernosa express in the AR gene in vitro in approximately the same levels, suggesting that there are factors in culture that up-regulate the AR mRNA. Our results are compatible with the assumption that the age-dependent decrease in AR in the rat penis is due to transcriptional regulation, although they do not exclude the less likely alternative of a selective effect on AR mRNA stability and support the use of this model for studying tissue-specific factors controlling the developmental expression of the AR gene.

Effect of 5-alpha-reductase inhibitor, 4-MAPC, on testicular descent in male rat.

Testicular descent has been reported to be a dihydrotestosterone (DHT) dependent event. To further elucidate the role of DHT in the process of testicular descent, a group of rats were treated with the 5-alpha reductase inhibitor, 4-MAPC, from birth to day 28 of age and the incidence of testicular descent as well as ventral prostate weight was noted at day 29 of age. It was determined that in the doses used, 4-MAPC failed to prevent testicular descent. Because 4-MAPC inhibited ventral prostate weight by only 53% (as compared to a 75% inhibition by castration), the failure of the 4-MAPC to prevent testicular descent could be due to its inability to completely inhibit tissue 5-alpha reductase activity. The results of this study do not mitigate against the role of other nonhormonal factors working in tandem with DHT in the induction of testicular descent in this animal model.

Effect of the antiandrogen, WIN 49,596, on the descent of the testis in the male rat.

Testicular descent in the male rat is believed to be an androgen dependent event with dihydrotestosterone the most likely active androgen. To provide further insight into the endocrinology of this important physiological event, we treated male rats with the antiandrogen, WIN 49596 (50 mg./kg./day), from day 1 to day 27 of age and evaluated its effect on the post-natal androgen-dependent events in this animal model. It was determined that while treatment with WIN inhibited the weights of the ventral prostate, seminal vesicles and penis when compared to those seen in castrate animals, the drug only caused a 19% (3/16) inhibition in the descent of the testes when compared to the control group (0/16; p = 0.112). These data together with those previously obtained in animals exposed to selective inhibitors of the 5-alpha reductase enzyme suggest that other factors possibly working in tandem with androgens play a predominant role in testicular descent in the rat.

Inhibition of fatty acid-supported mitochondrial respiration by cyclosporine.

We have shown that, in addition to inhibition of the succinate-supported energy pathway (5), CS inhibition of mitochondrial Complex II activity also limits fatty acid oxidation. These results are consistent with the participation of altered lipid metabolism in CS nephrotoxicity.

Long-term effects of prepubertal testicular vessel ligation on testicular function in the rat.

To determine the effects of unilateral testicular vein and artery ligation in the immature rat on the function and final location of the testis at adulthood, 10-day-old male rats underwent either a sham operation or unilateral ligation of these vessels of the still undescended testis. Testicular location, blood flow, size and histology as well as ventral prostate weights were measured 50 days later at adulthood. At age 60 days, it was determined that all testes were descended into the scrotum, and there were no differences in testis and ventral prostate weights, intratesticular sperm counts and mean seminiferous tubular area between the control and sham operated animals. However, there was an 18% reduction in testicular blood flow (ml. per 100 gm. per minute +/- standard error of mean) in the operated animals when compared to the sham (20.43 +/- 1.10 versus 16.69 +/- 0.74, p less than 0.02). These data indicate that although there is a slight but significant reduction in testicular blood flow at adulthood when the testicular artery and vein are ligated early in life, this diminution is not sufficient to alter the ultimate location, testicular weight and spermatogenic function of the testis. This would suggest that after ligation of the main testicular vessels to the immature testis, the collateral blood supply is able to compensate with time to allow normal growth and development of the testis. These experimental observations provide additional support for the 2-staged approach to the high undescended testis whereby the testicular vessels are initially ligated and a subsequent procedure is performed to place the undescended testis into the scrotum.

Kinetics of cyclosporine A-induced inhibition of succinate-coenzyme Q dehydrogenase in rat renal cortical mitochondria.

The kinetic mechanism of succinate-coenzyme Q dehydrogenase (Complex II) inhibition by cyclosporine A (CS) on rat renal cortical mitochondria was investigated. CS showed two modes of inhibition of Complex II of the mitochondrial electron transport system: (a) a mixed linear noncompetitive inhibition of resting succinate-limited and ADP-stimulated respirations suggesting that CS binds to Complex II at a different site than the substrate, affecting the dissociation constant for the enzyme-substrate complex and (b) a competitive inhibition of the DNP-stimulated electron transport system suggesting competition with the oxidized form of a component of Complex II. CS action to renal mitochondrial Complex II limits its function, an effect which may be related to CS nephrotoxicity.

Cyclosporine augments renal mitochondrial function in vivo and reduces renal blood flow.

