Biomedical subjects

N F Gonzalez-Cadavid

Publications and source records attributed to N F Gonzalez-Cadavid.

30 records · Page 2Linked to original sources

PubMed1996

Androgens are not major down-regulators of androgen receptor levels during growth of the immature rat penis .

This study was undertaken to investigate the prevalent hypothesis that androgens are responsible for the organ-specific down-regulation of penile androgen receptors (ARs) and decline of penile growth in the rat during sexual maturation. Sexually immature male rats (21 days old) were castrated and treated for 3 days ("short-term"), with high doses of: (a) testosterone and the alpha-reductase inhibitor finasteride (T/F); (b) dihydrotestosterone (DHT); or (c) finasteride alone (F). Intact and castrate controls received vehicle only. PolyA + RNA was analysed by Northern blot hybridization and ARs were estimated in the penis and ventral prostates by (3-H)R-1881 binding in the cytosol. Short-term castration, with or without F, increased penile AR mRNA, whereas high doses of T/F and DHT reduced it considerably. Although penile cytosol AR concentration in the control castrates, with or without F, paralleled the AR mRNA rise, treatment with androgens left cytosol AR content per organ and AR concentration above those of the intact rat penis despite the drop in AR mRNA. A "long-term" treatment (10 days) on 19-day-old rats with either medium or high doses of T/F and DHT also failed to down-regulate penile cytosol ARs below the intact controls. Western blot analysis of penile cytosol AR levels confirmed these results. Block of pituitary FSH and LH release by a GnRH antagonist in castrates receiving T/F or DHT at high doses did not modify the response. In the case of intact rats, high doses of T/F or DHT actually increased penile cytosol AR content. No difference was observed between T/F and DHT effects. In contrast to what occurs during sexual maturation, the prostate ARs and growth rate responded to all treatments in a similar way to what was observed in the penis. Our results suggest that increases in serum T or DHT are not major factors in the physiological down-regulation of ARs and androgen-dependent growth in the rat corpora cavernosa.

Androgens↗

PubMed1995

Restoration of normal adult penile erectile response in aged rats by long-term treatment with androgens.

In order to determine whether the aging-related impairment of penile erection can be corrected by exogenous androgens, 20-mo-old ("old") rats received implants of Silastic tubing containing either testosterone (T) or dihydrotestosterone (DHT). Untreated aged-matched rats were used as reference controls, and values were compared with those obtained previously for 5-mo old ("adult") rats. After 45 days, groups of six rats were submitted to electrical field stimulation of the cavernosal nerve (EFS), and the intracavernosal pressure and mean arterial pressure (MAP) were recorded. Compared to the untreated old rats, the old animals receiving either T or DHT showed a similar and significant (p < 0.01) increase (56%) in the maximal intracavernosal pressure (MIP) to a level slightly above that of the untreated adult rats. The response was sensitive to the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME). The MIP:MAP ratio in the treated animals was nearly double that found in the old untreated controls. Penile NOS activity was measured by the [3H]L-arginine-citrulline conversion, and neuronal NOS (nNOS) levels were estimated by a semiquantitative Western blot assay with antibodies against human nNOS. No significant variations were observed in either NOS levels or NOS activity between the treated and untreated old rats. These results suggest that aging-related erectile dysfunction in the intact rat may be responsive to androgen therapy and that this correction is not associated with an increase in the basal levels of penile NOS, in contrast to what occurs in castrated rats.

Aging↗

PubMed1995

Reduction of penile nitric oxide synthase in diabetic BB/WORdp (type I) and BBZ/WORdp (type II) rats with erectile dysfunction.

Erectile dysfunction occurs frequently in human diabetes, and it is sometimes associated with hypogonadism. These conditions also develop in a model of insulin-dependent (type I) diabetes, the BB/WORdp (diabetic prone) rat but have not yet been investigated in the model of insulin-resistant (type II) diabetes, the BBZ/WOR rat. It is also unknown whether diabetes-related impotence is due to reduced levels of the mediator of penile erection, nitric oxide, caused by a decrease of nitric oxide synthase (NOS) in the penis. To clarify these questions, groups (n = 5-6) of diabetic BB/WORdp (insulin-maintained) and BBZ/WOR rats were age-matched with diabetic-resistant BB/WORdr and non-diabetic BB/WORdp rats and submitted to determinations of serum glucose, testosterone, and penile reflexes (cups and flips). Erectile dysfunction was found in all of type I and in most of type II diabetic animals (glycemias of 25.0 and 31.1 mM), at the selected mean ages of 310 and 180 days old, respectively. This was evidenced by over 95% decreases of erectile reflexes in both types of diabetes and was accompanied by 75% reduction of serum testosterone. Soluble NOS activity was measured in penile tissue from the diabetic rats with impaired erectile reflexes and in the corresponding controls, by the (3H)-L-arginine/citrulline conversion assay. The neuronal NOS isoform (nNOS) content was determined by a semiquantitative western blot assay. Both types of diabetes showed a marked decrease of penile NOS activity (74 and 55%, respectively), and a lower reduction of penile nNOS content (47 and 33%, respectively). No endogenous NOS inhibitor was detected in the diabetic type I penile cytosol by cross-mixing NOS activity assays. Our data support a common etiology for erectile dysfunction present in rats with types I and II diabetes mellitus and suggest that the etiology is related to a decrease of penile NOS derived in part from serum androgen deficiency.

Animals↗

PubMed1991

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.

Animals↗

PubMed1989

Direct sequencing analysis of exon 1 of the c-K-ras gene shows a low frequency of mutations in human pancreatic adenocarcinomas.

We have applied direct dideoxy sequencing of DNA fragments amplified in vitro by the polymerase chain reaction to the detection of mutations in exon 1 of the c-K-ras gene in human pancreatic adenocarcinomas. Four fresh frozen primary tumors, one metastatic tumor, and twelve formalin-fixed paraffin embedded tumors were analysed. Only three cases showed a possible mutation in codon 12 in a small population of cells, in contrast to the high frequency reported for this alteration with the RNAase A protection assay and allele-specific oligoxynucleotide hybridization. No major difference in sensitivity was found between DNA sequence analysis, and the latter method. Our results suggest that if c-K-ras mutations are indeed present in pancreatic adenocarcinomas at the high frequency reported by others, they must be confined to a small fraction of the cell population to escape detection by direct sequencing. Such a phenomenon would have implications for c-K-ras mutations in the pathogenesis of pancreatic adenocarcinomas.

Adenocarcinoma↗

PubMed1978

Comparative effect of a family of substituted thiopseudoureas on protein synthesis by rat liver and Walker carcinoma ribosomes.

In order to assess the role played respectively by the pseudothiourea group and the alkylic chain in the inhibition of protein synthesis and tumour growth caused by compound AHR-1911, a series of eight related substances were studied. The blockade of protein synthesis on liver and Walker carcinoma ribosomes and on suspensions of Walker carcinoma cells, depended essentially on the length of the alkylic chain and the substitution in C-1. The minimum chain was 9 carbons and a plateau in the activity was reached at 11 carbons. Replacement of the thiourea group in C-1 by an NH2 group did not change the pattern. A double bond in the distal section of the chain (AHR-1911) increased inhibition on intact cells with a parallel decrease in cytotoxicity, and reduced the aggregation of ribosomes, protein synthesis factors and other proteins. The antitumor effect depends on the pseudothiourea group and is not caused primarily by interference with protein synthesis. Aminoacyl tRNA binding and transfer appeared to be targets of AH4-1911, but this did not affect significantly tRNA change or nascent peptide release. Drug binding to ribosomes and their subsequent aggregation can be regulated by K+ concentration and temperature. It is assumed that the inhibition of protein synthesis is caused by AHR-1911 effects on elongation factors, impairing their interaction with ribosomes.

Animals↗

PubMedDate not supplied

Molecular pathophysiology and gene therapy of aging-related erectile dysfunction.