The in vivo action of cyclosporine A (CS) on rat renal cortical mitochondria was investigated. CS (30 mg.kg-1.day-1) given orally to rats for 30 days caused an augmentation of renal mitochondrial oxidative phosphorylation. The ADP-stimulated respiratory rate was increased by 37.0% with glutamate plus malate as respiratory substrates (P less than 0.025) but not with succinate-supported respiration, indicating enhancement of mitochondrial complex I activity. This reaction may be a response to the 32.5% reduction of renal blood (P less than 0.005) in the CS-treated group, possibly serving to maximize ATP synthesis during ischemia. Ligation-induced decreases in renal blood flow also resulted in enhancement of mitochondrial complex I activity.

Effect of cyclosporine on steroidogenesis in rat Leydig cells.

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.

Two biochemically distinct populations of histaminocytes separated by isokinetic sedimentation of dispersed rat gastric cells.

Two populations of histaminocytes, with different sedimentation rates (SR), were separated by a computer developed isokinetic gradient using dispersed rat gastric mucosal cells. Histamine content, histidine decarboxylase (HDC) activity and incorporation of radiolabelled histidine metabolites were used to assess the migration of specific cells throughout the gradients. One histaminocyte population, with cells of lower SR, contained high HDC activity and undetectable levels of histamine, whereas the other population, with cells of higher SR, contained lower HDC activity and high concentration of histamine. Both types of histaminocytes incorporated 3H-histidine metabolites. Electron microscopy showed that the fractions containing histaminocytes with lower SR had 3.5 times more endocrine ECL cells than the original population of dispersed fundic cells and lacked A and D cells, whereas the fractions with histaminocytes of higher SR were associated with a 2.7 times higher concentration of A and D cells and with a 7.7 times higher ratio of a variety of partial cells with a distinct mitochondrial morphology. These results are consistent with prior novel information regarding the separation of two populations of rat histaminocytes using different sedimentation techniques.

Separation and characteristics of two histaminocytes from rat gastric mucosa.

To determine the properties of rat gastric cells involved in histamine metabolism (histaminocytes), fundic mucosa was enzymatically dispersed prior to separation by sedimentation methods. The distribution of histamine content, histidine decarboxylase (HDC) activity and incorporation of radioactive histidine metabolites were used to determine the characteristics of various populations of gastric cells. All activities measured, as well as most of the dispersed gastric cells, occurred in a narrow range of density between 1.083 and 1.091 g/ml. Velocity sedimentation showed that two populations of histaminocytes can be distinguished. One population has a higher sedimentation rate, suggesting a larger size, contains histamine, HDC activity and incorporates radioactive metabolites. Another population, in fractions with lower sedimentation rates, contains little histamine, has a higher HDC activity than the previous population and also incorporates radiolabelled histidine metabolites. For the first time, two populations of viable histaminocytes have been separated that differ in their biochemical properties.

Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and after injection of erythrocytes, carbohydrates, and other materials.

Agglutinin activity and total protein concentration in earthworm coelomic fluid were determined 24 hours after injecting different erythrocyte types and carbohydrates. Agglutinin titers increased from 14 to 1216 after a single injection of erythrocytes. At the same time, protein concentrations increased by variable amounts, suggesting an indirect correlation between increased agglutinin titer and protein concentration. To understand the nature of erythrocyte determinants responsible for inducing agglutinins and other proteins, separate groups of earthworms were injected with different carbohydrates. At 24 hours, coelomic fluid was assayed for agglutinin titer and protein concentration. The injection of different carbohydrates, amino acids, nucleotides and protein (BSA), like erythrocytes, also increased protein concentration and agglutinin titers. Changes in the protein concentration and composition of coelomic fluid were studied by standard and bidimensional immunoelectrophoresis. Different patterns and numbers of components were found before and after injection. At least two different components were present after injecting carbohydrates or erythrocytes, which may be related specifically to the agglutinins. Clearly, earthworms can synthesize and shed these molecules at rapid rates into the coelomic fluid. These molecules may be involved in earthworm defense.

Splenic and thymic histamine forming enzyme activity and weights following organ allograft and tumor cell transplantation.

Kinetic of the increasing activity of histamine forming enzyme--histidine decarboxylase in host spleen and thymus, following kidney and heart allografting and after tumor cells transplantation is presented.

Decreases in histamine forming enzyme activity of non-metastasizing fibrosarcomas in hamsters with progressive tumor growth.