Erectile dysfunction (ED) is a major public health problem that seriously affects the quality of life of patients and their partners. ED is mainly associated with vascular disease, diabetes, smoking, and radical prostatectomy, and its prevalence increases significantly with aging. Vasculogenic ED, specifically corporal veno-occlusive dysfunction (CVOD), is caused by the impairment of the relaxation of the smooth muscle in the penile corpora cavernosa and occurs in 2/3 of cases, whereas the less common neurogenic ED is due to a defective nitrergic neurotransmission triggered by the sexual stimulus, either at the central hypothalamic and spinal levels or at the penile nerves. Based on animal and cell studies, neurogenic ED is assumed to be caused mainly by: (a) an insufficient synthesis of nitric oxide (NO) due to a decrease in the levels of the penile neuronal nitric oxide synthase (PnNOS) or the impairment of its regulation by protein effectors (NMDA receptor, protein inhibitor of nNOS: PIN), occurring in the neuronal bodies or nerve terminals, or (b) a loss of the cells themselves by apoptosis caused by the induction of inducible NOS (iNOS) and the production of peroxynitrite. In contrast vasculogenic ED, although may involve endothelial damage and down-regulation of endothelial NOS (eNOS), appears to be mainly caused by the relative loss of smooth muscle cells and replacement by collagen fibers (fibrosis) that impairs tissue compliance. In this case, iNOS induction may not be deleterious, but a defense mechanism preventing excessive collagen deposition. Gene therapy to the penile corpora cavernosa of cDNAs expressing PnNOS or eNOS, or counteracting PIN, has been effective in ameliorating ED in the aging rat model that exhibits both neurogenic ED and CVOD. cDNA constructs for other genes involved in the control of penile erection have also been successfully tested. Gene transfer into the penis may soon translate to the clinic as a therapy aimed to cure the underlying conditions in ED, including fibrosis, as opposed to the facilitation of erection on demand offered by the current oral therapies.

Aged↗

PubMedDate not supplied

Therapeutic stimulation of penile nitric oxide synthase (NOS) and related pathways.

The advent of a novel oral treatment for erectile dysfunction appears to have reduced the urgency for seeking alternative therapeutic innovations. However, the currently available therapies can be viewed as simply palliative, requiring on-demand access and having a significant failure rate. Therefore, additional approaches for patients unresponsive to any type of medical treatment require investigation. The modulation of the synthesis of nitric oxide (NO), the main mediator of penile erection, is an attractive target for such an approach. Models for human erectile dysfunction related to aging, diabetes and endocrine disorders have been characterized in the rat. The affected animals exhibit a reduced erectile response to electrical stimulation of the cavernosal nerve and/or a loss of spontaneous erectile reflexes. In virtually all cases, the content and/or enzyme activity of penile NOS is significantly reduced, or NO synthesis may be insufficient to overcome the relatively poor compliance of the penile corpora cavernosa smooth muscle (aging). This led to the proposal that the stimulation of penile NO synthesis may be a viable therapy for erectile dysfunction. The upregulation of penile NOS activity may be achieved by: a) increase in NOS substrate concentration, b) stimulation of NOS activity through related pathways or c) blockade of endogenous NOS inhibitors. We have shown that approach a ameliorated the erectile dysfunction of aging rats by elevating intracavernosal NOS substrate through long-term oral administration of high doses of L-arginine. Evidence supporting approach b is based on our recent discovery that the main stimulating pathway for NOS, the NMDA receptor, is present in the penis. However, NMDA receptor antagonists unpredictably trigger cavernosal relaxation in vitro in a NO-independent pathway. Finally, in the case of approach c we have found that a protein inhibitor of NOS (PIN) is expressed in the corpora cavernosa. Penile NOS content may be pharmacologically increased by: d) induction of NOS expression, and e) gene therapy with NOS cDNA. Approach d applied to the penis ameliorated erectile dysfunction in aging rats, but may be clinically unfeasible because of the risk of side effects. Our laboratory cloned the inducible and neuronal NOS isoforms expressed in the penis (PiNOS and PnNOS, respectively), and showed that approach e, based on gene transfer of PiNOS into the rat corpora cavernosa improved the erectile response. Since PnNOS is a nNOS variant different from the one expressed in the CNS, gene therapy with PnNOS is promising in combination with viral vectors and tissue-specific promoters. The aim is to achieve a long-lasting increase in penile NOS that is responsive to sexual stimulation. Following validation in animal models, oral L-arginine, intracavernosal antagonists of NMDA receptor and PIN, and intracavernosal or systemic PnNOS, may eventually translate to clinical trials.

Journal Article↗

PubMedDate not supplied

Presence of NMDA receptor subunits in the male lower urogenital tract.

Some sexual responses in the male rat, specifically penile erection, are controlled by neural circuits in the brain and spine that are stimulated by the binding of excitatory amino acids (EAAs) to the postsynaptic N-methyl-D-aspartate receptor (NMDAR). In the hypothalamus, EAA/NMDAR interaction triggers the activation of neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO). The local synthesis of this neurotransmitter in the penile nerve terminals causes corpora cavernosal relaxation and erection. During sexual activity, NO is assumed to participate in seminal emission and ejaculation in the prostate and to inhibit voiding reflexes in the bladder. This study aimed to determine in vitro whether NMDAR is present in these organs and whether it affects the tone of tissue strips through an NO-dependent mechanism. We obtained penile, urinary bladder, and ventral prostate tissues from adult male rats and homologous surgical tissues from human male patients. We detected the NMDAR protein by Western blot and determined the binding of the NMDA antagonist, 3H-CGP. The NMDAR messenger ribonucleic acid (mRNA) was detected by reverse transcription/polymerase chain reaction and identified by cloning and sequencing. The in vitro response to NMDAR antagonists was measured in tissue strips that were precontracted with bethanechol or electrical field stimulation (in rat and human bladder), phenylephrine (in human corpora cavernosa), or norepinephrine (in human prostate). The NMDAR2B protein; ligand-binding activity; and NMDAR1, 2A, and 2B mRNAs were detected in all tissues studied. We found an NMDAR1 variant in rat prostate and penis and in human prostate that is larger than its cerebellar counterpart, but it encodes a 767-amino acid truncated protein (NMDAR1-T). The in vitro contraction of tissue strips was inhibited by NMDAR antagonists against the following sites: polyamine (with ifenprodil); ion channnel high affinity (with dizocilpine); ion channel low affinity (with memantidine, dextrometorphan, and ketamine); and, in an NO-independent, nonadrenergic-noncholinergic pathway that was only partially affected by EAAs. We conclude that, in vitro, all the essential NMDAR subunits are present in the lower urogenital tract, a novel variant of subunit 1 is expressed, the tissues bind an NMDAR ligand, and the NMDAR antagonists induce relaxation of tissue strips. Further work is necessary to determine whether the NMDAR subunits form a fully active receptor and participate in the control of organ tone that is relevant to male sexual activity.

Amino Acid Sequence↗

PubMedDate not supplied

Efficient malignant transformation of rat embryo fibroblasts by genomic DNA from Walker carcinoma cells.

DNA isolated from Walker carcinoma ascites cells was transfected into primary rat embryo fibroblasts (REF), selecting transformed cells by growth in soft agar after prolonged propagation in monolayer. Both high molecular weight genomic DNA and a partially purified mitochondrial DNA fraction were able to transform REF with high efficiency, whereas pure mitochondrial DNA failed to elicit a transformed phenotype. Hybridization experiments showed that the mitochondrial DNA fraction contained DNA species of presumably extramitochondrial origin. Colonies were cloned into morphologically transformed, foci-forming, immortalized cell lines, showing different degrees of chromosomal alterations, tumorigenicity, and production of cell growth factors. These results indicate that although REF are refractory to genomic neoplastic DNA or to single cloned oncogenes in the absence of enhancers, they can be efficiently transformed by chromosomal DNA from a highly malignant tumor under conditions selecting against the remaining normal cells.