A marked and progressive decrease in the activity of the histamine forming enzyme, histidine decarboxylase (HDC), of tumors was found to be associated with the progressive growth of SV-40 virus induced and transplanted syngeneic non-metastasizing fibrosarcomas in inbred LSH Syrian hamsters. Histamine forming enzyme activity was highest in the smallest tumors (p < .005) and in the tumors with the slowest growth rate (p < .005, r - 0.84). Tumor histamine forming enzyme activity was highest for each interval of animal exposure to inoculated tumor cells in those animals which had limited their tumor growth to the smallest tumor size. These findings suggested a local anti-inflammatory effect of progressive tumor growth. Induced local inflammation by repeated intratumor injections of bradykinin markedly elevated tumor histamine forming enzyme activity above expected levels for tumors of the same size in a small group of individual animals which were sampled at random from a larger group of animals which were being studied for the tumor growth kinetics effects of repeated intralesional injections of bradykinin. Tumor histamine forming enzyme activity was highest in those animals which were managed by the frequency of injection and dose schedules which were found in the tumor growth kinetics study to be most effective in limiting tumor growth. These findings suggested that the observed anti-inflammatory effects of progressive tumor growth may be reversed by locally induced inflammation at the tumor site with beneficial effects on tumor growth.

Histidine decarboxylase activity in fetal intrauterine growth-retarded rats.

Fetal intrauterine growth retardation was induced in nine rats at 17 days' gestation by ligating the blood supply to one of the uterine horn. The 27 festuses from the uterine vessel ligated side were the intrauterine growth retarded fetuses (IUGR) and the 37 fetuses from the nonligated side were controls. The mean weight of the IUGR fetuses was 3.4 +/- 0.3 g which was significantly smaller (P less than 0.005) than the mean weight of the control fetuses, 4.0 +/- 0.3 g. Whole body histidine decarboxylase (HDC) activity was assayed by a radioisotope method. The HDC activity per g tissue was not correlated to body weight in the control fetuses. This is in contrast to the IUGR festuses in which the HDC per g tissue was significantly and inversely related to body weight (r - 0.48, P less than 0.001).

Alterations in kinins of coronary blood from rat heart homografts.

Forty-eight heterotopic heart transplants (33 homografts and 15 isografts) were performed in inbred rats. Levels of kallikrein, prekallikrein, and kallikrein inhibitor were determined in coronary sinus venous blood of grafted hearts at various intervals postgrafting. Homograft observations were assessed against those in nonrejecting isografts. Significant increases in homograft prekallikrein level on day 4 were followed by significant increases in homograft kallikrein activity on day 6. Kallikrein inhibitor levels in homografts were consistently increased in relation to isograft levels. Kallikrein and inhibitor activity showed an inverse relationship in their respective changes. High levels of kallikrein and prekallikrein occurred in homografts ten days posttransplantation. Immunological rejecting homografts showed meaningful changes in the activity of kinin-forming substances known to participate in inflammation.

Accumulation of mononuclear cells in tumors with growth slowing and elevation in host splenic histidine decarboxylase activity following repeated tumor injections with bradykinin.

Repeated intratumor injections of SV-40 virus-induced and transplaned syngenic fibrosarcomsa in hamsters with bradykinin (BK) has produced markded slowing of tumor growth in comparison with control saline injections. Growth slowing was greatest when the injections were daily, with a decrease in growth slowing as injections became less frequent. The growth slowing also was dose dependent (greater with 250 mug BK injections than with 50 mug BK injections). BK-injected tumors, on histological study, were found to have marked infiltration with mononuclear cells. This was not encountered in noninjected or saline-injected tumors. Significant mononuclear cell infiltration of noninjected tumors was found in two tumor animals which had had one tumor injectecd with BK. Splenic histidine decarboxylase (HDC) activity was higher in BK tumor-injected animals than in saline tumor-injected animals. Splenic HDC activity was higher when studed nearer the period of daily intratumor injections. The findings of this study suggest a potential role of inportance for inter-related vasoactive substances which act as mediators of inflammation in the study and therapy of neoplasia.

Accumulation of 131I hippurate in rat heart allografts during rejection.

The accumulation of 131I hippurate in heterotopic (abdominal) rat heart allografts (Brown-Norway to Lewis) in comparison with native (autologous) hearts 6 and 7 days after transplantation was studied by noninvasive scintigraphic imaging and by direct isotope counting of excised hearts. Six hours after intravenous isotope injection, isotope accumulation in the allografted heart was clearly identified by scintigraphic study in comparison with the background, kidneys, urinary bladder, and native heart. Six and 7 days after transplantation, excised allografted hearts were significantly increased in weight in comparison with native hearts. A significant increase in isotope accumulation in allografted hearts in comparison with native hearts was found both per heart and per gram of heart. The findings of this study suggests the potential usefulness of 131I hippurate in the immunoinflammatory monitoring of organ allograft rejection by direct counting as well as by noninvasive scintigraphic study if sufficient time is allowed to permit clearance of high concentrations of isotope from the background areas, kidneys, and urinary bladder.