Animals↗

PubMedDate not supplied

Prevention of seminiferous tubular atrophy in a naturally cryptorchid rat model by early surgical intervention.

In an attempt to determine whether the seminiferous tubular atrophy of the cryptorchid testis is preventable by early surgical correction of the cryptorchid state, aberrantly developed gubernacula destined to result in a cryptorchid testis in the Long-Evans cryptorchid (LE/ORL) rat were surgically reimplanted to the bottom of the scrotum on day 10 to 12 of age. Testis descent was monitored and the changes in testicular histology and in the volumes of the seminiferous tubules and Leydig cells were examined at day 60. As expected, normal testis descent occurred on or about day 25. Compared to untreated undescended testes at day 60, relative seminiferous tubular volumes (volume: % +/- SEM) were significantly increased by early surgical reimplantation of the gubemacula (89 +/- 1 vs. 66 +/- 3; P < 0.01). Absolute seminiferous tubular volumes (microliter +/- SEM) were also significantly increased by early surgical intervention when compared to undescended nontreated testes (893 +/- 27 vs. 170 +/- 12; P < 0.01). The testes of the surgically corrected cryptorchid animals were similar in all respects to those found in the descended testes of the sham-operated controls. Relative Leydig cell volume (% +/- SEM) was increased in the untreated cryptorchid testes compared to the surgically corrected testes (5.2 +/- 0.6 vs. 1.2 +/- 1.0; P < 0.05). Relative Leydig cell volumes in the surgically corrected testes were not significantly different from those found in the sham-operated descended controls. A modest but significant (P < 0.05) increase in absolute Leydig cell volume was also noted in the cryptorchid testes when compared both to normal controls or surgically corrected cryptorchid testes. From these observations, we conclude that early gubernaculopexy reverses the histologic changes normally seen in the cryptorchid rat testis to a relatively normal histologic architecture. These data provide experimental evidence to support the value of orchiopexy in the treatment of cryptorchidism.

Animals↗

PubMedDate not supplied

Transforming growth factor-beta1 (TGF-beta1) in penile and prostate growth in the rat during sexual maturation.

The goal of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) may contribute to the arrest of penile growth and the down-regulation of androgen receptors (AR) that occur during sexual maturation in the rat penis. For this purpose, body, penis, and prostate weights were obtained from male rats of increasing ages, and penis and prostate TGF-beta1 concentrations were determined by a sandwich enzyme-linked immunosorbent assay. The cytosol fraction was obtained from the shafts and glandes of immature (19-day-old) and adult (90-day-old) rat penises, and ARs were measured by a western blot assay. The effect of exogenous TGF-beta1 on penile growth was examined in vivo in two groups of immature rats (21 and 27 days old) implanted with miniosmotic pumps delivering either human TGF-beta1 or vehicle only directly into the corpora cavernosa for 6 days. The penises, prostates, and testes were weighed, and the AR content was estimated by western blot. The growth rate of the penis declined after 8 weeks of age, whereas the ventral prostate growth rate increased until 14 weeks of age and then slowed down. The content of penile AR protein decreased seven-fold in the adult rats compared to the immature animals. Penile TGF-beta1 concentration increased nearly three-fold from the 19-day-old rats to a peak at 60 days of age and then decreased over the next 4 months to the initial levels. In contrast, TGF-beta1 concentration in the prostate was not significantly affected by age and remained below the lowest penile values in all age groups. Transforming growth factor-beta1 given locally to the penis reduced penile shaft weight by 38 and 22% in two groups of immature rats, while the weights of the penile glans, testis, and ventral prostate remained unaffected. Androgen receptor content was higher in the glans than in the shaft and was not changed by TGF-beta1 treatment. These results suggest that the increase of TGF-beta1 levels in the penis may reinforce growth arrest caused by the down-regulation of penile ARs, whereas the maintenance of a high content of ARs and a low TGF-beta1 concentration may allow prostate growth to continue.

Aging↗